beta-cyclodextrin conjugated magnetic nanoparticles for … · 2018. 1. 10. · beta-cyclodextrin...

BETA-CYCLODEXTRIN CONJUGATED MAGNETIC

NANOPARTICLES FOR BIO- AND ENVIRONMENTAL

APPLICATIONS

ABU ZAYED MD. BADRUDDOZA

NATIONAL UNIVERISTY OF SINGAPORE 2011

BETA-CYCLODEXTRIN CONJUGATED MAGNETIC

NANOPARTICLES FOR BIO-AND ENVIRONMENTAL

APPLICATIONS

ABU ZAYED MD. BADRUDDOZA (B.Sc., Bangladesh University of Engineering & Technology)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2011

To My Parents

With Love

Acknowledgement

i

Acknowledgements I would like to take this opportunity to express my sincere thanks and grateful

acknowledgement to my supervisors, Associate Professorial Fellow Mohammad

Shahab Uddin and Associate Professor Kus Hidajat, for their valuable guidance,

support and encouragement throughout the research program. Their meticulous

attentions to details, incisive but constructive criticisms and insightful comments have

helped me shape the direction of this thesis research to the form it is presented here.

I would also like to give my deepest appreciation to all the staff members in the

Department of Chemical and Biomolecular Engineering and all my colleagues in the

lab, who have given me great help in my research work.

I would also like to extend my eternal gratitude to my parents, wife and my baby

daughter for their love, support and dedication.

Finally, my thanks to National University of Singapore for providing the Research

Scholarship and to the Department of Chemical and Biomolecular Engineering for

providing the facilities to take this project through in its entirety.

Table of contents

ii

Table of contents Acknowledgements ........................................................................................................... i Table of contents .............................................................................................................. ii Summary .......................................................................................................................... v Nomenclature ................................................................................................................ viii List of figures ................................................................................................................... x List of tables .................................................................................................................. xvi Chapter 1 Introduction ................................................................................................ 1

1.1 General background ............................................................................................... 1 1.2 Research objective and significance ...................................................................... 3 1.3 Organization of the thesis ....................................................................................... 6

Chapter 2 Literature Review ....................................................................................... 7

2.1 Magnetic nanoparticles .......................................................................................... 7 2.1.1 Synthesis of superparamagnetic iron oxide nanoparticles .............................. 7 2.1.2 Properties of magnetic particles ...................................................................... 9 2.1.3 Surface modification of magnetic particles ................................................... 11

2.2 Magnetic Separation ............................................................................................. 15 2.2.1 Parameters of magnetic separator ................................................................. 17 2.2.1 Applications of magnetic separation ............................................................. 18

2.3 Cyclodextrins ....................................................................................................... 18 2.3.1 Basic properties of cyclodextrins .................................................................. 19 2.3.2 Cyclodextrin inclusion complexes ................................................................ 21 2.3.3 Characterization of cyclodextrin inclusion complexes ................................. 23 2.3.4 Applications of cyclodextrin ......................................................................... 25

2.4 Protein refolding ................................................................................................... 26 2.5 Pollutants removal from wastewater .................................................................... 31 2.6 Adsorption and Desorption .................................................................................. 39

2.6.1 Adsorption equilibrium ................................................................................. 39 2.6.2 Adsorption kinetics ....................................................................................... 40 2.6.3 Desorption study ........................................................................................... 42

2.7 Scope of the thesis ................................................................................................ 43 Chapter 3 Characterization Techniques .................................................................... 46

3.1 Transmission Electron Microscopy (TEM) Measurement ................................... 46 3.2 X-ray Diffraction (XRD) ...................................................................................... 46 3.3 Vibrating Sample Magnetometer (VSM) ............................................................. 47 3.4 Thermogravimetric Analysis (TGA) .................................................................... 48 3.5 Differential Scanning Calorimetry (DSC) ............................................................ 48 3.6 Fourier Transform Infrared (FTIR) ...................................................................... 48 3.7 Brunauer-Emmett-Teller (BET) Method ............................................................. 49 3.8 Zeta Potential Analyzer ........................................................................................ 49 3.9 X-ray Photoelectron Spectroscopy (XPS) ............................................................ 51 3.10 Circular Dichroism (CD) .................................................................................... 51 3.11 Fluorescence ....................................................................................................... 53

Table of contents

iii

Chapter 4 β–cyclodextrin bonded magnetic nanoparticles as solid-phase artificial chaperone in refolding of proteins1 ................................................................................ 54

4.1 Introduction .......................................................................................................... 54 4.2 Experimental ........................................................................................................ 56

4.2.1 Materials ........................................................................................................ 56 4.2.2 Preparation of mono-tosyl-β-cyclodextrin (Ts-β-CD) .................................. 57 4.2.3 Preparation of Fe3O4 magnetic nanoparticles ................................................ 57 4.2.4 Preparation of 3-aminopropyltriethoxy silane (APES) modified magnetic nanoparticles (APES-MNPs) .................................................................................. 58 4.2.5 Fabrication of Ts-β-CD modified Fe3O4 nanoparticles (CD-APES-MNPs) . 58 4.2.6 Protein refolding experiments ....................................................................... 59

4.3 Results and Discussion ......................................................................................... 61 4.3.1 Synthesis of β-CD bonded magnetic nanoparticles ....................................... 61 4.3.2 Characterization of magnetic nanoparticles .................................................. 63 4.3.3 CD-APES-MNPs assisted CA Refolding ...................................................... 68 4.3.4 Structural analyses of the refolded products by intrinsic fluorescence and far-UV circular dichroism ............................................................................................ 76

4.4 Conclusions .......................................................................................................... 80 Chapter 5 Selective recognition and separation of nucleosides using carboxymethyl-β-cyclodextrin functionalized hybrid magnetic nanoparticles2 ...................................... 82

5.1 Introduction .......................................................................................................... 82 5.2 Experimental ........................................................................................................ 85

5.2.1 Materials ........................................................................................................ 85 5.2.2 Preparation of carboxymethyl-β-cyclodextrin (CM-β-CD) .......................... 85 5.2.3 Fabrication of CM-β-CD modified APES-MNPs (CMCD-APES-MNPs) ... 86 5.2.4 Adsorption of nucleosides ............................................................................. 86

5.3 Results and discussions ........................................................................................ 87 5.3.1 Synthesis and characterization of magnetic nanoparticles ............................ 87 5.3.2 Adsorption of nucleosides onto CMCD-APES-MNPs ................................. 93 5.3.3 Selective adsorption of A and G ................................................................... 98 5.3.4 UV-vis spectra analysis and determination of binding constants ............... 100 5.3.5 Interactions of the nucleosides with cyclodextrins ..................................... 103

5.4 Conclusions ........................................................................................................ 106 Chapter 6 Adsorptive removal of dyes from aqueous water using carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbent3 ..................................................... 108

6.1 Introduction ........................................................................................................ 108 6.2 Experimental ...................................................................................................... 111

6.2.1 Materials ...................................................................................................... 111 6.2.2 Fabrication of CM-β-CD modified Fe3O4 nanoparticles [CMCD-MNP(C) & CMCD-MNP(P)] .................................................................................................. 111 6.2.3 Adsorption/desorption of methylene blue ................................................... 112

6.3. Results and Discussion ...................................................................................... 113 6.3.1 Synthesis and characterization of magnetic nanoparticles .......................... 113 6.3.2 Adsorption of MB onto CM-β-CD modified MNPs ................................... 122 6.3.3 Adsorption interactions ............................................................................... 135 6.3.4 Desorption and regeneration experiments ................................................... 137

6.4 Cost analysis ....................................................................................................... 139 6.5 Conclusions ........................................................................................................ 140

Table of contents

iv

Chapter 7 Carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbents for removal of copper ions from aqueous water4 ............................................................... 142


7.2.1 Materials ...................................................................................................... 145 7.2.2 Adsorption/ desorption of Cu2+ ions ........................................................... 145

7.3 Results and Discussion ....................................................................................... 146 7.3.1 Synthesis and characterization of magnetic nanoparticles .......................... 146 7.3.2 Adsorption of Cu2+ ions on CMCD-MNP(C) ............................................. 149 7.3.3 Adsorption interactions ............................................................................... 159 7.3.4 Desorption and regeneration studies ........................................................... 163

7.4 Conclusions ........................................................................................................ 164 Chapter 8 Multifunctional core-shell silica nanoparticles with magnetic, fluorescence, specific cell targeting and drug-inclusion functionalities ...................... 166


8.2.1 Materials ...................................................................................................... 169 8.2.2 Synthesis of multifunctional core-shell silica magnetic nanoparticles ....... 169 8.2.3 Cell culture and intracellular uptake study .................................................. 172 8.2.4 Cytotoxicity assay ....................................................................................... 172 8.2.5 Inclusion/adsorption of retinoic acid ........................................................... 173 8.2.6 Release study ............................................................................................... 173

8.3 Results and discussions ...................................................................................... 174 8.3.1 Synthesis and characterization of as-synthesized magnetic nanoparticles .. 174 8.3.2 Cytotoxicity assay ....................................................................................... 182 8.3.3 Confocal microscopy observations ............................................................. 182 8.3.4 Drug inclusion/adsorption and release studies ............................................ 184

8.4 Conclusions ........................................................................................................ 189 Chapter 9 Summary and Recommendations ........................................................... 190

9.1 Summary of findings .......................................................................................... 190 9.2 Recommendations for future work ..................................................................... 192 9.3 Research opportunities ....................................................................................... 199

References .................................................................................................................... 201 Appendix A: Supporting information for Chapter 5 .................................................... 229 Appendix B: Supporting information for Chapter 8 .................................................... 237 Appendix C .................................................................................................................. 241 List of publications ....................................................................................................... 250

Summary

v

Summary

Magnetic nanoparticles (MNPs) due to their high specific surface area,

biocompatibility, low toxicity and strong magnetic responsivity, have emerged as

excellent materials in many fields, such as biology, medicine, environment and material

science. In this work, we designed and fabricated β-cyclodextrin (β-CD) conjugated

MNPs which could be used in various bio- and environmental applications.

Cyclodextrins are natural oligosaccharides which have the molecular

inclusion/complexation capabilities through host-guest interactions with a wide variety

of organic and inorganic molecules. Tagging cyclodextrins with magnetic, stable

nanoparticles makes them magneto-responsive and may lead to a new generation of

adsorbents which will provide good opportunities for applications in the fields of bio-

separation/purification, contaminants removal from wastewater in environment

pollution cleanup and hydrophobic drug delivery. In this thesis work, tosyl-β-

cyclodextrin (Ts-β-CD) and carboxymethyl-β-cyclodextrin (CM-β-CD) conjugated

Fe3O4 MNPs were fabricated using different synthetic routes. Functionalized

nanoparticles were characterized with FTIR, TEM, XPS, XRD and TGA etc.

The use of Ts-β-CD grafted 3-aminopropyltriethoxysilane (APES) modified MNPs

(CD-APES-MNPs) as a solid-phase artificial chaperone to assist protein refolding in

vitro was demonstrated using carbonic anhydrase bovine (CA) as model protein. Our

refolding results show that a maximum of 85% CA refolding yield could be achieved

using these β-CD-conjugated magnetic nanoparticles which was at the same level as

that using liquid-phase artificial chaperone-assisted refolding. In addition, the

secondary and tertiary structures of the refolded CA were the same as those of native

Summary

vi

protein under optimal conditions. These results indicate that CD-APES-MNPs are

suitable and efficient materials (stripping agents) for solid-phase artificial chaperone-

assisted refolding due to easier and faster separation of these nanoparticles from the

refolded samples and also due to recycling of the stripping agents.

Selective recognition and separation of nucleosides (guanosine and adenosine) were

studied using CM-β-CD grafted APES modified MNPs (CMCD-APES-MNPs).

CMCD-APES-MNPs showed a higher adsorption ability and selectivity for guanosine

than adenosine under identical conditions. For better understanding to gain insights into

the molecular recognition mechanism of nucleosides and CM-β-CD, the inclusion

relation between the immobilized CM-β-CD and the guest substrates were investigated

through FTIR, UV-vis spectrophotometer and circular dichroism. Our results indicate

that this adsorbent would be a promising tool for easy and selective adsorption and

separation, analysis of nucleosides and nucleotides in biological samples. In fact, this

study provides a practical way to separate organic molecules based on the difference of

binding property by forming host–guest inclusion.

Adsorption behaviors of dyes (methylene blue) and heavy metals (Cu2+ ions) onto CM-

β-CD grafted magnetic nanocomposites were studied from equilibrium and kinetic

viewpoints. The grafted CM-β-CD on the iron oxides nanoparticles contributed to an

enhancement of the adsorption capacities because of the strong abilities of the multiple

hydroxyl/carboxyl groups and the inner cores of the hydrophobic cavity in CM-β-CD to

form complexes with metal ions and organic pollutants, respectively. The adsorption of

both pollutants onto CM-β-CD modified MNPs was found to be dependent on pH and

temperature. To gain insight into adsorption interactions, FTIR and XPS data were

introduced for better understanding. The regeneration and reusability studies suggest

Summary

vii

that CM-β-CD conjugated MNPs could be used as easily separable, recyclable and

effective adsorbents for the removal of organic/inorganic pollutants from aqueous

solution in environment pollution cleanup.

Highly uniform magnetic nanocomposite materials [Fe3O4@SiO2(FITC)-FA/CMCD

NPs] possessing an assortment of functionalities: superparamagnetism, luminescence,

cell-targeting, hydrophobic drug storage and delivery were also fabricated in this work.

Magnetic particle Fe3O4 is encapsulated within a shell of SiO2 that ensures

biocompatibility of the nanocomposite as well as act as a host for fluorescent dye

(FITC), cancer-targeting ligand (folic acid), and a hydrophobic drug storage-delivering

vehicle (β-CD). Inclusion/release of hydrophobic drug by these multifunctional

nanoparticles was studied using all-trans-retinoic acid (RA) as a model drug. Our

preliminary results suggest that such core-shell nanocomposite can be a smart

theranostic candidate for simultaneous bioimaging, magnetic control, cancer cell-

targeting and drug delivery.

Nomenclature

viii

Nomenclature Greek letters θ Half diffraction angle, (deg)

λ Wavelength of X-ray, (nm)

β Half width of XRD diffraction line (rad)

Hc Coercive field

Ms Saturation magnetization

Mr Remanent magnetization

d Particle size

ξ Zeta potential

TB Blocking temperature

Co Initial concentration (mg/ml)

Ce Equilibrium concentration (mg/ml)

qm Maximum adsorbed quantity (mg/g solid)

qe Adsorbed quantity at equilibrium (mg/g solid)

qt Adsorbed quantity at any time t (mg/g solid)

k1 Pseudo-first-order rate constant

k2 Pseudo-second-order rate constant

RL Dimensionless separation factor

Abbreviations CD Cyclodextrin

β-CD β-cyclodextrin

Ts-β-CD Mono-tosyl-β-cyclodextrin

Nomenclature

ix

CM-β-CD or CMCD Carboxymethyl-β-cyclodextrin

MNPs Magnetic nanoparticles

NPs Nanoparticles

SPIONs Superparamagnetic iron oxide nanoparticles

CA Carbonic anhydrase from bovine

G Guanosine

A Adenosine

MB Methylene blue

APES 3-aminopropyltriethoxy silane

CTAB Cetyltrimethylammonium bromide

TTAB Tetradecyltrimethylammonium bromide

SDS Sodium dodecyl sulfate

FITC Fluorescein isothiocyanate

FA Folic acid

RA All-trans-retinoic acid or vitamin A

BET Brunauer- Emmett-Teller method

CD Circular Dichroism

FTIR Fourier transform infrared spectroscopy

TEM Transmission electron microscopy

FESEM Field emission scanning electron microscopy

TGA Thermogravimetric analysis

VSM Vibrating sample magnetometer

XPS X-ray photoelectron spectroscopy

XRD X-ray diffraction

HPLC High performance liquid chromatography

List of Figures

x

List of figures Figure 2-1 Theoretical magnetization versus magnetic field curve for

superparamagnetic (SPM) and ferri- or ferromagnetic nanoparticles (FM) where the coercive field (HC), the saturation magnetization (MS) and the remanent magnetization (MR) parameters are indicated [27]. ............................................... 10

Figure 2-2 Variation of the coercivity (HC) of magnetic nanoparticles with size [27]. . 11 Figure 2-3 (a) Ligand exchange; (b) ligand addition; and (c) inorganic coating. F

represents functional chemical group that can be used for further conjugation [29]. ................................................................................................................................ 12

Figure 2-4 Schematic diagram of the magnetic separation of non magnetic targets ..... 16 Figure 2-5 Structures and molecular dimensions of α, β, γ- cyclodextrins. ................... 19 Figure 2-6 Functional structure of cyclodextrin ............................................................. 20 Figure 2-7 Schematic representation of cyclodextrin inclusion complex formation: p-

Xylene is the guest molecules; the small circles represent the water molecules. .. 22 Figure 2-8 Processes for the recovery of inclusion body proteins [61]. ........................ 26 Figure 2-9 Different approaches of protein refolding; (A) dilution mode (B) dilution-

additive assisted protein refolding (C) artificial chaperone assisted protein refolding. ................................................................................................................ 28

Figure 2-10 A typical high-gradient magnetic separation facility [121]. ....................... 34 Figure 2-11 Schematic diagram of the superconducting HGMS system [123]. ............ 35 Figure 4-1 Preparation steps for fabricating β-CD bonded magnetic nanoparticles. ..... 63 Figure 4-2 FTIR spectra of (a) Uncoated Fe3O4 MNPs, (b) APES-MNPs, (c) CD-APES-

MNPs, and (d) β-CD. ............................................................................................. 64 Figure 4-3 Typical TEM images and hydrodynamic size distribution of APES-MNPs

(a) &(c) and CD-APES-MNPs (b) & (d), respectively. ......................................... 65 Figure 4-4 (i) XRD patterns of (a) uncoated magnetic nanoparticles (MNPs), (b) APES

modified magnetic nanoparticles (APES-MNPs), and (c) β-CD modified magnetic nanoparticles (CD-APES-MNPs).(ii) Magnetization curves for uncoated and β-CD coated Fe3O4 magnetic nanoparticles measured by VSM at room temperature. .... 67

Figure 4-5 Schematic diagram of CD-APES-MNPs assisted protein refolding in-vitro.

................................................................................................................................ 69

List of Figures

xi

Figure 4-6 Time course of refolding yield of CA (■) and residual CTAB concentration (●) in refolded samples in presence of 40 mg/ml CD-APES-MNPs. The CTAB concentration in the capturing step was set at 2mM and final CA concentration was 0.029 mg/ml. .......................................................................................................... 70

Figure 4-7 Relative changes in the recovered enzymatic activity of the assisted

renatured CA as a function of detergent type and the solid phase quantity. Protein-detergent (CTAB, □: TTAB, Δ and SDS, ○) complex solution (2.7 ml, with a detergent concentration of 2mM in Tris–Sulfate buffer, pH 7.75) was added to 1.3 ml suspension containing various amounts of β-CD bonded magnetic nanoparticles. The final protein concentration was 0.029 mg/ml. The residual CTAB concentration (●) in the supernatant of the suspension was also measured. ................................................................................................................................ 72

Figure 4-8 Proposed mechanism by Hanson and Gellman for cyclodextrin-induced

folding from a protein–detergent complex [187]. U–dn, protein–detergent complex generated during the capture step (contains only one protein molecule); U, unfolded protein molecule; d, one detergent molecule; U–dn–m, protein–detergent complex from which detergent has been partially stripped away; (U–dn–m)p, partially stripped protein–detergent complex that has self-associated to form a species containing multiple protein molecules; F, first detergent-free form of the protein (extent of folding unspecified); N, native state of the protein. .................. 73

Figure 4-9 Refolding yield of thermally denatured CA in the presence of different

concentrations of β-CD in liquid phase artificial chaperone assisted refolding approach. The denatured CA (0.043 mg/ml) was diluted with water containing different concentration of β-CD (0-16 mM). The error bars represent standard error of mean for three independent measurements. .............................................. 74

Figure 4-10 The effect of CA concentration and the amount of CD-APES-MNPs on CA

renaturation yield. The CTAB concentration in the capturing step was set at 2 mM. The protein concentration in the captured state were 0.043 (●), 0.075 (■) and 0.15 (▲) mg/ml. ............................................................................................................. 75

Figure 4-11 Effect of various concentrations of CD-APES-MNPs on the intrinsic

fluorescence emission of refolded CA samples. (a) Intrinsic fluorescence spectra of refolded samples in the presence of different amounts of CD-APES-MNPs (0 mg/ml, curve 1; 10 mg/ ml, curve 2; 20 mg/ml, curve 3; 30 mg/ml, curve 4; 40 mg/ml, curve 5 and 50 mg/ml, curve 7). Curve 6 represents the native CA. (b) Maximum emission wavelength peak point of the refolded CA in presence of various concentrations of CD-APES-MNPs. ......................................................... 77

Figure 4-12 ANS fluorescence spectra of native (6) CA and refolded CA in the presence

of 0 (curve 1), 10 (curve 2), 20 (curve 3), 30 (curve 4), 40 (curve 5), 50 (curve 7) and 60 (curve 8) mg/ml of CD-APES-MNPs added in the solution. The final protein concentration was 0.029 mg/ml for all samples. Samples were excited at 360 nm. ................................................................................................................... 78

Figure 4-13 Far-UV circular dichroism spectra of native (curve 1) and the refolded CA

samples in the presence of 10 (curve 4), 30 (curve 3) and 40 (curve 2) mg/ml of the

List of Figures

xii

CD-APES-MNPs added in the solution. Each spectrum was obtained at 25C with a 1mm path length cell and is the average of two scans. All necessary corrections were made for background absorption. .................................................................. 80

Figure 5-1 Chemical structure of adenosine (A) and guanosine (G) ............................. 84 Figure 5-2 Schematic illustration of the fabrication of the carboxymethyl-β-cyclodextrin

modified magnetic nanoadsorbents and mechanism for selective separation of nucleosides. ............................................................................................................ 88

Figure 5-3 FTIR spectra of as-synthesized magnetic nanoparticles-(a) uncoated MNPs,

(b) APES-MNPs, (c) CMCD-APES-MNPs and (d) CM-β-CD. ............................ 90 Figure 5-4 (a) TEM micrograph and size distribution of CMCD-MNPs measured by

TEM and (b) hydrodynamic size distribution by DLS. .......................................... 91 Figure 5-5 TGA curves for (a) uncoated MNPs, (b) APES-MNPs and (c) CMCD-

APES-MNPs. .......................................................................................................... 92 Figure 5-6 Zeta potentials of uncoated, APES and CM-β-CD modified magnetic

nanoparticles at different pH. ................................................................................. 93 Figure 5-7 Effect of pH on the adsorption of A and G by CMCD-APES-MNPs:

Conditions: Initial A and G concentration: 1.0 g/l, temperature: 25oC. ............... 94 Figure 5-8 Effect of contact time on nucleoside adsorption by CMCD-APES-MNPs.

Conditions: Initial A or G concentration: 1.0 g/l, pH: 5, temperature: 25oC, adsorbent: 110 mg. ................................................................................................. 95

Figure 5-9 Effects of initial concentrations on the adsorption of A and G onto CMCD-

APES-MNPs. Conditions: Initial A and G concentration: 0.1,0.2,0.4,0.6,0.8,1.0g/l, pH: 5, temperature: 25oC, adsorbent: 110 mg. ....................................................... 96

Figure 5-10 Langmuir plot illustrating the linear dependence of Ce/qe on qe for A and G.

................................................................................................................................ 97 Figure 5-11 Selective adsorption of A and G mixture solutions using (a) CMCD-APES-

MNPs and (b) APES-MNPs. Conditions: pH: 5, temperature: 25oC, adsorbent: 110mg. .................................................................................................................. 100

Figure 5-12 Absorption spectra of (a) adenosine and (b) guanosine (1x10–5 mol l–1) in

pH 5.0 buffers containing various concentrations of CM-β-CD. Concentration of CM-β-CD: (1) 0, (2) 1.0x10–3, (3) 2.0x10–3, (4) 2.5x10–3 and (5) 3.5x10–3 mol l–1. Insets: Double reciprocal plot for nucleoside complexes with CM-β-CD. .......... 103

Figure 5-13 FTIR spectra of adenosine and guanosine and their inclusion complexes

with β-CD. ............................................................................................................ 106 Figure 6-1 An illustration for the carboxymethylation and binding of cyclodextrin onto

Fe3O4 nanoparticles by two different methods. .................................................... 115

List of Figures

xiii

Figure 6-2 FTIR spectra of (a) uncoated MNPs, (b) CMCD-MNP(P), (c) CMCD-MNP(C) and (d) CM-β-CD. ................................................................................. 116

Figure 6-3 TEM micrographs and hydrodynamic diameter distributions for CMCD-

MNP(C) (a) & (b); and CMCD-MNP(P) (c) & (d) respectively. ......................... 118 Figure 6-4 TGA curves for (a) bare Fe3O4 MNP, (b) CMCD-MNP(C) and (c) CMCD-

MNP(P). ............................................................................................................... 119 Figure 6-5 (i) XRD patterns and (ii) magnetization curves (a) uncoated Fe3O4 MNPs,

(b) CMCD-MNP(C) and (c) CMCD-MNP(P). .................................................... 120 Figure 6-6 Zeta potentials of bare Fe3O4 MNP, CMCD-MNP(C) and CMCD-MNP(P).

.............................................................................................................................. 121 Figure 6-7 The schematic representation of the selective adsorption and separation of

MB by CM-β-CD modified magnetic nanoparticles. ........................................... 123 Figure 6-8 UV-vis spectra of methylene blue (0.5 mg/ml) at pH~12 upon addition of (a)

0 mg MNPs; (b) 50 mg uncoated MNPs; (c) 50 mg CMCD-MNP(C); (d) 50 mg CMCD-MNP(P). .................................................................................................. 124

Figure 6-9 The molecular structure of MB. ................................................................. 125 Figure 6-10 Effect of pH on the adsorption of MB by CM-β-CD modified magnetic

nanoparticles. Conditions: (Initial MB concentration: 2 mg/ml, temperature: 25ºC, adsorbents: 120 mg). ............................................................................................ 125

Figure 6-11 Effect of contact time on the adsorption of MB onto CMCD-MNP(P) (a)

and CMCD-MNP(C) (b) and pseudo-second order kinetics of MB adsorption onto CMCD-MNP(P) (c) and CMCD-MNP(C) (d). Conditions: (Initial MB concentration: 0.25, 0.50 and 2 mg/ml, pH: 8, temperature: 25ºC). .................... 127

Figure 6-12 Equilibrium isotherm for the adsorption of MB on CMCD-MNP(P) and

CMCD-MNP(C) at 25ºC. ..................................................................................... 131 Figure 6-13 Langmuir plot illustrating the linear dependences of Ce/qe on Ce (a) and

Freundlich plot illustrating the linear dependences of lnqe on lnCe (b). .............. 132 Figure 6-14 Separation factor for MB onto CM-β-CD modified magnetic nanoparticles

at pH 8 and 25ºC. ................................................................................................. 134 Figure 6-15 FTIR spectra of CMCD-MNP(P) of before (a) and after (b) adsorption of

MB, and only MB (c). .......................................................................................... 136 Figure 6-16 (a) Desorption of MB from CMCD-MNP(P) as a function of acetic acid

concentration in methanol, and (b) performance of CMCD-MNP(P) by multiple cycles of regenaration. Adsorption conditions: (CMCD-MNP(P): 120 mg; MB concentration: 2 mg/ml; temperature: 25ºC; pH: 8; contact time: 2 hr). Desorption

List of Figures

xiv

conditions: (CMCD-MNP(P): 120 mg; temperature: 25ºC; contact time: 2 hr; desorption eluent: methanol with 5 % acetic acid) .............................................. 138

Figure 7-1 (a) TGA curves for uncoated Fe3O4 MNPs and CMCD-MNP(C); (b) Effect

of the amount of CM-β-CD in solution on the grafted CM-β-CD contents onto magnetic nanoparticles. ........................................................................................ 147

Figure 7-2 Zeta potentials of uncoated and CM-β-CD coated magnetic nanoparticles

and copper(II) nitrate solution at different pH. .................................................... 148 Figure 7-3 Effect of pH on the adsorption of Cu2+ ions by CMCD-MNP(C) at 25ºC.

Open points: qe vs. initial pH values; solid points: equilibrium pH values vs. initial pH values. ............................................................................................................. 150

Figure 7-4 (a) Effect of contact time on Cu2+ adsorption by CMCD-MNP(C); (b)

pseudo-second-order kinetics for adsorption of Cu2+. (Initial pH: 6, temperature: 25ºC). .................................................................................................................... 153

Figure 7-5 Equilibrium isotherms for Cu2+ adsorption by CMCD-MNP(C) at different

temperature and by uncoated Fe3O4 nanoparticles (inset) at 25ºC and initial pH 6. .............................................................................................................................. 155

Figure 7-6 The Langmuir (a) and Freundlich (b) isotherm plots for Cu2+ ions adsorption

by CMCD-MNP(C) at pH 6. ................................................................................ 157 Figure 7-7 van’t Hoff plot for the adsorption of Cu2+ by CMCD-MNP(C). ................ 159 Figure 7-8 FTIR spectra of CMCD-MNP(C) before (a) and after (b) adsorption of Cu2+

ions. ...................................................................................................................... 160 Figure 7-9 XPS spectra of CMCD-MNP(C): (a) C1s before adsorption; (b) C1s after

adsorption; (c) O1s before adsorption; and (d) O1s after adsorption; (e) Cu(2p3/2) core-level spectrum of Cu-loaded CMCD-MNPs. ............................................... 162

Figure 7-10 (a) Kinetics of desorption of Cu2+ from Cu-loaded CMCD-MNP(C) in

0.1M citric acid solution; (b) performance of CMCD-MNPs by multiple cycles of regeneration. ......................................................................................................... 164

Figure 8-1 Synthesis of CM-β-CD conjugated fluorescein-doped magnetic mesoporous

silica nanoparticles [Fe3O4@SiO2(FITC)-FA/CMCD NPs]. ............................. 175 Figure 8-2 UV–vis spectra of (a) pure FITC, (b) pure folic acid, (c) Fe3O4@SiO2, (d)

Fe3O4@SiO2(FITC), and (e) Fe3O4@SiO2(FITC)–FA/NH2 NPs in water. ......... 176 Figure 8-3 Excitation (dashed line, recorded at 515 nm emission) and emission (solid

line, recorded at 480 nm excitation) spectra of Fe3O4@SiO2(FITC)-FA/CMCD NPs dispersed in water. Insets: visual photographs of (a) Fe3O4@SiO2(FITC) NPs dispersion in water under room light; (b) the green emission of these MNPs in water and (c) in response to a NdFeB magnet under the 365 nm excitation portable UV lamp. .............................................................................................................. 178

List of Figures

xv

Figure 8-4 TEM mages of (a) OA-MNPs, (b) Fe3O4@SiO2(FITC) NPs and (c) Fe3O4@SiO2(FITC)-FA/CMCD NPs. .................................................................. 179

Figure 8-5 (A) Zeta potentials of as-synthesized nanoparticles at pH 7.4; (B) TGA

curves of (a) Fe3O4@SiO2(FITC)-FA/NH2 and (b) Fe3O4@SiO2(FITC)-FA/CMCD MNPs. ................................................................................................................... 180

Figure 8-6 In vitro cell viability of HeLa cells incubated with Fe3O4@SiO2(FITC)-

FA/CMCD MNPs at different concentrations for 6 and 24 h at 37oC. ................ 182 Figure 8-7 CLSM images of cells incubated with Fe3O4@SiO2(FITC)-FA/CMCD NPs

at 37oC: (A) HeLa cells (FA-positive) and (C) MCF-7 cells (FA-negative). Bright field images of HeLa cells (B) and MCF-7 cells (D) were also supplied. The nanoparticle concentration is 50 µg/ml and the incubation time is 1 h. ............... 183

Figure 8-8 (a) Absorption spectra of RA (7.3x10–6 mol l–1) in pH 7.4 buffers containing

various concentrations of CM-β-CD. Concentration of CM-β-CD: (1) 0, (2) 7.5x10–5, (3) 1.1x10–4, (4) 1.6x10–4, (5) 2.3x10–4, (6) 5.6x10–4 and (7) 3.75x10–3 mol l–1. Inset: Double reciprocal plot for RA complexes with CM-β-CD. .......... 185

Figure 8-9 Effects of nanoparticle dosage on adsorption/inclusion of retinoic acid. ... 187 Figure 8-10 Release profile of retinoic acid from Fe3O4@SiO2(FITC)-FA/CMCD NPs.

.............................................................................................................................. 188 Figure 9-1 Schematic presentation of complexation of CMCD-MNPs magnetic

nanoparticles with both organic and inorganic pollutants. ................................... 196 Figure 9-2 Schematic of the proposed multistage countercurrent continuous contact

[313]. .................................................................................................................... 197 Figure 9-3 Schematic description of the overall process: in the first part (left), the target

solute is extracted from a large volume using modified magnetic nanoparticles. In the second part (right), the loaded magnetic nanoparticles are washed (enrichment) and release the solute into a small volume. The moving solids (the true moving bed, i.e. the nanoparticles) links the two tank systems and transports the nanoparticles-bound solute against the concentration gradient between the large tank (left, low concentration) and the small tank (right, high concentration) [314]. .............................................................................................................................. 198

List of Tables

xvi

List of tables Table 2-1 Materials used for coating or encapsulating iron oxide magnetic nanoparticles

and their bio- and environmental applications. ...................................................... 14 Table 2-2 List of magnetic separation applications [12]. ............................................... 18 Table 2-3 Properties of α, β, γ- cyclodextrin .................................................................. 21 Table 2-4 Protein refolding-dilution additive mode ....................................................... 29 Table 2-5 Liquid phase artificial chaperone-assisted protein refolding ......................... 30 Table 2-6 Solid-phase artificial chaperone-assisted protein refolding ........................... 31 Table 2-7 Adsorption capacities and experimental conditions of functionalized

magnetic nanoparticles for various heavy metals and dye removal from wastewater ................................................................................................................................ 36

Table 2-8 Adsorption capacities and experimental conditions of cyclodextrin based

materials for various dye and heavy metals removal from wastewater ................. 38 Table 4-1 Data on chemical analysis of magnetic nanoparticles modified with APES

and β-CD. ............................................................................................................... 66 Table 5-1 Adsorption isotherm parameters for A and G at pH 5 and 25oC ................... 98 Table 6-1 Characteristics of Methylene Blue (MB) dye .............................................. 111 Table 6-2 Adsorption kinetic parameters of MB onto the CM-β-CD modified magnetic

nano-adsorbents at pH 8 and 25ºC ....................................................................... 129 Table 6-3 Adsorption isotherm parameters for MB adsorption onto CM-β-CD modified

magnetic nanoparticles at 25ºC ............................................................................ 133 Table 6-4 Maximum adsorption capacities (qm in mg/g) for MB by some other

adsorbents reported in literature ........................................................................... 133 Table 7-1 Adsorption kinetic parameters of Cu2+ onto CMCD-MNP(C). ................... 154 Table 7-2 Adsorption isotherm parameters for Cu2+ ions adsorption on uncoated and

CM-β-CD coated magnetic nanoparticles at pH 6. .............................................. 156 Table 7-3 Comparison of maximum adsorption capacity of CMCD-MNP(C) with those

of some other magnetic adsorbents reported in literature for Cu2+ ions adsorption. .............................................................................................................................. 157

List of Tables

xvii

Table 7-4 Thermodynamic parameters for the adsorption of Cu2+ ions onto CMCD-MNP(C). ............................................................................................................... 159

Table 7-5 FTIR absorption frequencies (cm-1) of CMCD-MNP(C) before and after

adsorption and their possible assignments ........................................................... 160 Table 8-1 Surface composition of Fe3O4@SiO2(FITC), Fe3O4@SiO2(FITC)-FA/NH2

and Fe3O4@SiO2(FITC)-FA/CMCD nanoparticles obtained from XPS spectra. 181

Chapter: 1 Introduction

1

Chapter 1 Introduction

1.1 General background

Recently nano-sized materials, due to their unique size as well as physico-chemical

properties have attracted significant interest and have now become one of the most

important research and development frontiers in modern science. Among them,

magnetite (Fe3O4) nanoparticles due to their good biocompatibility, superior

superparamagnetic property, nontoxicity and easy preparation process, are becoming

very popular and promising materials now-a-days [1]. They have attracted increasing

attention and enormous interest in various fields, such as environmental and biomedical

applications including enzyme immobilization, protein separation, magnetic resonance

imaging (MRI), hyperthermia, targeting drug delivery system [2,3].

Magnetite nanoparticles with superparamagnetism can be easily magnetized with an

external magnetic field and demagnetized immediately once the external magnetic field

is removed, not retaining any magnetism [4]. However, due to high specific surface

energy and anisotropic dipolar attraction, magnetite nanoparticles tend to aggregate

together into larger clusters which lead to a possible loss of superparamagnetism and

limit their applications. Therefore, the surface modification of nanoparticles is

indispensable and the particle surface can be modified by inorganic or organic coating.

Surface functionalization of MNPs endows the particles some important properties that

the bare particles lack. Modification of the surface of MNPs not only prevents

aggregation/agglomeration of the particles, leading to colloidal stability, but also

renders them with water-solubility, biocompatibility, non-toxicity, nonspecific

adsorption to cells, and bioconjugation. Considerable efforts have been made to modify

the surface of magnetic nanoparticles and the preparation of organic–inorganic


2

nanocomposites. The combination of inorganic and organic components in a single

particle at the nano-sized level has made accessible an immense area of new functional

materials [5]. Inorganic materials (silica, gold etc.) [2], natural [6,7] or synthetic

polymers [5,8] are frequently employed as coating materials to impart surface

reactivity. Natural polymers include chitosan, dextran, gelatin, starch, cyclodextrin etc.

and synthetic polymers are polyacrylic acid, polyvinyl chloride, poly vinyl alcohol etc.

Cyclodextrins (CDs) are a group of naturally cyclic oligosaccharides, with six, seven,

or eight glucose subunits linked by α-(1, 4) glycosidic bonds in a torus shaped structure

and are denominated as α-, β-, and γ-CD, respectively. Attention has recently been

focused on cyclodextrin based polymeric materials in a wide variety of applications due

to their unique sorption properties [9]. The sustained interest in the research and

application of cyclodextrin-based copolymer materials is attributed to the ability of

forming inclusion compounds with a wide range of organic molecules through host-

guest interactions: the interior cavity of the molecule provides a relatively hydrophobic

environment into which an apolar organic compound can be trapped. Recently, a

number of insoluble cyclodextrin polymer or co-polymers have been widely used for

various application such as contaminants removal from wastewater, protein refolding,

drug delivery etc. [10,11]. However, the difficulty in separating those powdery

cyclodextrin-based adsorbents, except high speed centrifugation, from treated effluent

limits their practical applications. Magnetic assisted adsorption separation technology

provides an alternative method to separate powdery adsorbents from solution

effectively.

Magnetic separation has been considered as an effective method for solid–liquid phase

separation techniques [12]. It has drawn more and more attention due to its merits of


3

speed, accuracy, simplicity and effectiveness. Magnetic separation has numerous

applications in biotechnology and biomedical diagnostics such as cell sorting, nucleic

acid detachment, enzyme immobilization, drug delivery and protein adsorption and

purification [2,5,13-16]. Magnetic separation involves magnetic particles, carrier

liquids, complexes and target molecules. Magnetic separation has been applied in many

areas to remove, isolate and/or concentrate the desired components from a sample

solution. The principle of the magnetic separation process is, at first the magnetic

particles are combined with the intermediates (polymers, ligands, surfactant etc.) to

form a complex. These complex particles can interact with the target molecules and

then by using gradient magnetic field the target is separated from the mixture. The

basic concept is to utilize the physical interactions between magnetic complex particles

and target molecules and as well as the specific chemical interactions between the

particles and target molecules.

1.2 Research objective and significance

Many researchers focused on synthesis of nano-sized magnetic particles with various

surface reactivity such as natural or synthetic polymers (i.e. chitosan, polyacrylic acid,

poly-N-isopropylacrylamide etc.). However, no work so far has been carried out on the

preparation of organic-inorganic nanocomposite materials through covalent binding

between MNPs and CDs and their use as a tool for bioseparation/purification and in the

cleanup of environmental pollutants. The incorporation of inclusion/complexation

property of CDs and the superparamagnetic property of magnetite (Fe3O4) in one nano-

system is considered to be a promising tool for wide variety of applications such as bio-

separation, drug delivery, molecular recognition, environmental pollution control etc.


4

In bio-applications, particularly in artificial chaperone-assisted protein refolding

approach [see Section 2-4], soluble β-CD or some insoluble β-CD polymers have been

used as stripping agents for detergent molecules. However, excessive soluble β-CD and

β-CD/detergent complexes are to be removed from the refolded products for

downstream purification. The insoluble stripping agents also suffer lower protein

refolding yield than the corresponding values obtained from the liquid-phase artificial

chaperone assisted method and separation inconvenience. Therefore, efforts are still

required to carry out investigation for new promising striping agent which can act as a

solid-phase artificial chaperone.

The use of β-CD as an adsorbent with molecular recognition function for the selective

removal of biomolecules from the aqueous stream is limited due to its inherent water

solubility. We presume that the adsorbent formed by anchoring β-CD to magnetite

would improve its molecular recognition ability and additionally, the reaction converts

β-CD into an insoluble matrix which in turn facilitate the separation. As far as we

know, cyclodextrin bonded magnetic nanoparticles for selective recognition and

separation of biomolecules has not been reported yet. Furthermore, functionalization of

the magnetic nano-surface by β-CD in well-defined host-guest interactions may also

lead to a generation of nanocarriers for hydrophobic drug delivery applications.

β-CD has potentials applications in pollutants removal from industrial wastewater as it

can facilitate the incorporation of various organic compounds such as dyes,

pharmaceutical waste chemicals, phenolic compounds etc. into its hydrophobic cavity

through host-guest interaction. It is also reported that β-CD has the ability to complex

heavy metals such as cadmium, lead, mercury etc. through the interactions between the

metal ions and multiple hydroxyl groups on it. Hence, β-CD is a procedure of choice


5

for decontaminating technique. Considering the complexation property of β-CD and the

separation convenience of magnetic Fe3O4 nanoparticles, grafting of β-CD on the

magnetic nanoparticles is expected to be a promising alternative to develop magnetic

nano-adsorbent for the efficient removal of both organic and inorganic pollutants from

wastewater. To evaluate their effectiveness in clean up of environmental pollutants

adsorption equilibrium, adsorption kinetics and effects of various parameters (such as

pH, temperature etc.) on adsorption need to be studied in details.

The desired objectives of this work are summarized as follows:

1. Grafting of β-CD derivatives on magnetic nanoparticle surface using different

synthetic approaches and characterization of as-synthesized magnetic particles using

different analytical tools i.e. FTIR, TEM, XPS, TGA and elemental analysis.

2. Evaluation of the effectiveness of β-CD conjugated magnetic nanoparticles as solid-

phase artificial chaperone in refolding of protein in-vitro.

3. Individual and selective adsorption study of nucleosides (guanosine and adenosine)

onto CM-β-CD conjugated magnetic nanoparticles having molecular recognition and

magnetic properties.

4. Adsorption behaviors of organic dye (methylene blue) using CM-β-CD conjugated

magnetic nanoparticles as nano-adsorbents.

5. Adsorption of copper ions onto CM-β-CD conjugated magnetic nano-adsorbents and

study of adsorption interactions.

6. Synthesis and characterization of CM-β-CD grafted multifunctional silica core-shell

nanoparticles with the magnetic, fluorescent, specific cell targeting and inclusion


6

functionalities and investigation of the possibility of the applications of these

nanoparticles in biomedical research fields, particularly for imaging, specific cell

targeting and hydrophobic drug inclusion/delivery etc.

1.3 Organization of the thesis

The present thesis is organized into eight chapters. Chapter 1 gives a brief introduction

of magnetic nanoparticles with magnetic separation technique and β-CD coated

magnetic nanoparticles, the objectives of the thesis and a structural organization of the

whole thesis. A review of related literature is presented in chapter 2. Chapter 3 presents

various characterization techniques that were used throughout my thesis work. Chapter

4 presents the characterization results of magnetic nanoparticles and also the results for

refolding of carbonic anhydrase (CA) using β-CD bonded magnetic nanoparticles as

solid-phase artificial chaperone. Chapter 5 shows the characterization results of as-

synthesized magnetic nano-adsorbents, adsorption behaviors of nucleosides (guanosine

and adenosine) at different experimental conditions and the adsorption mechanism. In

Chapter 6, the results of adsorption, desorption and adsorption interactions of organic

pollutant (methylene blue) with the nano-adsorbents are described where two types of

CD-conjugated magnetic nanoparticles were used as nano-adsorbents. Experimental

results on adsorptive removal of heavy metals (Cu2+ adsorption) from aqueous solution

using the same nanoadsorbents have been detailed in Chapter 7. Chapter 8 represents

the synthesis and characterization results of CM-β-CD grafted multifunctional silica

core-shell nanoparticles that combine the magnetic, fluorescent, specific cell targeting

and hydrophobic drug-inclusion functionalities. Overall conclusions together with

recommendations are given in Chapter 9.

Chapter: 2 Literature Review

7

Chapter 2 Literature Review

In Chapter 2, literature reviews on magnetic nanoparticles, their properties and surface

functionalization, cyclodextrin and its structural properties and applications, protein

refolding, pollutants removal from wastewater, adsorption and desorption processes are

presented.

2.1 Magnetic nanoparticles

The special and superior properties of nanomaterials have attracted much attention in

the past two decades. Particularly, magnetic nanoparticles (MNPs) with inherent

magnetic properties and high surface-to-volume ratio have continued to draw

considerable interest because of their diverse potential applications in biological,

environmental and medical diagnostic fields. One of the rapidly emergent research

subjects involving MNPs is their application in biological systems, including their

application in magnetic resonance imaging (MRI), targeted drug delivery, rapid

biological separation, biosensors, and magnetic hyperthermia therapy [2,3]. The most

commonly used magnetic particles are magnetite (Fe3O4) and maghemite (γ- Fe2O3).

Other types of magnetic particles are pure metal (Fe and Co) and spinel type

ferromagnets (MeO.Fe2O3, where M = Ni, Co, Mg, Zn, Mn).

2.1.1 Synthesis of superparamagnetic iron oxide nanoparticles

Due to unique size, biocompatibility, low toxicity and superparamagnetic properties,

magnetic nanoparticles are emerging as promising tools in various fields such as

physics, medicine, biology, environment and material science [1]. Several types of iron

oxides have been investigated recently in the field of nano-sized magnetic particles

(mostly maghemite, γ-Fe2O3, or magnetite, Fe3O4, single domains of about 5–20 nm in


8

diameter), among which Fe3O4 is a very promising and popular candidate since its

biocompatibility have already proven [1]. In last decades, numerous synthetic methods

have been developed to synthesize iron oxide nanoparticles including coprecipitation

[17], sol-gel synthesis [18], microemulsion synthesis [19], sonochemical reaction [20],

hydrothermal reaction [21], thermal decomposition [22], laser pyrolysis [23] etc.

Among these reported methods, the chemical co-precipitation may be the most

promising one because of its simplicity and productivity. In a chemical co-precipitation

method, aqueous FeCl3 and FeCl2 solutions are mixed at a concentration ratio of Fe3+

:

Fe2+

=2:1 in an aqueous ammonia solution, yielding Fe3O4 NPs with d = 3–15 nm. The

control of size, shape and composition of nanoparticles depends on the type of salts

used (chlorides, sulfates, nitrates, perchlorates), Fe2+

and Fe3+

ratio, pH, temperature and

ionic strength of the media [24,25]. In general, the overall reaction may be written as

follows [26]:

Fe2+ + 2Fe3+ + 8OH- Fe3O4 + 4H2O (2-1)

This method would critically affect the physical and chemical properties of the

nanosized magnetic particles. In addition, Fe3O4 NPs are not very stable under ambient

conditions and are easily oxidized to Fe2O3 or dissolved in an acidic medium. In order

to avoid the possible oxidation in the air, the synthesis of Fe3O4 NPs must be done in an

anaerobic condition. In this method, the reaction temperature is limited by the boiling

point of water, and iron oxide nanoparticles synthesized under these conditions exhibit

usually a low degree of crystallinity and large polydispersity

Compared to the co-precipitation method, the organic phase synthesis which relies on

pyrolysis of iron precursors with the presence of surfactants, allows better control over


9

the size and monodispersity of Fe3O4 nanoparticles. Two of the most widely used iron

precursors in organic phase iron oxide nanoparticle synthesis are Fe(acac)3 and

Fe(CO)5. Sun et al. showed that high-temperature reaction of Fe(acac)3 in the presence

of alcohol, oleic acid, and oleylamine can be used to produce size-controlled

monodisperse Fe3O4 nanoparticles [22]. The advantage of this method is that the

synthesis is not limited to simple iron oxide nanoparticles but can be extended to

various types of ferrite nanoparticles as well.

The microemulsion (water in oil: W/O) method using water droplets as nanoreactors in

a continuous phase (oil) in the presence of surfactant molecules is reported to be an

alternative and more controlled method [19]. The size of the NPs can be controlled by

controlling the size of the water droplets. Surfactants, which are responsible for

micellisation, can be utilised for the dispersion of iron oxide NPs.

2.1.2 Properties of magnetic particles

In contrast to bulk iron oxide, which is a multi-domain ferromagnetic material (exhibits

a permanent magnetization in the absence of a magnetic field), iron oxide magnetic

nanoparticles smaller than approximately 20–30 nm in size contain a single magnetic

domain with a single magnetic moment and exhibit superparamagnetism [4]. A material

in a paramagnetic phase is characterized by randomly oriented (or uncoupled) magnetic

dipoles, which can be aligned only in the presence of an external magnetic field and

along its direction. This type of material has no coercivity nor remanence, which means

that when the external magnetic field is switched off, the internal magnetic dipoles

randomize again, no extra energy is required to demagnetize the material and hence the

initial zero net magnetic moment is spontaneously recovered, see Figure 2-1. A

nanoparticle with such magnetic behavior is superparamagnetic (SPM).


10

Figure 2-1 Theoretical magnetization versus magnetic field curve for superparamagnetic (SPM) and ferri- or ferromagnetic nanoparticles (FM) where the coercive field (HC), the saturation magnetization (MS) and the remanent magnetization (MR) parameters are indicated [27].

The size reduction of magnetic materials shows interesting advantages that make them

more suitable for therapeutic and diagnostic techniques compared to their bulk

counterparts. Magnetic parameters such as the coercivity of the nanoparticles can be

finely tuned by decreasing their size. Moreover, a further reduction of the size below a

certain value of the radius, the so-called superparamagnetic radius (rSP), induces a

magnetic transition in particles where both ferro- and ferrimagnetic nanoparticles (FM)

become superparamagnetic and, as previously said, high magnetic moments are

observed under the effect of a magnetic field, but no remanent magnetic moment will

be present when the external magnetic field is removed. Superparamagetism is a

property strictly associated to nanostructured magnetic materials and arises when the

thermal energy is sufficiently high to overcome the magnetic stabilization energy of the

particle. Figure 2-2 explains how the coercivity of the nanoparticles varies (at a certain

temperature) when their size is decreased, until the superparamagnetic state is reached.


11

With increasing size, the particles will become blocked as thermal energy becomes

insufficient to allow the free rotation of spins. Superparamagnetic particles are usually

ordered below a blocking temperature, TB (blocking temperature is the transition

temperature between the ferrimagnetic and superparamagnetic state and is directly

proportional to the size of the particles).

Figure 2-2 Variation of the coercivity (HC) of magnetic nanoparticles with size [27].

2.1.3 Surface modification of magnetic particles

Control of the surface chemistry of superparamagnetic iron oxide nanoparticles

(SIONPs) is essential to develop SIONPs for bio-related applications. The stability of

SPIONs in suspension is controlled by three principal forces: (a) hydrophobic–

hydrophilic, (b) magnetic and (c) van der Waals [28]. Pristine SIONPs tend to

aggregate into large clusters, in suspension due to the attractive van der Waals forces in

order to minimize the total surface or interfacial energy. The resulting large

agglomerates reduce intrinsic superparamagnetic properties. Modification of the surface

of SIONPs not only prevents aggregation/agglomeration of the particles, leading to

colloidal stability, but also renders them with water-solubility, biocompatibility,


12

nonspecific adsorption to cells, and bioconjugation. Several approaches to modify the

surfaces of SIONPs have been investigated for the preparation of water-soluble SIONPs

which can be roughly divided into three categories: ligand exchange, ligand addition,

and inorganic coating [29] as shown as Figure 2-3.

Figure 2-3 (a) Ligand exchange; (b) ligand addition; and (c) inorganic coating. F represents functional chemical group that can be used for further conjugation [29].

In the first approach, ligand exchange, the native monolayer of hydrophobic surface

ligands is exchanged with ligands containing head groups that bind the magnetic

nanoparticle surface and hydrophilic tails that interact with aqueous solvent [3,13,30].

The most common molecules used are ligands such as oleic acid, lauric acid, alkane

sulphonic acids, and alkane phosphonic acids. Silanes were employed to exchange the

hydrophobic ligands on ferrite magnetic nanostructures [30]. The end group of silanes,


13

including isocyanine, acrylate, thiol, amino, and carboxylic groups, offer extensive

chemistry for the modification of nanostructures. Surfactant addition is achieved

through the adsorption of amphiphilic molecules that contain both a hydrophobic

segment and a hydrophilic component. The hydrophobic segment forms a double layer

structure with the original hydrocarbon chain, while hydrophilic groups are exposed to

the outside of the NPs, rendering them water soluble [31,32].

The third route for magnetic NP surface modification is the fabrication of an inorganic

shell, typically consisting of silica. Silica coatings are formed either via the Stöber

process [33] or through a microemulsion synthesis [34]. Silica has been widely used to

protect the core nanoparticles from the external environment, thereby improving the

stability of the NPs. In addition, silica is highly biocompatible and its surface can be

easily modified with amines, thiols, and carboxyl groups, which enables covalent

modification of the particle surfaces with biological molecules.

There are various kinds of materials that can be chosen for coating nanoparticles. Table

2-1 presents a list of materials which have been used as stabilizing agents during the

synthesis of iron oxide nanoparticles. These materials can be chemically anchored or

physically adsorbed on magnetic nanoparticles to form a single or double layer, which

creates repulsive (steric repulsion) forces to balance the van der Waals attractive forces

acting on the nanoparticles, and the magnetic particles are stabilized in the process.


14

Table 2-1 Materials used for coating or encapsulating iron oxide magnetic nanoparticles and their bio- and environmental applications.

Materials used

Size and size distribution

Applications Advantages Reference

Silica 10-200 nm, narrow

Cellular MRI, heavy metal removal from wastewater, drug delivery, fluorescence

Improves biocompatibility, hydrophilicity and chemical stability. Prevents aggregation of particles in liquid. Provides further functionalization

[35,36]

Polyethylene glycol (PEG)

20-40 nm, narrow

MRI contrasting

Improves the biocompatibility, blood circulation time and easy to functionalize

[3]

Chitosan 10-200 nm, broad

Heavy metals separation, drug delivery

Natural polysaccharide with hydrophilicity, biocompatibility, biodegradability, antibacterial properties and remarkable affinity for many biomacromolecules. Used in food, biotechnology, biomedicine, food ingredients, cosmetics, water treatment

[37,38]

Dextran 10-50 nm, narrow

Biomedical applications

Enhances the blood circulation time, stabilizes the colloidal solution

[6,39]

Polyacrylic acid

8-20 nm, broad

Heavy metals removal, dye removal, enzyme recovery

Increases the stability and biocompatibility of the particles and also helps in bioadhesion

[8,40]

Gum arabic 13-67 nm, narrow

Heavy metals removal

Natural, harmless and environment friendly polymer, wide applications as a stabilizer, thickening agent and hydrocolloid emulsifier, mostly used in food and pharmaceutical industries

[41,42]


15

Table 2-1 (Continued) Poly(3,4-ethylenedioxythiophene) (PEDOT)

11 nm, narrow


Promising conducting polymers because of its high conductivity, excellent environmental stability, and simple acid/base doping/ dedoping chemistry

[43]

Humic acid 10-190 nm, broad


Enhances the stability of nanodispersions by preventing their aggregation

[44]

Poly-N-isopropyl acrylamide (PNIPAAm)

10-25 nm, broad

Hyperthermia, protein separation, controlled drug release

Thermoresponsive polymer, surface hydrophilicity or hydrophobicity can be varied easily

[15,45,46]

Dimercapto- succinic acid (DMSA)

~6 nm Heavy metals removal

Excellent chelating agent for heavy metals

[47]

2.2 Magnetic Separation

Chromatography is a powerful technology for the purification of biological substances

in both analytical and preparative scales. However, packed bed chromatographic

column is prone to clogging so that the method is unable to process particulate feed

stocks such as whole fermentation broth, cell disruptions and unclarified biological

extracts. To overcome this drawback, various alternative separation techniques have

been developed, including fluidizing bed adsorption, expanded bed adsorption and

magnetic separations [12]. These techniques offer great opportunities for process

integration by achieving particulate removal and desired product capture in a single

operation.

Over the last three decades, magnetic separation technology has emerged as one of the

most promising separation technologies [12]. In this method, colloidal particles are

manipulated by mismatches in their magnetization. Magnetic separation has been

developed as a technique to separate mainly magnetic materials, but recent development


16

has made possible the separation of non magnetic materials also. Magnetic separation

involves the transportation of magnetic or magnetically susceptible particles in a

gradient magnetic field. Generally, magnetic separation could be divided into two parts:

1. Separation of magnetic materials and 2. Separation of non-magnetic materials. In the

first method, magnetic separation of the target molecule could be achieved without

further modification of magnetic materials. For example separation of colored

magnetic impurities from kaolin clay, magnetic particulates from stack gases, magnetic

impurities from wastewater treatment, magnetic materials in mineral beneficiations, and

magnetotactic bacteria containing magnetic particles inside their cells. Most of the

application of magnetic fractionation can be classified in the second type. The principle

of this separation process is to use magnetic particles modified with some

intermediates, such as surfactant, polymer and ligand to adsorb the target molecule,

which can be separated by applying magnetic field gradient. The whole process of

separation of non magnetic target by magnetic separation method is illustrated in Figure

2-4. The interaction mechanisms between the non-magnetic targets and the

intermediates, coated in advance on magnetic particles, could be either electrostatic

interaction, hydrophobic interaction, and/or ligand-specific interaction.

Figure 2-4 Schematic diagram of the magnetic separation of non magnetic targets

P

I

Magnetic particle

Intermediate

Preparation

P I P I: : T P I T

Surface Preparation Coupling Release

TargetMolecule

Magnetic field gradient


17

2.2.1 Parameters of magnetic separator

The magnetic force (Fm) acting on a magnetizable particle of volume Vp and particle

magnetization Mp, placed in a magnetic field H can be expressed as,

Fm = µ0VpMpgrad H (2-2)

where µ0 denotes permeability constant of the vacuum.

The particle magnetization (Mp) may be expressed by the magnetic volume

susceptibility χ and the magnetic field strength H where the volume susceptibility is a

constant for diamagnetic and paramagnetic substances and a function among others of

particle shape and size as well as field strength for the ferromagnetic or ferrimagnetic

substances.

Mp = χH (2-3)

The most significant external forces that compete with the magnetic force in a magnetic

separator are the force of gravity, centrifugal force, and the hydrodynamic drag. For a

spherical particle of density ρp the gravitational force (Fg) is given by

Fg = (ρp – ρf)Vpg (2-4)

where ρf and g are the density of the fluid medium and the acceleration due to gravity,

respectively.

The centrifugal force (Fc) can be expressed as

Fc = (ρp – ρf)ωVpr (2-5)


18

where r is the radial position of the particle and ω is the angular velocity.

The hydrodynamic drag force (Fd) can be obtained from Stokes’ equation

Fd = 6πηb(vf – vp) (2-6)

where η is the dynamic viscosity of the fluid and vf and vp are velocities of the fluid and

particle respectively.

2.2.1 Applications of magnetic separation

In the past decades, magnetic separation has shown to be useful in many promising

applications in various areas of chemical and biotechnological processes. Table 2-2

summarizes the applications of magnetic separations.

Table 2-2 List of magnetic separation applications [12].

Areas Application of magnetic separation Chemical processes Ores–mineral beneficiation Kaolin (clay) decolorization Water treatment and metal removal Food Industries Biotechnological processes Enzyme immobilization Cell sorting DNA and protein separation and purification Nucleic acid extraction Drug delivery Biocatalysis and diagnostics

2.3 Cyclodextrins

Cyclodextrins (CDs) are cyclic torus-shaped oligosaccharides consisting of six (α-

cyclodextrin), seven (β-cyclodextrin), eight (γ-cyclodextrin) or more glucopyranose

units linked by α-(1,4) bonds [9,48,49] (Figure 2-5). They are also known as

cycloamyloses, cyclomaltoses and Schardinger dextrins. They are produced as a result


19

of intramolecular transglycosylation reaction from degradation of starch by

cyclodextrin glucanotransferase (CGTase) enzyme [9].

Figure 2-5 Structures and molecular dimensions of α, β, γ- cyclodextrins.

2.3.1 Basic properties of cyclodextrins

The three major cyclodextrins are crystalline, homogeneous and non-hygroscopic

substances, which are of a torus-like macro ring shape, built up from glucopyranose

units. Based on the number of glucopyranose units, cyclodextrins are commonly

classified as α (6), β (7), γ (8)- cyclodextrins. β-CD is the most accessible, the lowest-

priced and generally the most useful. Cyclodextrins contain a relatively hydrophobic

internal cavity, which can include various inorganic and organic molecules and exhibits

regio- and stereoselectivity, and a hydrophilic surface, which has primary and

1.69nm 1.53nm

0.78nm

1.37nm

0.95nm 0.57nm

0.79nm


20

secondary hydroxyl groups. The main properties of those cyclodextrins are given in

Table 2-3.

In cyclodextrin molecules glucose units situated in the classical 4C1 conformation of

chains are linked through α-1,4 bonds. The interior of the cavity, which contains two

rings of C-H groups with a ring of glycosidic oxygen in between, is relatively,

hydrophobic, while the external faces with hydroxyl groups are hydrophilic. All

secondary hydroxyl groups (C2-OH and C3-OH) are situated on the wider end of the

cavity, whereas all primary hydroxyls (C6-OH) are situated on the narrower end, as

shown in Figure 2-6. The internal hydrophobic cavity is the key structural feature of the

cyclodextrins. It provides their ability to complex and holds a wide variety of inclusion

molecules. To bind with cyclodextrins, the inclusion molecule must have a size that fits, at

least partially, into the cavity, creating the complex. The inclusion compound, however,

does not have to be completely contained in the cavity. Complexes can be formed by the

insertion of some specific functional groups or part of the molecule to bind in the

hydrophobic cavity.

Figure 2-6 Functional structure of cyclodextrin


21

In cyclodextrins, the non-bonding electron pairs of the glycosidic oxygen bridges are

directed towards the inside of the cavity, giving it high electron density and some

Lewis-based characters. The C2-OH group of one glucopyranose unit can form a

hydrogen bond with the C3-OH group of the adjacent glucopyranose unit

intramolecularly. A complete secondary belt is formed by these H-bonds to give β-CD

molecules a rigid structure. Cyclodextrins are readily soluble in water, because all the

free hydroxyl groups are located on the outer surface of the ring [9]. Among them, β-

CD is observed to have the lowest solubility. The hydrogen belt in α-cyclodextrins is

incomplete because one glucopyranose unit is in a distorted position and only four out

of six possible H-bonds can be formed. γ-CDs have a non-coplanar and more flexible

structure so it is the most soluble among the three common cyclodextrins.

Table 2-3 Properties of α, β, γ- cyclodextrin

Properties α β γ

Number of glucose units 6 7 8

Molecular weight (g/mol) 972 1135 1297

Solubility in water at 25oC (g/100ml) 14.5 1.85 23.2

Volume of cavity (Å3)

Cavity diameter ( Å)

174

4.7-5.3

262

6-6.5

427

7.5-8.3

Outer diameter ( Å) 14.6 15.4 17.5

Height of the torus ( Å) 7.9 7.9 7.9

2.3.2 Cyclodextrin inclusion complexes

The most important property of cyclodextrin for both practical application and scientific

research is the ability to selectively form inclusion complexes with other molecules,

ions, and radicals [49]. Inclusion complexes are molecular compounds in which one


22

compound (host molecule) spatially encloses another (guest). The driving forces of the

formation of the inclusion complexes are: (1) van der Waals (or hydrophobic)

interactions between hydrophobic “guest” molecules and the CD cavity; (2) hydrogen

bonds between polar functional groups of “guest” molecules and CD hydroxyl groups.

The role of hydrogen bonds is not universal, because “guest” molecules such as

benzene, which cannot form hydrogen bonds, also form stable inclusion complexes; (3)

substitution of high energy (high enthalpy) water molecules in the CD cavities in the

complexation process. In aqueous solutions nonpolar CD cavities are occupied by water

molecules, which is energetically unfavorable (polar–nonpolar interactions), and,

therefore, they can be easily replaced by suitable molecules of the “guests” less polar

than water (Figure 2-7); (4) the release of the strain energy in the ring structure of CD

molecules; (5) charge-transfer interactions [49,50].

Figure 2-7 Schematic representation of cyclodextrin inclusion complex formation: p-Xylene is the guest molecules; the small circles represent the water molecules.

Complex formation is a dimensional fit between host cavity and guest molecule. The

ability of a cyclodextrin to form an inclusion complex with a guest molecule is a

function of two key factors [51]. The first is steric and depends on the relative size of

the cyclodextrin to the size of the guest molecule or certain key functional groups

within the guest. If the guest is of wrong size, it will not fit properly into the


23

cyclodextrin cavity. For example, the bulky anthracene can only fit into γ-CD.

Propionic acid is relatively small so it is compatible with α-CD but its fitting with large

cavity of γ-CD is unsatisfactory. The second critical factor is the thermodynamic

interactions between the different components of the system (cyclodextrin, guest,

solvent). For a complex to form there must be a favorable net energetic driving force

that pulls the guest into the cyclodextrin.

The stability of inclusion complexes also depends on the polarity of the “guest”

molecules. Highly hydrophilic molecules and strongly hydrated and ionized groups are

not, or weakly complexable with cyclodextrin. Only compounds less polar than water

can form inclusion complexes with cyclodextrin. The hydrophobic character of the

substituent determines the stability of the complexes. The stability of inclusion complex

increases with an increase in the electron donor nature of the “guest”.

2.3.3 Characterization of cyclodextrin inclusion complexes

Inclusion complexes can be characterized by a variety of spectrometric methods. UV

spectroscopy has been used to determine the stability constant and thermodynamic

parameters for interaction of guests with CD in aqueous solution [52]. The

complexation causes a change in the absorption spectrum of a guest molecule. During

the complexation, the chromophore of the guest molecule is transferred from an

aqueous medium to the non-polar cyclodextrin. These changes must be due to a

perturbation of the electronic energy levels of the guest caused either by direct

interaction with the CD, by the exclusion of solvating water molecules or by a

combination of these two effects [53]. Hypsochromic or bathochromic shift or increase

in the absorption intensity without change in the λmax has been considered as evidence

of the formation of the complex. Hydrogen bonding can be considered as the main force


24

behind the inclusion complex formation. As hydrogen bonding lowers the energy of ‘n’

orbitals, a hypsochromic shift (blue shift) is observed. Cleavage of the existing

hydrogen bonds in the compound can lead to a bathochromic shift due to complexation

[54,55]. An increase or decrease in the absorption intensity of UV band without change

in its λmax is also reported in certain inclusion complexes. For example, the absorption

intensity was increased when benzene was complexed with β-CD [52].

Circular Dichroism is a useful method to detect cyclodextrin inclusion complexes in

aqueous solution [56]. When an achiral guest molecule is included within the

asymmetric locus of the cyclodextrin cavity, new circular dichroism bands can be

induced in the absorption bands of the optically inactive guest. Not only achiral guest

molecules but also chiral guest molecules may show changes in circular dichroism

spectra upon the formation of inclusion complexes with cyclodextrin [56].

Infra-Red spectroscopy is also used to estimate the interaction between CD and the

guest molecules in the solid state. CD bands often change only slightly upon complex

formation and if a part of guest molecules is encapsulated in CD cavity, bands which

could be assigned to the included part of the guest molecules are easily masked by the

bands of the spectrum of cyclodextrin. Generally IR techniques are not suitable for the

detection of inclusion compounds because the resultant spectra have a superposition of

host and guest bands [55].

Recently, thermal analysis has also been used to detect the inclusion compounds. In

some cases no melting or decomposition peaks of the guests are observed on the

thermogram obtained by DTA or DSC after the formation of complexes [55,57]. The


25

thermal decomposition of inclusion compounds was studied only hardly. It was

observed that the decomposition of the complexed guest occurs above the

decomposition temperature of the uncomplexed guest molecule [53]. Apart from the

techniques described above, there are methods used to characterize or detect the

inclusion complexes such as, powder X-day diffraction [54,55,57], nuclear magnetic

resonance (1H-NMR) [54-57], fluorescence spectroscopy [53] etc.

2.3.4 Applications of cyclodextrin

The unique structure and properties of cyclodextrins have made them a popular choice

for a wide range of applications over the past few decades [51,58]. There are numerous

applications for cyclodextrins in the pharmaceuticals field [59,60]. Cyclodextrins can be

used as drugs either for complexation or as auxiliary additives such as carriers, diluents,

solubilisers or tablet gradients. The physical and chemical properties of the drugs can be

modified through the inclusion complex formation. These modifications are desirable in

the formation of oral drugs and have the potential to improve their physical and

chemical stability, as well as enhance the bioavailability of poorly soluble drugs. In

food and cosmetics industries, cyclodextrins can be used for molecular encapsulation of

flavors and fragrances based on their water retention properties. The major advantages

of cyclodextrins in this application include the protection of active ingredient against

various chemical reactions and elimination or reduction of undesirable tastes/odors and

microbiological contamination. Furthermore, cyclodextrins are widely used in chemical

technology [51]. For example, cyclodextrins can modify the reactivity of the guest

molecules through the alteration of the reaction pathways, enhancement of the

hydrophilicity and reactivity in aqueous systems, chiral recognition and reduction of the

free, uncomplexed guest substance. In the chemical industry, cyclodextrins are widely


26

used to separate isomers and enantiomers, to catalyse reactions, to aid in various

processes and to remove or detoxify waste materials.

2.4 Protein refolding

Protein refolding is an important downstream process in the field of biotechnology.

Many of these proteins are currently produced by Escherichia coli in the form of

insoluble, inactive aggregates often termed as inclusion bodies. The general strategy

used to recover active protein from inclusion bodies involves three steps: inclusion

body isolation and washing; solubilization of the aggregated protein; and refolding of

the solubilized protein (Figure 2-8). Although the efficiency of the first two steps can be

relatively high, renaturation yields may be limited by the accumulation of inactive

misfolded species as well as aggregates. Such aggregates must be separated from cell

debris and solubilized by exposing them to a strong denaturant, such as urea or

guanidine (GdmCl) or detergent sodium dodecyl sulphate (SDS) and n-cetyl

trimethylammonium bromide (CTAB).

Figure 2-8 Processes for the recovery of inclusion body proteins [61].


27

In conventional methods, refolding of unfolded polypeptide chains to their native state

is initiated through decreasing the denaturant concentration by direct dilution or dialysis

[62]. The main disadvantages of dilution refolding for commercial applications are the

need for larger refolding vessels and additional concentration steps after renaturation. In

addition, protein aggregates formed due to hydrophobic interactions between partially

folded polypeptide chains leads to low yields of biologically functional protein [63]. On

the other hand, renaturation yields using these membrane-based dialysis methods can be

significantly affected by protein binding to the membranes. The difference between

protein folding and aggregation is described as kinetic competition between

intramolecular reaction (folding) or inter molecular reaction (aggregation) by the

following reactions:

U → N (2-7)

U + U → A2 (2-8)

where U, N, and A2 represent the unfolded protein, native protein, and a dimer,

respectively [64].

Therefore, many strategies for in vitro protein refolding have focused on suppression of

aggregation along with improved renaturation yields. An efficient strategy to suppress

aggregation is the inhibition of the intermolecular interactions leading to aggregation by

the use of low molecular weight additives. This strategy is often termed as ‘dilution

additive approach’ (Figure 2-9). However, scaling up dilution processes leads to large

volumes and low protein concentration. A wide variety of additives has been used in

protein refolding (Table 2-4).


28

Recently, Rozema and Gellman developed a two-step protein refolding procedure using

CTAB as capturer to bind denatured protein, and β-CD as stripper to dissociate the

CTAB–protein complex so that the individual protein may fold into the native form

[65-68]. This method is also referred to as artificial chaperone-assisted protein refolding

which presents a useful way to inhibit the formation of protein aggregates. In addition

to applying this method to different proteins research into new “capturers” and

“strippers” has attracted growing attention and efforts in recent years (Table 2-5 and

Table 2-6).

Figure 2-9 Different approaches of protein refolding; (A) dilution mode (B) dilution-additive assisted protein refolding (C) artificial chaperone assisted protein refolding.

Denatured protein

Protein-detergent complex

Native protein (+some aggregates)

Capture step Stripping Step

Aggregate (inactive)

Dilution

Native protein (+some aggregates)

Dilute +additive

A

B

C


29

Table 2-4 Protein refolding-dilution additive mode

Model proteins (denaturant used)

Additives References

Glycosylated recombinant human deoxyribonuclease (rhDNAse) (Urea, GdmCl)

Polyethylene glycol (0.1-0.4 g/l)

[69]

Bovine Carbonic anhydrase (GdmCl)

α-CD (20-100 mM) [70]

Bovine Carbonic anhydrase II (GdmCl)

Detergents (CHAPS, Triton X-100, n-hexanol, n-pentanol, cyclohexanol)

[71]

β–lactamase (GdmCl)

Temperature-sensitive polymer (PNIPAAm)

[72]

Sulfertranferase enzyme rhodanese (Urea)

Detergent-phospholipids mixtures [73]

Rhodanese (GdmCl) Wide range of detergents used: ionic, zwitterionic, nonionic etc.

[74,75]

Calf intestinal alkaline phosphatase (CIP) (GdmCl)

Magnesium ions (<3mM) [76,77]

Rabbit muscle creatine kinase (GdmCl)

Proline, glycerol and heparin sodium [78]

Rabbit muscle creatine kinase (GdmCl)

Osmolytes, including dimethysulfoxide, glycine, proline and sucrose

[79]

Hen egg-white lysozyme(GdmCl) Proline (>1.5 M)

[80]

Hen egg-white lysozyme (GdmCl)

Acetone, acetoamide, or urea derivatives (1.4M)

[81]

Hen egg-white lysozyme(GdmCl denatured), bovine carbonic anhydrase (urea )

A varieties of cyclodextrins [82,83]

hen egg-white lysozyme(GdmCl) L-Arginine (0.4–1 M)

[84,85]

Yeast inorganic pyrophosphatase

Sugars (Trehalose, glycerol) [86]

α-amylase(GdmCl)

α-CD (100 mM)

[87,88]

Hen egg-white lysozyme (GdmCl)

Acetamide (1-3M), acetone (0.5-1.5M), thiourea (0.2-0.6M), l-arginine (0.4-1M) or glycerol (3.2-6.5M)

[89]

Hen egg white lysozyme, recombinant human lysozyme from E. coli BL21 (Urea)

CTAB [90]

Hen egg white lysozyme (GdmCl)

GdmCl (0.5-1.25M), Arginine (0.4-1M) [91]


30

Table 2-5 Liquid phase artificial chaperone-assisted protein refolding

Model proteins(denatured state)

Capturers Striping agents Refolding yields

References

Lysozyme (GdmCl)

CTAB β-CD, methyl-β-CD

[67]

Bovine carbonic anhydrase (CAB) (GdmCl/thermally)

Several types of detergents used: CTAB, SDS, STS, POE(10)L etc.

α-CD, β-CD, and methyl-β-CD

~80% (max.) [65]

Porcine heart citrate synthase (CS) (GdmCl/urea )

Several types of detergents used: CTAB, SDS, Triton X-100, ZW 3–14, POE(10)L etc.

β-CD ~65% (max.) [66]

Rabbit muscle creatine kinase (MM-CK) (Thermally)

SDS Hydroxypropyl-β-CD ~75% [92]

ALP (Urea) α –amylase (urea) CAB (thermally)

CTAB, DTAB, TTAB, SDS, STS, SHS

Alginate ALP~ 85% α –amylase-78% CAB-89%

[93]

CAB (GdmCl) Porcine heart citrate synthase ((GdmCl)

cholesterol group-bearing pullulan (CHP) nanogels

α-CD, β-CD, and methyl-β-CD, HP- β-CD

CAB~65% CS~76%

[94]

CAB (Thermally)

cholesterol group-bearing pullulan (CHP) nanogels

α-CD, β-CD, and methyl-β-CD, HP-β-CD

~90% [95]

Xylanase (GdmCl & urea)

CTAB, TTAB etc.

Methyl-β-CD

50~60% [96]

HCAB (GdmCl) Lysozyme (GdmCl)

CTAB Linear dextrins

HCAB-77% Lys-60%

[97]

α–amylase (GdmCl) CTAB α-CD ~75% [98]

Porcine heart citrate synthase (GdmCl) CAB(GdmCl) Lysozyme(GdmCl)

A wide variety of detergents

Cycloamylose ~90% [99]

Lysozyme CTAB β-CD-grafted-PNIPAAm(β-CD-g-PNIPAAm)

~70% [100]

ALP from calf intestine (Urea) CAB (thermally) α –amylase (Urea)

Detergents Detergents ~78-88% [101]

CAB (GdmCl) Lysozyme(urea)

Dextran-grafted-PNIPAAm

[102]


31

Table 2-6 Solid-phase artificial chaperone-assisted protein refolding Model proteins(denatured state)

Capturing agents

Solid-phase stripping agents

Refolding yield References

CAB (SDS denatured)

SDS β -Cyclodextrin-Polyurethane Polymer

~75% [103]

α-glucosidase (urea) SDS POLY RING-DEX B (β-cyclodextrin-epichlorohydrin copolymer)

50-60% [104]

Lysozyme (GdmCl) CAB ( GdmCl)

CTAB β -cyclodextrin-acrylamide copolymer beads

Lysozyme~65% CAB~80%

[105]

CAB (thermally denatured)

CTAB, TTAB, SDS

β-cyclodextrin-epichlorohydrin copolymer

~75% [106]

2.5 Pollutants removal from wastewater The removal of heavy metal pollutants and dyes from industrial wastewater has become

an important issue because of their adverse affects on human health and environment

[107,108]. Dyes are widely used in many industries in order to color their products,

such as cosmetics, leather, paper, printing textile finishing industries. The presence of

these dyes in water, even at very low concentrations, is highly visible and undesirable.

Most of the used dyes are stable to photodegradation, bio-degradation and oxidizing

agents [109]. However, heavy metals, unlike the organic contaminants, are not

biodegradable and tend to accumulate in living organisms. Due to the toxic and

carcinogenic properties of both inorganic and organic pollutants, it is necessary to treat

the pollutant containing water before being released into the environment.

Numerous approaches have been developed for the removal of these contaminants from

wastewater. Traditional heavy metal and dye removal methods include chemical

precipitation, ion exchange, liquid-liquid extraction, membrane filtration, adsorption


32

and biosorption [110,111]. Among these methods, adsorption has increasingly received

more attention due to its merits of effectiveness, efficiency and economy [41,112-115].

The common adsorbents primarily include activated carbons, zeolites, clays, industrial

by-products, agricultural wastes, biomass and polymeric materials [110,116]. However,

these adsorbents described above suffer from low adsorption capacities and separation

inconvenience. Therefore, efforts are still required to carry out investigation for new

promising adsorbents.

Adsorption using nano-sized adsorbents is becoming more popular as it possesses better

performance when compared to sub-micron to micron sized particles which need

internal porosities to ensure adequate surface area for adsorption. However, the

existence of intraparticle diffusion may lead to the decreases in the adsorption rate and

available capacity, particularly for macromolecules [40]. Therefore, to develop an

adsorbent with large surface area and small diffusion resistance has significant

importance in practical use [117]. Now-a-days, magnetic nanoparticles with tailored

surface chemistry have been widely used in various fields of applications. The

functional groups on the surface play an important role in determining the effectiveness,

capacity, selectivity, and reusability of these adsorbents. The basic properties of

nanoparticles- extremely small size, the absence of internal diffusion resistance and

high surface area to volume ratio, provide better kinetics for the adsorption of

contaminants from aqueous solutions [40]. Magnetic nano-adsorbents have the

advantages of both magnetic separation techniques and nano-sized materials, which can

be easily recovered or manipulated with an external magnetic field. The main advantage

of this technology consists in its capability of treating large amount of wastewater

within a short time and producing no contaminants. Magnetic materials, particularly

superparamagnetic Fe3O4 nanoparticles, provide a novel biomedical and environmental


33

purification technique because of their specific magnetic separation characteristics. A

list of magnetic materials that have been used as adsorbents so far for the removal of

heavy metals and dyes from wastewater is summarized in Table 2-7.

Attention has recently been focused on cyclodextrin based non-magnetic materials as

adsorbents. The most characteristic feature of CDs is the ability to form inclusion

compounds with various organic molecules through host-guest interactions: the interior

cavity of the molecule provides a relatively hydrophobic environment into which an

apolar pollutant can be trapped. The insoluble cyclodextrin polymer or immobilization

of derivatives of cyclodextrin onto inert support materials such as silica, have been

widely used for contaminants removal from wastewater [10,118]. It has been shown

that these materials could be effectively used for the removal of phenolic compounds

and dyes from wastewater. The list of cyclodextrin based materials is summarized in

Table 2-8. However, less work has been done for heavy metal removal from wastewater

using cyclodextrin based magnetic adsorbents. Combining the advantages of both

materials, a superior adsorbent could be developed for wastewater treatment.

When the magnetic particles containing pollutants move through a magnetic field, they

are rapidly and efficiently separated or captured. The capture of particles depends

strongly on the creation of these large magnetic field gradients, as well as on the

magnetic properties. There are three categories of separators based on magnet type:

permanent magnet-based (field of less than 1 T), electromagnet-based (max. 2.4 T) and

superconducting magnet-based (2 to 10 T). The high gradient magnetic separation

(HGMS), commonly used term in magnetic separations has been of scientific and

technological interest over the past decades [12,119]. The technique has shown great

advantages in ferromagnetic material separation and it has been industrialized, such as


34

magnetic separation in steel industry, steel making, steel rolling, and circulating cooling

water in thermal power plant and so on. An HGMS device comprises of a bed of

magnetically susceptible wires placed between two poles of an electromagnet, as shown

in Figure 2-10. When a magnetic field is on, the wires dehomogenize the magnetic field

in the column, producing large field gradients around the wires that attract magnetic

particles to their surfaces and trap them there. Recent research showed that the capture

of particles depends on particle sizes and aggregates of nanoparticles larger than 50 nm

showed significantly high capture efficiency in HGMS operations [120].

Figure 2-10 A typical high-gradient magnetic separation facility [121].

The superconducting HGMS is a new kind of separation technology was also used

many researchers. Instead of copper coil, the high gradient magnetic field of more than

10 T can be generated using superconducting coil in this separator [122]. Moreover, this

technology has many advantages such as low investment, energy saving, low operating

cost, easy handling, automatic control. Recently Kondo and Miura used magnetic

mesoporous carbon to remove the dissolved organic matter from wastewater efficiently

with the help of superconducting HGMS technique as shown in Figure 2-11[123].


35

Figure 2-11 Schematic diagram of the superconducting HGMS system [123].


36

Table 2-7 Adsorption capacities and experimental conditions of functionalized magnetic nanoparticles for various heavy metals and dye removal from wastewater

Adsorbent Adsorbate Adsorption capacity (mg/g)

pH Temperature (oC)

Kinetic model Isotherm References

Chitosan-coated Fe3O4 NPs modified with alpha-ketoglutaric acid

Cu(II) 96.15 6 22 Langmuir, Freundlich

[108]

Humic coated Fe3O4 NPs Hg(II) Pb(II) Cd(II) Cu(II)

97.70 92.40 50.40 46.30

6 20 - Langmuir [44]

Glutaraldehyde modified Fe3O4 NPs (GA-APTES-NPs)

Cu(II) 61.07 4 20 - Langmuir, Freundlich

[124]

Calcium alginate modified Fe3O4 NPs Cu(II) 60.00 5 20 Pore diffusion model

Langmuir [125]

Gum Arabic modified Fe3O4 NPs Cu(II) 38.50 5.1 27 - Langmuir, Freundlich

[41]

Amino functionalized Fe3O4@SiO2 core-shell NPs

Cu(II) Pb(II) Cd(II)

29.85 76.66 22.48

6 25 - Langmuir [126]

Chitosan bound MNPs Cu(II) 21.50 5 27 - Langmuir [37] Amino-functionalized MNPs Cu(II)

Cr(VI)12.43 11.24

5 2

25 Langmuir [8]

Carbon encapsulated MNPs Cu(II) Cd(II) Co(II)

3.21 1.77 1.23

9 r.t. - - [127]

Dimercaptosuccinic acid (DMSA) coated Fe3O4 NPs

Hg(II) 227.00 8.1 r.t. - Langmuir [47]

Magnetic (γ-Fe2O3) hydrogel Cr(VI) 205.00 4-6 23 - Langmuir [128] Carbon encapsulated MNPs (CEMNPs)

Cu(II) 3.21 (at initial conc. 20 mg/l)

8 20 Freundlich [129]

Cross-linked magnetic chitosan–diacetylmonoxime Schiff’s base resin (CSMO)

Cu(II) Co(II) Ni(II)

95 ± 4 60 ± 2

47 ± 1.5

5 28 Pseudo-first order model,

pseudo-second-order model

Langmuir, Freundlich and

Tempkin

[130]

Chitosan-magnetite composite Pb(II) Ni(II)

63.33 52.55

6 r.t. - Langmuir [38]


37

Table 2-7 (Continued)


pH Temperature (oC)


Dendrimer conjugated MNPs Zn(II) 24.30 7 25 Langmuir, Freundlich

[131]

Fe3O4 hollow spheres Neutral red 105.00 6 25 Pseudo-second-order model

Langmuir, Freundlich

[132]

Cross-linked magnetic chitosan–diacetylmonoxime Schiff’s base resin (CSMO)

Cu(II) Co(II) Ni(II)

95 ± 4 60 ± 2

47 ± 1.5

5 28 Pseudo-first order model,


Langmuir, Freundlich and

Temkin

[130]

γ-Fe2O3 Acridine orange (AO)

59.00 4.5 24 Pseudo-second-order model


[133]

Magnetic silica modified with amino groups

Acid Orange 10 (AO10)

61.33 3 25 Pseudo-first order model,



[134]

Carboxymethyl chitosan-conjugated Fe3O4 NPs

Crocein orange G (AO12)

Acid green 25 (AG25)

94.16

73.16

3

25 Pseudo-first order, pseudo-

second-order and intraparticle

diffusion model


[135]

Polyacrylic acid-bound Fe3O4 MNPs Methylene blue (MB)

199.00 9 25 - Langmuir [40]

MWCNTs filled with γ-Fe2O3 NPS Methylene blue Neutral red

42.30 77.50

6 25 - Freundlich

[136]

Fe3O4-activated carbon nanocomposite

Methylene blue 321.00 - 25 - Langmuir [137]

SDS coated Fe3O4 NPs Safranin O 769.23 3 r.t. Pseudo-second-order model


[138]

Carboxylic functionalized superparamagnetic mesoporous silica microspheres

Acridine orange Methylene blue

112.70 109.80

10 25 - Langmuir, Freundlich

[139]

Amine-functionalized silica magnetite (NH2/SiO2/Fe3O4 adsorbents)

Cu(II) Reactive black 5

10.41 217.00

5.5 3

25 Pseudo-first order model,



[140]


38

Table 2-8 Adsorption capacities and experimental conditions of cyclodextrin based materials for various dye and heavy metals removal from wastewater


pH Temperature (oC)


Cyclodextrin/carboxymethyl cellulose crosslinked polymers

C.I. Basic Green 4

(Malachite Green)

91.9 8 25 Pseudo-first order, pseudo-second-order and intraparticle diffusion model


[10]

β-CD polymer crosslinked by citric acid

Methylene blue 105 Deionized water

30 - Langmuir [11]

Cyclodextrin/carboxymethyl cellulose crosslinked polymer

Methylene blue 56.4 8 25 Pseudo-first order model, pseudo-second-order

model

Freundlich, Langmuir,

Temkin and generalized

[141]

Cyclodextrin/carboxymethyl cellulose crosslinked polymer

Astrazone blue Crystal violet Rhodamine B

53.2 42.4 35.8

8 25 pseudo-second-order and

intraparticle diffusion model


[113]

Cyclodextrin-starch based polymer

Congo red 36.2 5.8 25 - Langmuir, Freundlich

[142]

β-CD/chitosan copolymer Pb(II) 434.78 4,5 30 Pseudo-first order model, pseudo-second-order

model


[143]

Pyromellitic dianhydride (PMDA) grafted β-CD

Methylene blue Neutral red

497.5 555.6

- 25 Pseudo-first order model, pseudo-second-order

model


[144]

β-CD polymer (HMDI as crosslinking agent)

Evans Blue Chicago Sky Blue

10.59 12.09

2 25 - Langmuir, Freundlich

[145]

β-CD/citric acid copolymer Pb(II) Cd(II) Zn(II)

263.2 107.5 82.6

5 20 - Langmuir [146]


39

2.6 Adsorption and Desorption

Adsorption is one of the most important parts of separation. It has been widely used in

the chemical, biological, analytical, and environmental fields. In most cases, the

adsorbents have diameters in the range of sub-micron to micron and have large internal

porosities to ensure adequate surface area for adsorption. However, the diffusion

limitations within the particles lead to decrease in the adsorption rate and the available

capacity, particularly for macromolecules such as proteins and DNA. To understanding

adsorption more precisely we investigated the adsorption equilibrium and the

parameters affecting adsorption. However, desorption is also important for the recovery

of target molecules from the surface of the particles. Therefore, suitable adsorption

desorption parameters should be developed.

2.6.1 Adsorption equilibrium a) Langmuir Model

The Langmuir model assumes that the adsorption process takes place on a surface

composed of a fixed number of adsorption sites of equal energy, with one molecule

adsorbed per adsorption site until a monolayer coverage is obtained. The Langmuir

model is described by the equation [147]:

m ee

L e

q Cq

K C

(2-9)

The linear form of this equation can be represented as follow:

1e e

e m m L

C C

q q q K (2-10)


40

where Ce is the adsorbate liquid-phase equilibrium concentration, qe is the adsorbate

surface concentration, qm is the maximum adsorbate binding capacity and KL is

Langmuir constant, which represents the affinity between adsorbate and adsorbent. The

values of qm and KL can be obtained from the slope and intercept of the linear plots of

Ce/qe versus Ce.

b) Freundlich Model

This isotherm is an empirical equation to describe multilayer adsorption with

interaction between adsorbed molecules [148]. This model predicts that the solute

concentrations on the adsorbent will increase as long as there is an increase of the solute

concentration in the solution. Usually it applies to adsorption onto heterogeneous

surfaces with a uniform energy distribution and reversible adsorption. The Freundlich

equation suggests that the adsorption energy decreases exponentially on the completion

of the adsorption centers of an adsorbent. This equation is given as follows:

qe=KFCe1/n (2-11)

The commonly used linear form of the equation is:

lnqe = (1/n) lnCe + lnKF (2-12)

where KF and n are the Freundlich equilibrium constant and the Freundlich isotherm

power term (also called the heterogeneity factor), respectively. The values of KF and 1/n

can be determined from the slope and intercept of the linear plot of lnqe versus lnCe.

2.6.2 Adsorption kinetics In order to investigate the mechanism of adsorption and potential rate controlling steps

such as mass transport and chemical reaction processes, kinetic models have been used


41

to test experimental data. The adsorption of pollutants from aqueous solution plays an

important role in wastewater treatment; hence it is important to know the sorption rate

at which the pollutants are removed from aqueous solutions in order to design the

appropriate sorption treatment plants [149]. Several kinetic models have been reported

to describe the adsorption of pollutants (dyes, heavy metals, phenolic compounds etc.)

[149-152]. These kinetic models included the pseudo-first order equation, the pseudo-

second order equation and intraparticle diffusion model.

The pseudo-first-order equation of Lagergren is generally expressed as follows [150]:

1( )te t

dqk q q

dt (2-13)

Integrating this equation for the boundary conditions t = 0 to t = t and q = 0 to q = qt,

gives:

ln(qe – qt) = lnqe – k1t (2-14)

where qe and qt refer to the adsorption capacity at equilibrium and at any time, t (min),

respectively, (mg/ml) and k1 is the equilibrium rate constant of pseudo-first-order

sorption (min-1). The slope of the linear plot of ln(qe - qt) versus t is used to determine

the first-order rate constant, k1.

The pseudo-second order kinetic rate equation is expressed as follows [149]:

22 ( )t

e t

dqk q q

dt (2-15)


and rearranging the variables, gives:

22

1 1

t e e

tt

q k q q (2-16)


42

where k2 is the equilibrium rate constant of pseudo-second-order adsorption (g mg-1

min-1). If pseudo-second order kinetics is applicable, the plot of t /qt against t of

equation [2-16] should give a linear relationship, from which qe and k2 can be

determined from the slope and intercept of the plot.

The dye sorption is governed usually by either the liquid phase mass transport rate or

the intraparticle mass transport rate. Hence diffusive mass transfer is incorporated into

the adsorption process. In diffusion studies, the rate can be expressed in terms of the

square root time. The mathematical dependence of qt versus t1/2 (known as the Weber-

Morris plot) is obtained if the process is considered to be influenced by diffusion in the

particles and convective diffusion in the solution. According to this model proposed by

Weber and Morris [153], the root time dependence may be expressed by Equation [2-

17]:

qt = kidt1/2 + C (2-17)

where qt is the amount of solute on the surface of the sorbent at time t (mg/g), kid the

intraparticle diffusion rate constant (mg/g min1/2), t the time (min), and C is the

intercept (mg/g). The kid values are found from the slopes of qt versus t1/2 plots.

2.6.3 Desorption study

Desorption study is important to explore the possibility of recyclability of the magnetic

nano-adsorbent. Desorption of solutes from interfaces depends essentially on the

conditions under which they have been adsorbed. Therefore, suitable desorbing agents

should be selected to release the adsorbed pollutants from magnetic particles. For heavy

metal desorption from magnetic nanoparticles, acidic solution (HCl or HNO3) could be

used as eluant [154-156]. EDTA which is a strong hexadentate chelating agent has also


43

been used as eluant for metals desorption [151,154]. For desorption of dye molecules,

methanol or ethanol solution has been shown to be effective eluant [40,157].

2.7 Scope of the thesis

The aim of this project is to synthesize and characterize β-CD coated MNPs (either bi-

functional or multifunctional) and their applications in biomolecule separation,

biomolecule refolding, drug delivery, organic/inorganic pollutants removal from

wastewater. Although nanoparticles have diverse applications in various fields of

bioscience, biotechnology and environment, only limited work has been published on

the preparation of organic-inorganic nanocomposite materials using β-CD coated

nanoparticles and their use as a tool for bio/chemical separation, drug delivery etc.

Based on the literature review the scopes of the present project are:

(a) Synthesis and characterization of β-CD coated magnetic nanoparticles

MNPs are synthesized by chemical precipitation method using FeCl3 and FeCl2 with

ammonia under nitrogen environment in a ratio of 2:1. These nanomagnetic particles

are then functionalized with β-CD derivatives e.g. mono-tosyl-β-CD (Ts-β-CD) and

carboxymethyl-β-CD (CM-β-CD) using different synthetic routes. Characterization of

the chemical, physical and magnetic properties of the as-synthesized nanomagnetic

particles are carried out using FTIR, XPS, BET, TEM, XRD and VSM.

(b) Application of Ts-β-CD bonded MNPs in biomolecule refolding

Ts-β-CD conjugated MNPs are used as a solid-phase artificial chaperone in refolding of

bovine carbonic anhydrase (CA) as model protein. CA is a Zn containing

(metalloenzyme). It is expected that with cumulative effects of chaperoning function of

Ts-β-CD and superparamagnetism of iron oxide, Ts-β-CD conjugated MNPs would


44

have potential applications in biomolecule i.e. protein refolding. To evaluate the

efficiency of TS-β-CD conjugated MNPs as solid-phase artificial chaperone in protein

refolding, results are compared with that using only β-CD as liquid-phase chaperone.

Also the structural changes of the refolded protein are studied. In the refolding study,

only Ts-β-CD grafted on magnetic particles is used which acts as protein refolding aid

whereas other CD derivative for example, CM-β-CD induces serious protein

aggregation [83].

(c) Application of CM-β-CD bonded MNPs in biomolecule recognition and separation

CM-β-CD conjugated core-shell APES-MNPs are used for individual and selective

adsorption of nucleosides. Adenosine (A) and guanosine (G) were selected as model

target guest molecules. For better understanding to gain insights into the molecular

recognition mechanism of nucleosides and β-CD, the characteristics of inclusion

complexes prepared are also investigated through UV-vis spectrophotometer, FTIR,

circular dichroism, DSC etc.

(d) Application of CM-β-CD bonded MNPs in wastewater treatment

CM-β-CD conjugated MNPs are used as nano-adsorbent for the removal of organic and

inorganic pollutants from wastewater. In organic pollutant removal study, methylene

blue (MB) is used as model contaminant because of their extensive use in various

industries. Methylene blue is a cationic dye which is toxic and carcinogenic. On the

other hand, the adsorption behavior of these adsorbents is investigated using Cu2+

(inorganic pollutant) as the target metal contaminant because of its extensive

environmental impacts. The CM-β-CD grafted onto Fe3O4 nanoparticles contributes to

an enhancement of the adsorption capacity of Fe3O4 nanoparticles because of the strong


45

complexation abilities of the multiple hydroxyl/carboxyl groups in CM-β-CD with

metal ions and of the hydrophobic cavity with organic contaminants. The adsorption

interactions are also investigated towards the removal of these pollutants.

(e) CM-β-CD grafted multifunctional core-shell silica MNPs for biomedical applications In this study, fluorescein dye-doped silica coated magnetic nanoparticles with core-shell

architecture is synthesized and then the silica surface is further functionalized with folic

acid and CM-β-CD. These as-synthesized nanoparticles has multiple functional

moieties- (i) silica shell which increases the stability and biocompatibility of these

nanoparticles, (ii) incorporated dye conjugate, (iii) folic acid which is a common anti-

cancer targeting ligand, and (iv) CM-β-CD which has encapsulation ability for

hydrophobic drugs. The possibility of the applications of these multifunctional

nanoparticles in biomedical fields, particularly for imaging, specific cell targeting and

anti-cancer drug inclusion/delivery is investigated.

It is believed that the present work on simple fabrication and applications of β-CD

conjugated magnetic nanoparticles for biomolecule refolding/separation, drug

inclusion/delivery, contaminants removal from wastewater can provide useful

information on the extension of these as-synthesized bifunctional or multifunctional

magnetic nanoparticles with combined properties of superparamagnetism and

complexation/ molecular recognition functions to multiple potential applications.

Chapter: 3 Characterization Techniques

46

Chapter 3 Characterization Techniques

3.1 Transmission Electron Microscopy (TEM) Measurement

A bright field transmission electron microscopy (TEM) was used for the size

measurement and size distribution of the magnetic nanoparticles. TEM images were

recorded on a JEOL 2010 transmission electron microscope at an accelerating voltage

of 200 kV. The TEM specimens were prepared by placing a drop of the nanoparticle

suspension on a carbon-coated copper grid (200 mesh and cover with formvar/carbon).

The copper film was then dried at room temperature for 24 h before the measurement.

3.2 X-ray Diffraction (XRD)

The structural properties and crystallographic structure of Fe304 powders were analyzed

by X-ray diffraction with shimadzu XRD-6000 diffractometer using a

monochromatized X-ray beam from nickel-filtered CuKα radiation (40 KV/ 30 mA,

wavelength λ=0.1504 nm) at a scanning rate of 2o min-1. The XRD patterns change was

plotted by intensity vs. 2θ in the range of 20o -70o. The average size of the crystals was

estimated using Debye-Scherrer’s formula:

cosCDB

(3-1)

where DC is the average crystalline diameter, Κ = particle shape factor (for spherical

particles, Κ = 0.9), λ is the X-ray wavelength, B is the angular line width of half-

maximum intensity and θ is the Bragg’s angle in degree.


47

3.3 Vibrating Sample Magnetometer (VSM)

The VSM is the basic instrument for characterization of magnetic materials as a

function of magnetic field and temperature. It is based on vibrating a sample in a

magnetic field to produce an alternating electromagnetic field in the pickup coils.

Sample is placed between two electromagnet poles which are attached to pickup coils.

The electromagnets produce a high magnetic field (~90 kOe) to saturate the sample.

The sample vibrates vertically and the oscillation induces an AC signal (i.e. e.m.f.

according to the Faraday’s law) in the pickup coils. The amplitude of this signal is

proportional to the magnetic moment of the sample. A plastic cylinder cell containing

the sample was attached on a rod in the applied magnetic field from -15,000 to 15,000

Oe, in which the rod was vibrating in a certain rate. The magnetization curve of the

magnetic particles at room temperature was then plotted with the changes of magnetic

field strength and its direction. The magnetization curve (M-H hysteresis loop) of the

magnetic particles at room temperature is obtained by slowly sweeping the applied field

from a maximum positive field, through zero, to a maximum negative field and

reversing to the maximum positive field.

Wet magnetic particles were freeze-dried (Edwards freeze-dryer, ESM 1342) for 24 h

before VSM measurement. In this work, VSM (Lakeshore, Model 665) was used to

determine the saturation magnetization (MS) of the nanoparticles. VSM experiments

were carried by placing 10-20 mg dry powder samples (wrapped with aluminum foil) in

a VSM sample holder and then scanned from 2T to -2T (Tesla, 1 T = 104 Gauss) at the

room temperature. A small piece of nickel was used as a standard sample to calibrate

the VSM before all the VSM measurements.


48

3.4 Thermogravimetric Analysis (TGA)

To measure the changes in the weight loss of the sample as a function of temperature

and time thermal analysis technique is used. The thermogravimetric analysis (TGA)

was performed on a thermal analysis system (Model: TA 2050). For TGA

measurements, 7-15 mg of the dried sample was loaded into the system and the mass

loss of dried sample was monitored under N2 at temperatures ranging from room

temperature to 800oC at a rate of 10oC/ min.

3.5 Differential Scanning Calorimetry (DSC)

DSC is mainly concerned with the amount of heat absorbed or released by a sample as

it is heated or cooled or kept at constant temperature (isothermal). In actual practice the

sample and reference materials are simultaneously heated or cooled at a constant rate.

The thermogram is plotted as mW vs T. DSC is a fast and relatively inexpensive

technique to characterize cyclodextrin inclusion complexes. The DSC records were

obtained with a Mettler Toledo DSC 882 apparatus. Between 4 and 10 mg of sample

was crimped in a sealed aluminium pan and heated at 10oC/ min in the range of room

temperature to 300oC using an empty sealed pan as a reference. Dry nitrogen was used

as purge gas and the N2 flow rate was 80 ml/min.

3.6 Fourier Transform Infrared (FTIR)

FTIR is an analytical technique for the identification of organic (and in some case for

inorganic) materials. It measures the absorption of various infrared light wavelengths by

the target sample, which can identify specific molecular components and structures.

Absorption bands in the range of 4000-1500 wavenumbers are typically due to

functional groups (e.g. –OH, C=O, N-H, and CH3, et al.). The region between 1500-400

wavelength numbers is considered as the fingerprint region, which is highly specific for


49

each molecule and no two molecules have the same spectrum at this region. FTIR

measurements were performed in a transmission mode using a Shimadzu infrared

spectrometer (Model 8400) with KBr as background over the range of 4000-400 cm-1.

Samples were prepared by following steps: (1) First 1-2 mg particles/samples were

mixed with 150 mg KBr powder (highly transparent substance in IR range) using

spatula. (2) Then the resulting mixture was pounded with a pestle. (3) Finally a thin

pallet was formed under a press.

3.7 Brunauer-Emmett-Teller (BET) Method

The specific surface area was measured by BET analyzer (Model: Nova 3000). Surface

cleaning (degassing) of the dried solid magnetic particles were carried out by placing

the sample in a glass cell and heating under vacuum before the measurements. Once

clean, small amounts of gas molecules were admitted in steps into the sample chamber

and then stick to the surface of solid particles, which was carrying out in an external

bath maintained by liquid nitrogen. As the equilibrium adsorbate pressures approach

saturation, the surfaces of magnetic particles become completely occupied by adsorbate.

Knowing the density of the adsorbate, it is easy to calculate the specific area of the

magnetic particles.

3.8 Zeta Potential Analyzer

Zeta potential measurement is an easy and powerful way to investigate the charge

properties of particles and colloidal suspensions. According to the electrical double

layer (EDL) theory, the zeta potential represents the surface charge at the shear plane

which separates the Stern layer, attached to the particle, from the diffusive layer. It is

the most relevant parameter


50

regarding electrostatic and electrokinetic properties of the particles, because the ions in

the Stern layer merely effect a static charge compensation. The zeta potential ζ is

usually deducted from the measurement of the electrophoretic mobility μe, based on the

Smoluchowski equation:

μe = ε. (ζ/η) (3-2)

where ε is the permittivity constant of the liquid involved and η is the viscosity of this

liquid. In experiments ζ is investigated as a function of the solution pH and the ionic

strength. If all the particles in suspension have a large negative or positive zeta potential

then they will tend to repel each other and there will be no tendency for the particles to

come together (flocculate). However, if the particles have low zeta potential values then

there will be no force to prevent the particles coming together and flocculating. The

general dividing line between stable and unstable suspensions is generally taken at

either +30 or -30 mV. Particles with zeta potentials more positive than +30 mV or more

negative than -30 mV are normally considered stable. The most important factor that

affects zeta potential is pH. In this thesis work, the zeta potentials of as-synthesized

uncoated or coated Fe3O4 magnetic nanoparticles (0.1 mg/ml) were measured in 10−3M

NaCl aqueous solution at different pH using a Malvern Zetasizer Nano-ZS model

ZEN3600 equipped with a standard 633 laser. The pH was adjusted with diluted HNO3

or NaOH solution. Hydrodynamic size and size distribution of the nanoparticles was

also measured using this same equipment by the dynamic light scattering (DLS)

technique. Hydrodynamic size (dH) is estimated from the following equation:

(3-3)

where k is the Boltzmann constant, T is the absolute temperature, D is the translational

diffusion coefficient, and η is the viscosity of the liquid. This method is based on the


51

measurement of the speed at which particles are diffusing because of Brownian motion.

A light source, usually a laser, illuminates a sample cell containing the particles, and

fluctuations in the scattered intensity provide information on the particles’ dynamics.

3.9 X-ray Photoelectron Spectroscopy (XPS)

XPS is well established technique for surface analysis of particles, in which surfaces are

irradiated with soft X-ray and the emitted photoelectrons energy are analyzed. The

difference between X-ray energy and the photoelectron energies give the binding

energies (BEs) of the core level electrons, an atomic characteristic. XPS measurements

were made on an AXIS HSi spectrometer (Kratos Analytical Ltd.) using a

monochromatized Al Kα X-ray source (1486.6 eV photons) at a constant dwell time of

100 ms and a pass energy of 40 eV. The sample was mounted on the standard sample

studs by means of double sided adhesive tape. The core-level signals were obtained at a

photoelectron take-off angle 90° (with respect to the sample surface). The anode

voltage was 15 kV, and the anode current was 10 mA. The pressure in the analysis

chamber was maintained at 6.7 x10-6 Pa or lower during each measurement. All binding

energies (BEs) were referenced to the C1s neutral carbon peak at 284.6 eV. The spectral

deconvolution was performed using the curve-fitting program with the subtraction of

Shirley background; the line-width (full width at half-maximum) of the Gaussian peaks

was maintained constant for all components in a particular spectrum. Surface elemental

compositions were determined from XPS peak area ratios, after correction with the

experimentally determined sensitivity factors.

3.10 Circular Dichroism (CD)

Circular dichroism (CD) is the difference in absorption between left and right handed

circularly polarized light. Proteins contain elements of asymmetry and thus exhibit


52

distinct circular dichroism signals. CD spectroscopy was used to measure the

conformational change of the refolded protein with respect to the native one. Solutions

of the native protein and the refolded protein were scanned over the wavelength range

200-250 nm by Jacob J810 spectropolarimeter, using 0.1 cm quartz cylindrical cell. All

circular dichroism measurements were carried out at 25C with the help of a

thermostatically controlled cell holder. Spectra were recorded at 1 nm intervals, with a

scan speed of 20 nm/min. Each spectrum was the average of two scans and noise in the

data was smoothed using the software. In all tests, circular dichroism spectra were

corrected for the background absorbance. The CD spectra measurements were

processed by Jasco’s spectra manager in millidegrees and were smoothed using the in-

built binomial smoothing algorithm. The collected data expressed in millidegrees

(mdeg) could be converted to mean molar ellipticity (deg cm2/dmol) using the relation:

θ mrd = θd (M/10CLNr)

(3-4)

where θmrd is the mean molar ellipticity per residue at 208 nm (deg cm2/dmol), θd is the

ellipticity in units of mdeg which is the experimentally measured value, M is the

molecular weight of the protein, C is the concentration of the sample in mg/ml, d is the

optical path length of the cuvette in centimeters and Nr is the number of amino residues.

In our refolding experiment, the secondary structures of native protein and refolded

protein were evaluated by comparing the α-helix contents which may be estimated

according to the following equation [158]:

% 100% (3-5)


53

3.11 Fluorescence

Fluorescence is the phenomenon in which absorption of light of a given wavelength by

a fluorescent molecule is followed by the emission of light at longer wavelengths. The

distribution of wavelength-dependent intensity that causes fluorescence is known as the

excitation spectrum, and the distribution of wavelength-dependent intensity of emitted

energy is known as emission spectrum. The fluorescence measurements of the native

protein and the refolded protein by different desorption agent were carried out with

fluorescence spectrometer (Quantamaster, GL-3300 & GL-302 laser systems).

Tryptophan (Trp) is one of the three naturally occurring aromatic amino acid residues

that fluoresce when excited with UV light. When Trp residue is located in a

hydrophobic environment, the fluorescence is blue shifted relative to the emission when

it is located in a hydrophilic environment. When protein changes its conformation, the

exposure of Trp residues to the solvent may change and thereby affect fluorescence.

That’s why Trp fluorescence can be used for monitoring structural changes of proteins.

For the intrinsic protein fluorescence spectra of native and refolded CA samples, the

excitation wavelength was set at 280 nm and the emission was scanned from 290 to 400

nm for all samples. The scan rate was 1.0 nm/min-1 with a 10 nm bandpass for both

excitation and emission.

Chapter: 4

54

Chapter 4 β–cyclodextrin bonded magnetic nanoparticles as solid-phase artificial chaperone in refolding of proteins1

4.1 Introduction

The production of biologically active recombinant proteins in heterologous systems

often encounters the formation of inactive aggregates known as inclusion bodies [61].

Protein aggregation, which is a kinetically competitive process with protein folding

leads to low yields of biologically functional protein. Such aggregates must be

separated from cell debris and solubilized by exposing them to a strong denaturant, such

as urea or guanidine (GdmCl). Refolding of unfolded polypeptide chains to their native

state is initiated through decreasing the denaturant concentration by dialysis or direct

dilution. Several approaches have been developed to prevent protein aggregation and

also to facilitate protein refolding. One of the extensively used approaches is dilution

method, in which small molecules are added to mask or to eliminate the intermolecular

hydrophobic interactions among denatured proteins, thus prevent misfolding and/or

aggregation and promote refolding of proteins. Examples of such additives include

sugars [159,160], amino acids [159,161], acetoamide [162], alcohols [160,161],

polyethylene glycol [163], metals [164,165], detergents [166,167], and cyclodextrins

[168]. Cyclodextrins (CDs) are non-toxic, stable, cyclic oligosaccharides consisting of α

(1→ 4) linked D-glucose units which can be visualized as toroidal, hollow truncated

1. A.Z.M. Badruddoza, K. Hidajat, M.S. Uddin, Synthesis and characterization of beta-cyclodextrin-

conjugated magnetic nanoparticles and their uses as solid-phase artificial chaperones in refolding of

carbonic anhydrase bovine, J. Colloid Interface Sci. 346(2) (2010) 337-346.

Chapter: 4

55

cones with hydrophilic rims, and hydrophobic internal cavity. The ability of

cyclodextrin to sequester hydrophobic moieties makes them interesting and valuable in

many areas of research such as enantiomeric separation [169], targeted drug delivery

[60], removal of contaminants from wastewater [170] etc.

More recently, a systematic approach referred to as “artificial chaperone-assisted

refolding” has been developed by Rozema and Gellman [65-68] that effectively

promotes the renaturation yield of denatured proteins by suppressing the aggregate

formation. This method consists of two steps: in the first (capturing) step, the protein is

captured by a detergent and a stable protein–detergent complex is formed. The

detergent molecules shield the hydrophobic regions of denatured protein, thus prevent

aggregation of protein. In the second (stripping) step, β-CD is added which strips the

detergent molecules from the complexes with concomitant refolding of the protein.

Several other molecules like linear dextrin [171], cycloamylose [172] or alginate [173]

have also been reported which could replace cyclodextrin to act as the stripping agents.

However, with such systems, it is necessary to remove the detergent-stripping agent

complex and excessive stripping agents from the refolded products for downstream

purification which in turn leads to process complexity and increased production cost.

Now-a-days, magnetic nanoparticles with tailored surface chemistry have been widely

used in various fields of biotechnology. Due to unique size, biocompatibility and

superparamagnetic properties, magnetic nanoparticles are emerging as promising tools

for various fields of applications [174]. In the present work, we use β-CD conjugated

magnetic nanoparticles (CD-APES-MNPs) as stripping agents for protein refolding.

With cumulative effects of chaperoning behavior of β-CD and superparamagnetism of

iron oxide, CD-APES-MNPs is expected to have potential applications in protein

Chapter: 4

56

refolding. Because, the use of solid phase CD-APES-MNPs would offer the following

potential improvements over the soluble artificial chaperone assisted refolding: (1) steps

to remove the detergent-cyclodextrin complex and excess cyclodextrin can be omitted

and hence minimizes the cost; and (2) the magnetic nanoparticles bonded with β-CD

can be handled from the suspension easily by using low gradient magnetic field and

recycled after simple washing. Taking into the consideration of the recycling potential

of the solid phase, CD-APES-MNPs might be a suitable and efficient candidate for

protein refolding system.

A few insoluble solid adsorbents, such as β-CD-epichlorohydrin copolymer [175-177],

β-CD–acrylamide copolymer beads [177], β-CD-polyurethane polymer [103], β-CD

bonded silica particles [178] were previously used as host molecules for detergents in

artificial chaperone-assisted (ACA) refolding of various denatured proteins. Although

there were some previous work carried out on solid phase assisted protein refolding, no

or limited work on nanosized magnetic particles assisted protein refolding has been

published to the best of our knowledge. In the present study, β-CD conjugated magnetic

nanoparticles (CD-APES-MNPs) are synthesized and characterized. These synthesized

CD-APES-MNPs are then used as solid phase artificial chaperone in refolding of

bovine carbonic anhydrase (CA) as model protein.

4.2 Experimental

4.2.1 Materials

The following chemicals were used for synthesizing β-CD conjugated magnetic

nanoparticles: iron (II) chloride tetrahydrate (99%) [Alfa Aesar], iron (III) chloride

hexahydrate (98%) [Alfa Aesar], 3-aminopropyltriethoxysilane (APES, 98%) [Alfa

Aesar], ammonium hydroxide (25%) [Merck], β-cyclodextrin (99%) [Tokyo Kasie

Chapter: 4

57

Kogyo (Japan)], p-toluenesulfonyl chloride (98%) [Lancaster (UK)], carbonic

anhydrase (CA) from bovine erythrocyte [Sigma], 4-nitrophenyl acetate (pNPAc, 99%)

[Fluka], cetyltrimethylammonium bromide (CTAB, 99%) [Sigma],

tetradecyltrimethylammonium bromide (TTAB, 99%) [Sigma], sodium dodecyl sulfate

(SDS, 99%) [Sigma], cetyltrimethylammonium monohydrogen sulfate (CTAHS, 99%)

[Sigma], 1-anilinonaphtalene-8-sulfonate (ANS) [Sigma]. All other chemicals were of

analytical grade and were used as received without further purification. All protein

refolding experiments were generally performed in 20 mM Tris sulfate buffer, pH 7.75,

prepared using double distilled water.

4.2.2 Preparation of mono-tosyl-β-cyclodextrin (Ts-β-CD)

Mono-tosyl-β-cyclodextrin (Ts-β-CD) was synthesized according to the following

method. A solution of β-CD (18g, 16mmol) in 100 ml dry pyridine was prepared and

2.5g of p-toluenesulfonyl chloride (12 mmol) was added to this solution. The reaction

was carried out at 2-4C for a period of 8 h, and then 2 days at room temperature [179].

After concentrating the solution under vacuum, the mixture was poured into diethyl

ether. A white precipitate was collected and purified by repeated crystallization from

water. This product was finally washed by acetone and dried under vacuum at 60ºC.

Chemical composition for Ts-β-CD (C49H75O37S): C, 45.68%; H, 5.83%; S, 2.49%.

Found from elemantal analysis: C, 45.60%; H, 5.71%; S, 2.42%. IR (KBr): 3387 (ν,

OH), 2927 (ν, C-H), 1636 (δ, OH), 1368 (ν, SO2), 1157 (ν, C-O-C), 1029 (ν, C-O).

4.2.3 Preparation of Fe3O4 magnetic nanoparticles

Fe3O4 magnetite nanoparticles were prepared by chemical co-precipitation method

maintaining a molar ratio of Fe2+: Fe3+ = 1: 2 under a non-oxidizing environment and

alkaline condition [14]. To obtain 1g Fe3O4 precipitate, 0.86 g of FeCl2.4H2O and 2.36 g

Chapter: 4

58

of FeCl3.6H2O salts were reacted under a nitrogen atmosphere in 40 ml of de-aerated

Milli-Q water with vigorous stirring. 5 ml of NH4OH (25%) was added after the

solution was heated to 80ºC. The reaction was carried out further for another 30 min at

80ºC under constant stirring to ensure the complete growth of the nanoparticle crystals.

The resulting particles were then washed with Milli-Q water to remove any unreacted

chemicals and dried by freeze-drying for 24 h.

4.2.4 Preparation of 3-aminopropyltriethoxy silane (APES) modified magnetic nanoparticles (APES-MNPs)

To synthesize APES-MNPs, 3 g of Fe3O4 nanoparticles was suspended in 90 ml ethanol

and 1 ml H2O and sonicated for 30 min in the ultrasonic bath. The mixture was then

vigorously stirred in a 250 ml three neck flask equipped with a reflux condenser for 40

min. 10 ml of APES was then added slowly to the flask. The reaction was carried out

for 3 h at the refluxing temperature under nitrogen atmosphere and mechanical stirring.

The resulting nanoparticles were washed with water, then acetone, and these washings

were repeated for one more time. The resulting APES-MNPs were finally dried in the

air.

4.2.5 Fabrication of Ts-β-CD modified Fe3O4 nanoparticles (CD-APES-MNPs)

50 ml of DMF containing 2 g of APTS-MNPs was stirred in a three neck flask equipped

with a reflux condenser at room temperature. 0.25 g of Ts-β-CD dissolved in 10 ml of

DMF was added to this reaction mixture (pH 7~8). The temperature was increased to

60ºC and the reaction was carried out for 7 h under nitrogen atmosphere and

mechanical stirring. The reaction mixture was left overnight at room temperature. The

resulting particles were then collected using a permanent magnet and washed with DMF

and acetone to remove any unreacted chemicals and dried in the air.

Chapter: 4

59

4.2.6 Protein refolding experiments

4.2.6.1 Determination of protein concentration

The protein concentration of the native carbonic anhydrase solution was determined by

measuring the absorbance at 280 nm with an extinction coefficient (ε280) of 5.7×104

mol−1 cm−1 [180].

4.2.6.2 CA denaturation/ renaturation and assay

In a typical thermal denaturation of carbonic anhydrase (CA), 0.043 mg/ml of CA in 20

mM Tris sulfate (pH 7.75) containing 2 mM of each of detergents (CTAB, TTAB,

SDS) were heated to 70°C for 10 min in thermostatically controlled water bath

according to Rozema and Gellman [65]. Protein inactivation was confirmed by activity

determination as well as by fluorescence analysis. After cooling for 10 min, the CA-

detergent complex solutions were diluted with addition of certain amount of stock

solutions of suspended CD-APES-MNPs or β-CD (for liquid phase artificial chaperone

assisted refolding) to make solutions of 0.029 mg/ml CA, 1.35 mM detergent, 13.5 mM

Tris sulfate, pH 7.75. After incubation for overnight, the supernatant was collected by

applying magnetic field and assayed for enzymatic activity. In each CA refolding study,

3 ml of aliquots was used. For refolding kinetic analysis, the enzymatic activities were

monitored immediately after addition of CD-APES-MNPs to the CA solutions.

The enzymatic activities of renatured CA solutions were determined based on pNPAc

esterase activity of the enzyme according to Pocker and Stone [181]. Briefly, 45 μl of

52 mM pNPAc in dry acetonitrile was added to 0.45 ml of CA solution to make a

solution of 0.0264 mg/ml CA, 12.3 mM Tris-sulfate, pH 7.75, and 4.7 mM pNPAc.

After 10 s of mixing, the increase in the hydrolysis product of p-nitrophenolate was

Chapter: 4

60

monitored by measuring the increase in absorbance at 400 nm as a function of time. The

absorbance of the solution was measured every second for 30–60 s using a Model UV-

1601 Shimadzu spectrophotometer. The yield of the refolded CA was determined by

comparing the resulting initial rate with the initial rate of pNPAc hydrolysis by the

native enzyme under identical conditions and expressed as percentage relative to the

activity exhibited by the native enzyme.

4.2.6.3 Structure analyses using Fluorescence Spectra and Far-UV circular dichroism

Fluorescence experiments to observe the tertiary structural changes of refolded CA

were performed using a fluorescence spectrometer (Quantamaster, GL-3300 & GL-302

laser systems) at 25C, with a 10 mm path length sample cuvette. For ANS

fluorescence studies, to the refolded CA solutions, a concentrated stock of ANS (10

mM) was added to get a final concentration of 250 μM. The dye 1-anilinonaphthalene-

8-sulfonate (ANS) which shows enhanced fluorescence in hydrophobic environments

has been used as hydrophobic reporter probe to monitor protein conformational changes

by binding to the hydrophobic regions of a protein. The fluorescence intensity of ANS

increases when the dye binds to the hydrophobic regions of a protein. All samples were

incubated with this hydrophobic reporter probe for 30 min at 25C and the changes of

ANS fluorescence spectra were recorded between 400 nm and 600 nm using excitation

wavelength of 360 nm. For the intrinsic protein fluorescence spectra of native and

refolded CA samples, the excitation and emission wavelengths were set at 280 and 290–

400 nm range, respectively. The secondary structural changes of the CA samples were

evaluated by far-UV circular dichroism spectroscopy. Measurements were recorded

over wavelength range of 200–250 nm using a Jacob J810 spectropolarimeter with a 0.1

cm path length sample cell. All circular dichroism measurements were carried out at

Chapter: 4

61

25C with the help of a thermostatically controlled cell holder. Spectra were recorded at

1 nm intervals, with a scan speed of 20 nm/min. Each spectrum was the average of two

scans and noise in the data was smoothed using the software. The final CA

concentration after renaturation was set to 0.15 mg/ml. In all tests, circular dichroism

spectra were corrected for the background absorbance.

4.2.6.4 Detergent (CTAB) quantification

After completion of the refolding process, the residual CTAB concentration in the

renaturation buffer was measured based on ANS fluorescence according to Mannen et

al. [175]. The refolded samples in solid-phase artificial chaperone assisted refolding

system were incubated with 100 μM ANS (final) and the fluorescence emission was

measured at 480 nm after 30 min incubation at room temperature using a 360 nm

excitation wavelength with 1 cm path length quartz cell. Fluorescence intensity was

plotted against the final detergent concentration and the percent of residual detergent

measured according to fluorescence intensity of each of the refolded samples.

4.3 Results and Discussion

4.3.1 Synthesis of β-CD bonded magnetic nanoparticles

Fe3O4 magnetic nanoparticles with a mean diameter of about 9.5 nm were synthesized

by co-precipitation method from ferrous and ferric ion solutions with a molar ratio of

1:2 [14,15]. The magnetic nanoparticles prepared by this method had significant

numbers of hydroxyl groups on the surface from contacting with the aqueous phase. For

grafting Ts-β-CD, the amino groups were preliminarily bonded onto the surface of the

magnetic nanoparticles by the reaction with 3–aminopropyltriethoxysilane (APES)

[182]. The APES hydrolyzes in the presence of water to form silanol groups which

coupled with the surface metal hydroxyl groups, forming Si-O-Fe bonds upon

Chapter: 4

62

dehydration. Thus –NH2 groups remain reactive on the surface through direct silanation

of APES on magnetic particles. It is known that the homogeneous reaction between

amino chemicals and tosyl-containing compounds occurs at 60−70ºC in an aqueous

medium with pH 11.5 [183]. However, in our experiment, the grafting of β-CD on the

surface of APES-MNPs was carried out at 60ºC between aminopropyl iron and Ts-β-

CD for 7h in DMF at pH 7-8 because of the poor solubility of Ts-β-CD in water. At

higher pH (pH>11) aminopropyl iron considerably collapsed and no attachment of β-

CD was observed. Similar phenomenon was observed on the synthesis of β-CD bonded

silica particles prepared by Belyakova et al. [28]. The lower reaction temperature was

chosen in order to avoid the side reactions that may occur due to the interactions of

surface amino groups with DMF [28]. The concentration of β-CD in reaction medium

was varied to see its effect on grafting on the surface of magnetic nanoparticles and it

was observed that the amount of grafted β-CD was 0.042, 0.038 and 0.036 mmolg-1

when 0.25, 0.50 and 0.75 g of β-CD was used, respectively. The more β-CD added in

the reaction, the less β-CD was immobilized on the surface of magnetic nanoparticles.

The reaction procedures for fabricating CD-APES-MNPs are shown step by step in

Figure 4-1.

Chapter: 4

63

Figure 4-1 Preparation steps for fabricating β-CD bonded magnetic nanoparticles.

4.3.2 Characterization of magnetic nanoparticles

The reaction pathway for grafting β-CD on surfaces of magnetic nanoparticles was

verified by FTIR studies. Figure 4-2 shows the FTIR spectra of MNPs, APES-MNPs,

CD-APES-MNPs and β-CD in the 400-2000 cm-1 wave number range. It shows the

characteristic absorption band of Fe–O bonds in the tetrahedral sites is 586 cm-1. The

spectrum of APES-MNPs shows peak at 997 and 1110 cm-1 which can be attributed to

siloxane bonds (Si-O-H) and Si-O-Si groups, respectively due to APES deposition on

the MNPs’ surface. The broad band at 1626 cm-1 is due to –NH stretching vibrations.

The spectrum of β-CD shows the characteristic peaks at 947, 1028, and 1159 cm−1. The

APES-MNPs

Chapter: 4

64

peak at 947 cm−1 is due to the R-1,4-bond skeleton vibration of β-CD, and the peaks at

1028 and 1159 cm−1 corresponded to the antisymmetric glycosidic νa (C-O-C)

vibrations and coupled ν (C-C/C-O) stretch vibration [16,184]. All the significant peaks

of β-CD in the range of 900–1200 cm−1 are present in the spectrum of CD-APES-MNPs

with a small shift. Thus, all the above results indicate that β-CD has been grafted

successfully on APES-MNPs.

Figure 4-2 FTIR spectra of (a) Uncoated Fe3O4 MNPs, (b) APES-MNPs, (c) CD-APES-MNPs, and (d) β-CD.

Typical TEM images and size distribution of APES-MNPs and CD-APES-MNPs are

shown in Figure 4-3. Spherical or ellipsoidal magnetic nanoparticles are observed. TEM

image of β-CD bonded magnetic nanoparticles is almost the same as that of magnetic

nanoparticles coated with APES. The mean diameter of both APES-coated and β-CD

400 800 1200 1600 2000

% T

rans

mis

sion

(a.u

.)

Wavenumber, cm-1

947

10281159

9481001 1029

997

1110

(a)

(c)

(b)

(d)

586

586

586

1626

1626

Chapter: 4

65

functionalized nanoparticles is about 11.5 nm. No detectable layer of β-CD can be

observed on CD-APES-MNPs due to the small amount of β-CD attached on the surface

(0.042 mmolg-1 solid from elemental analysis). It is known that magnetic particles with

size less than about 30 nm with zero coercivity and zero remanence exhibits

superparamagnetism [185]. Therefore, both the magnetic particles coated with APES

and β-CD show superparamagnetic properties which are further evidenced by VSM

studies. Figure 4-3(c) and 4-3(d) also show the hydrodynamic diameter distribution of

APES-MNPs and CD-APES-MNPs in water determined by the DLS technique. The

mean hydrodynamic diameters were found to be 38.6 and 51.8 nm, respectively.

Figure 4-3 Typical TEM images and hydrodynamic size distribution of APES-MNPs (a) &(c) and CD-APES-MNPs (b) & (d), respectively.

0

5

10

15

20

8 9 10 11 12 13 14 15 16 17

Particle diameter (nm)

Fre

quen

cy (

%)

(a) (b)

(c) (d)

0

5

10

15

20

25

30

7 8 9 10 11 12 13 14 15 16 17


Fre

quen

cy (

%)

Chapter: 4

66

Elemental analyses shown in Table 4-1 provide further evidence of the successful

immobilization of β-CD onto magnetic nanoparticle surface. The grafted amounts of

aminopropyl groups and β-CD moieties on the surface of magnetic nanoparticles (BET

area~109 m2/gm) calculated from elemental analyses are as 0.812 mmol/g and 0.042

mmol/g of solid, respectively. The concentration of surface amino groups (-NH2) is

0.812 mmol/gm (7.44 µmol/m2). It can be concluded that not all –NH2 groups on the

surface of APES-MNPs are bonded with tosyl groups of Ts-β-CD as the tosylation

reaction occurs 1:1 stoichiometic ratio. Taking into account the molecular area of Ts-β-

CD (=3.41 nm2) [28], the maximum quantity of Ts-β-CD grafted on the surface of

aminopropyl iron cannot exceed 0.054 mmol/g. Thus, the surface β-CD coverage ratio

of Fe3O4 magnetic nanoparticles was about 79%. Probably, the incompleteness (on

monolayer adsorption basis) of the surface coating is owing to the incompleteness of

surface tosylation and also due to existence of spatial resistance or the steric hindrance

of the β-CD moieties.

Table 4-1 Data on chemical analysis of magnetic nanoparticles modified with APES

and β-CD.

As-synthesized MNP samples %C %N %H

APES-MNPs 3.12 1.12 0.47

CD-APES-MNPs 4.96 1.09 0.73

The XRD patterns of MNPs, APES-MNPs and CD-APES-MNPs indicate five

characteristic peaks at 2θ = 30.1, 35.5, 43.1, 53.4, 57, and 63.1 as shown in Figure

4-4(i). These are related to their corresponding indices (220), (311), (400), (422), (511),

and (440), respectively. This reveals that the resultant nanoparticles are pure Fe3O4 with

a spinel structure and the modification of magnetic nanoparticles surface with APES or

β-CD does not result in the phase change of Fe3O4. The crystal size of CD-APES-MNPs

Chapter: 4

67

-80

-60

-40

-20

0

20

40

60

80

-15 -10 -5 0 5 10 15

M, e

mu/

g F

e 3O

4

H, kOe

Fe3O4 MNPs

CD-APES-MNPs

determined from the XRD pattern by using Scherrer’s equation is found to be 12.8 nm,

consistent with that observed from the TEM image.

Figure 4-4 (i) XRD patterns of (a) uncoated magnetic nanoparticles (MNPs), (b) APES modified magnetic nanoparticles (APES-MNPs), and (c) β-CD modified magnetic nanoparticles (CD-APES-MNPs).(ii) Magnetization curves for uncoated and β-CD coated Fe3O4 magnetic nanoparticles measured by VSM at room temperature.

(i)

(ii)

Chapter: 4

68

The magnetic properties of the uncoated and β-CD coated Fe3O4 nanoparticles were

measured by VSM at room temperature. The VSM technique is used to measure the

magnetization value of magnetic particles under applied magnetic field. For particles

with superparamagnetic property, when the applied magnetic field is removed, they

should exhibit no coercivity and remanence. In Figure 4-4(ii), the hysteresis loops that

are characteristic of superparamagnetic behavior can be clearly observed for both

MNPs and CD-APES-MNPs. Superparamagnetism is the responsiveness to an applied

magnetic field without retaining any magnetism after removal of the applied magnetic

field. The properties of superparamagnetism and high saturation magnetization value of

magnetic nanoparticles are the two important parameters in biomedical applications.

From M vs. H curve, the saturation magnetization value (Ms) of uncoated MNPs was

found to be 75 emu g−1 which is much less than its bulk counterpart (92 emu g−1) [186].

For CD-APES-MNPs, the magnetization obtained at same field was 64 emu g-1, lower

than that of uncoated Fe3O4. This is mainly attributed to the existence of non-magnetic

materials (APES and β-CD layer) on the surface of nanoparticles. This feature allows

the as-synthesized nanoparticles to be used even under relatively low external magnetic

field. The combination of magnetic property of iron oxide with chaperoning behavior of

β-CD suggests that these materials have great a potential to be used in protein refolding.

4.3.3 CD-APES-MNPs assisted CA Refolding As mentioned earlier, Rozema and Gellman [65,68] had developed an approach for

protein renaturation in which the aggregation of protein was prevented by detergents

which could shield the hydrophobic regions of denatured proteins. The detergent

molecules were then slowly stripped off the protein surface by cyclodextrins, allowing

the protein to refold. This approach requires an efficient detergent stripping agent and

further steps needed to remove detergent-CD complex and excess β-CD are necessary

Chapter: 4

69

in downstream purification process. But using solid phase CD-APES-MNPs as

stripping agent, further purification steps can be omitted. In addition, this solid phase

can be easily separated by using low gradient magnetic field and re-used it after simple

washing. A schematic diagram of the protein refolding process using β-CD conjugated

magnetic nanoparticles is shown in Figure 4-5.

Figure 4-5 Schematic diagram of CD-APES-MNPs assisted protein refolding in-vitro.

Figure 4-6 shows the kinetic properties of CA refolding at concentration of 0.029

mg/ml CA using 40 mg/ml of CD-APES-MNPs. We also measured the residual CTAB

concentration in refolded solution using ANS fluorescence probe at different time

intervals to check whether the refolding occurred concomitantly with the detergent

stripping steps. The CTAB concentration in the capturing step was set at 2mM which

Chapter: 4

70

was higher than the CMC of CTAB. The CMC of CTAB in the refolding buffer was

measured to be 0.63 mM, similar value was reported in literature [65]. As shown in

Figure 4-6 that only about 30-40 mins was required for the maximum refolding yield to

be reached in CD-APES-MNPs assisted CA refolding. Within this time, most of CTAB

molecules were stripped away by the CD-APES-MNPs and the residual CTAB

concentration also reached a plateau (~20%). Here, the stripping of CTAB form the

protein-detergent complexes was greatly limited by the amount of CD immobilized on

the solid surface. More CTAB could be stripped away using higher amount of CD-

bonded magnetic nanoparticles (Figure 4-7), but the refolding yield would decrease. To

solve this problem, one approach would be to speed up the stripping of detergent

molecule from the protein-detergent complexes by increasing the number of CD

molecules per unit volume or unit mass of solid matrix. However, this result indicates

that both detergent stripping process and refolding of CA are very fast processes and

occur concomitantly.

Figure 4-6 Time course of refolding yield of CA (■) and residual CTAB concentration (●) in refolded samples in presence of 40 mg/ml CD-APES-MNPs. The CTAB concentration in the capturing step was set at 2mM and final CA concentration was 0.029 mg/ml.

10

20

30

40

50

60

70

80

90

100

0

20

40

60

80

100

0 40 80 120 160

% R

esid

ual

CT

AB

Ref

old

ing

yiel

d,

%

Time, min

Chapter: 4

71

It was previously reported that the minimum detergent concentration i.e. the minimum

structural arrangement between CA and detergent is required to prevent CA aggregation

at the capture step [187,188]. This minimum structural arrangement is apparently

achieved at a detergent (CTAB, TTAB and SDS) concentration of 2 mM. Under these

conditions, the competent mixed micelles are formed [65]. Formation of competent or

incompetent protein-detergent complex strongly depends on the final concentration of

detergent used in the system. For example, CA–CTAB complexes generated with 0.2–

0.4 mM final CTAB concentration are ‘incompetent’: they do not lead efficiently to

refolded protein upon stripping, even though the amount of detergent is sufficient to

prevent detectable protein aggregation at the point of denaturant dilution [187]. It was

predicted that competent complexes contain only one protein molecule, whereas

incompetent protein–detergent complexes contain, on average, more than one protein

molecule per complex. So, in stripping step, the rate of detergent stripping which is

determined by the amount of β-CD (or β-CD : detergent ratio) is a key factor in

artificial chaperone assisted refolding approach [187]. Regarding this criteria, the

effects of CD-APES-MNPs on refolding yield of thermally denatured CA were

investigated. Figure 4-7 shows the refolding yield of CA upon addition of CD-APES-

MNPs (10–60 mg/ml) and as a function of detergent type. The maximum refolding

yield of CA of 85%, 74% and 48% were obtained in CD-APES-MNPs assisted

refolding for CTAB, TTAB and SDS, respectively. The order of refolding yields

(CTAB> TTAB> SDS) could be explained by the hydrocarbon chain length of the

detergent which influences the rate of detergent stripping from the detergent-protein

complexes [176,189]. It was reported that the detergent stripping, in the liquid-phase

ACA technique, is the rate-determining step of the whole process and the rate of this

step are controlled by β-CD/detergent binding constants [176,188,189].

Chapter: 4

72

Figure 4-7 Relative changes in the recovered enzymatic activity of the assisted renatured CA as a function of detergent type and the solid phase quantity. Protein-detergent (CTAB, □: TTAB, Δ and SDS, ○) complex solution (2.7 ml, with a detergent concentration of 2mM in Tris–Sulfate buffer, pH 7.75) was added to 1.3 ml suspension containing various amounts of β-CD bonded magnetic nanoparticles. The final protein concentration was 0.029 mg/ml. The residual CTAB concentration (●) in the supernatant of the suspension was also measured.

From Figure 4-7, it is also shown that the optimal refolding yield is achieved at

concentration of 40, 50 and 50 mg/ml of CD-APES-MNPs for CTAB, TTAB and SDS,

respectively which we refer to it as the optimal value and the refolding yield decreases

beyond these values for each of the three detergents. The reason for the decrease in

refolding efficiency at higher than optimal concentrations of the CD-APES-MNPs is

not clear. Yazdanparast et al. claimed that it was possibly due to the fact that solid-

phase ACA approach faces difficulties due to mass transfer limitation and/or the low

substitution content of β-CD in the CD-bearing solid phase [176]. Alternatively, there is

the possibility of ‘‘inappropriate’’ removal rate of the detergent molecules from the

detergent–protein complexes [103]. It could be better explained by the mechanism

proposed by Hanson and Gellman [187] where product-determining step occurs after a

0

20

40

60

80

100

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70

% R

esid

ual

CT

AB

Ref

old

ing

yiel

d,%

Conc. of CD bonded magnetic nanoparticles, mg/ml

Chapter: 4

73

portion of the detergent has been stripped from the protein complex U–dn–m as shown

in Figure 4-8. Under such a condition, the stripped and unfolded protein molecules will

form intermolecular hydrophobic interactions which will finally lead to aggregation

and/or misfolding. This view could be evaluated through analyzing the

microenvironment of the refolded and the partially refolded intermediates by using the

ANS fluorescence probe [190]. Use of too much striping agent might affect the

detergent stripping rates which in turn will lead to the exposure of hydrophobic surfaces

of the stripped and the unfolded intermediates which are quite suited for intermolecular

interactions and aggregate formations [103]. The lower yield obtained using higher

amount of magnetic nanoparticles values might also possibly be due to non-specific

binding of the folded or refolded enzymes to these nanoparticles. Non-specific

adsorption might occur due to residual amine (NH2) groups on the surface of magnetic

nanoparticles due to electrostatic interactions [191].

Figure 4-8 Proposed mechanism by Hanson and Gellman for cyclodextrin-induced folding from a protein–detergent complex [187]. U–dn, protein–detergent complex generated during the capture step (contains only one protein molecule); U, unfolded protein molecule; d, one detergent molecule; U–dn–m, protein–detergent complex from which detergent has been partially stripped away; (U–dn–m)p, partially stripped protein–detergent complex that has self-associated to form a species containing multiple protein molecules; F, first detergent-free form of the protein (extent of folding unspecified); N, native state of the protein.

Chapter: 4

74

In order to compare the solid-phase ACA refolding results with that of using liquid-

phase ACA refolding, renaturation of denatured CA was carried out in presence of

soluble β-CD (0-16 mM). The maximum refolding yield (85%) obtained using solid-

phase CD-APES-MNPs was almost in the same level as that for the liquid-phase ACA

refolding method using soluble β-CD (90%) as shown in Figure 4-9. Moreover, our

refolding results could be compared when using same protein concentration level with

those using other solid-phase ACA method. For example, Esmaeili et al. obtained CA

refolding yields of 80% and 54% using at 0.030 mg/ml β-CD-polyurethane polymer and

β-CD bonded silica particles at same CA concentration (0.030 mg/ml) in two different

studies [103,178]. Yamaguchi et al. also obtained same refolding yield (80%) using β-

CD–acrylamide copolymer hydrogel beads [177]. Our result indicates that CD-APES-

MNPs are suitable, effective and recyclable stripping agents for solid-phase ACA

technique. Moreover, use of solid phase CD-APES-MNPs in ACA method would

eliminate further tedious purification steps associated with liquid-phase ACA method.

Figure 4-9 Refolding yield of thermally denatured CA in the presence of different concentrations of β-CD in liquid phase artificial chaperone assisted refolding approach. The denatured CA (0.043 mg/ml) was diluted with water containing

0

20

40

60

80

100

0 4 8 12 16

Ref

old

ing

yiel

d,

%

Cyclodextrin, mM

Chapter: 4

75

different concentration of β-CD (0-16 mM). The error bars represent standard error of mean for three independent measurements.

It was previously observed that the refolding yield decreases with an increase in the

concentration of the protein to be refolded [63,103,176]. When refolding was performed

at high protein concentrations, inactive aggregates formed by intermolecular interaction

were the predominant products. Our data presented in Figure 4-10, clearly indicate that

a similar pattern also exist in solid-phase artificial chaperone assisted technique. As

shown in Figure 4-10, the maximum refolding yield for CA (85%) was obtained using

40 mg/ml CD-APES-MNPs at CA concentration of 0.043mg/ml in the captured step

(final CA concentration was 0.029 mg/ml). At higher protein concentrations (0.15

mg/ml in the captured step), the renaturation yields of CA dropped to 44.8%.

Figure 4-10 The effect of CA concentration and the amount of CD-APES-MNPs on CA renaturation yield. The CTAB concentration in the capturing step was set at 2 mM. The protein concentration in the captured state were 0.043 (●), 0.075 (■) and 0.15 (▲) mg/ml.

0

20

40

60

80

100

0 10 20 30 40 50 60

Ref

oldi

ng y

ield

, %

Conc. of CD bonded magnetic nanoparticles, mg/ml

0.043 mg/ml

0.075 mg/ml

0.15 mg/ml

Chapter: 4

76

4.3.4 Structural analyses of the refolded products by intrinsic fluorescence and far-UV circular dichroism

Fluorescence spectroscopy is a useful tool to study the structural and conformational

changes of protein molecules in solution. Intrinsic fluorescence analysis can perform

this kind of studies by differentiating the micro-environmental changes of tryptophan

residues upon excitation at 280 nm. Tertiary structure of the refolded (CA) products

was assessed by recording the fluorescence emission spectra as shown in Figure 4-

11(a). The sub-optimal concentration of CD-APES-MNPs causes variation of

fluorescence intensity of refolded samples implying that the tertiary structures are

different from those of native sample. However, the addition of optimal quantity of CD-

APES-MNPs (40 mg/ml, curve 5) to the captured solution caused a remarkable change

in the tertiary structure of the refolded sample in such a way that the pattern of its

tertiary structure became very similar to the native conformation. Figure 4-11(b) shows

the maximum emission peak point of the refolded CA upon addition of various

concentrations of CD-APES-MNPs. The fluorescence emission maximum for native

CA is at 340 nm, while the fluorescence emission maximum for refolded CA in

unassisted refolding method is at 346 nm, which is typical of water-exposed tryptophan

[67]. This implies that the unassisted refolded product is still in inactive CA-CTAB

complex form where the tertiary structure is lost. Detergent-protein complex shows

much higher fluorescence intensity than native CA, suggesting that the fluorescing side

chains (presumably tryptophan) are less subject to internal quenching in the detergent

complex than in the native folded state. However, with addition of various

concentrations of CD-APES-MNPs from 10 to 40 mg/ml, the fluorescence emission

maximum blue shifted from 346 to 340 nm. This indicates that CD-APES-MNPs at 40

mg/ml concentration are maximally assisting the captured CA to refold to its native

conformation.

Chapter: 4

77

Figure 4-11 Effect of various concentrations of CD-APES-MNPs on the intrinsic fluorescence emission of refolded CA samples. (a) Intrinsic fluorescence spectra of refolded samples in the presence of different amounts of CD-APES-MNPs (0 mg/ml, curve 1; 10 mg/ ml, curve 2; 20 mg/ml, curve 3; 30 mg/ml, curve 4; 40 mg/ml, curve 5 and 50 mg/ml, curve 7). Curve 6 represents the native CA. (b) Maximum emission wavelength peak point of the refolded CA in presence of various concentrations of CD-APES-MNPs.

The ANS fluorescence spectra of the native and the refolded CA in the presence of

various amounts of CD-APES-MNPs were also recorded in the wavelength range of

400-600 nm. All refolded samples were incubated at room temperature for 30 min in

0

20

40

60

80

100

120

140

160

180

305 320 335 350 365 380 395

Rel

ativ

e fl

uor

esce

nce

inte

nsi

ty

Wavelength, nm

1

2

3

4

7

5,6

(a)

338

340

342

344

346

348

0 20 40 60

Em

ax(n

m)

CD bonded magnetic nanopaticles, mg/ml

(b)

Chapter: 4

78

the presence of ANS solution (100 μM). The excitation wavelength was 360 nm. As

shown in Figure 4-12, at low CD-APES-MNPs concentrations, most of the released

protein molecules remained in their non-native states with exposed hydrophobic areas

attracting the ANS molecules leading to high fluorescence emission intensities [103].

However, at higher appropriate concentration of CD-APES-MNPs (40 mg/ml), more

efficient refolding has taken place which is evident by decrease in ANS fluorescence. It

is surprising to find that the ANS intensities of the samples with higher than 40 mg/ml

of CD-APES-MNPs were even smaller than that of the native CA. This might be caused

by various types of interactions among β-CD and the protein molecules [103] which

have limited the accessibility of ANS molecules to the proteins. Also, it could be

explained by the inappropriate CTAB stripping rate caused by using too-much stripping

agents which would lead to the exposure of hydrophobic surfaces of the stripped and

the unfolded intermediates suited for intermolecular interactions and aggregate

formations [103].

Figure 4-12 ANS fluorescence spectra of native (6) CA and refolded CA in the presence of 0 (curve 1), 10 (curve 2), 20 (curve 3), 30 (curve 4), 40 (curve 5), 50 (curve 7) and 60 (curve 8) mg/ml of CD-APES-MNPs added in the solution. The final

0

5

10

15

20

25

30

35

400 440 480 520 560 600

Wavelength, nm

Rel

ativ

e A

NS

flu

ores

cnce

inte

nsity

1

8

7

23

45

66

Chapter: 4

79

protein concentration was 0.029 mg/ml for all samples. Samples were excited at 360 nm.

Finally, to investigate whether the secondary structure of CA was successfully

recovered in solid phase CD-APES-MNPs assisted refolding method, the far-UV

circular dichroism spectra of refolded CA using different amounts of CD-APES-MNPs

were measured and compared with that of native CA. Circular dichroism measurements

are difficult with CTAB containing solutions, because of absorption by bromide ion;

therefore, cetyltrimethylammonium hydrogen sulfate (CTAHS) was used in these

studies. Control studies indicated very similar behavior for CTAB and CTAHS as

artificial chaperones [65]. Figure 4-13 shows the far-UV circular dichroism spectra in

200-250 nm range for native CA (curve 1) and for the refolded samples (curves 2, 3 and

4). From the figure, it can be seen that at lower concentration of CD-APES-MNPs (10

mg/ml), the far-UV circular dichroism spectra of the refolded CA have more negative

ellipticity which are attributed to higher α-helix content [65]. However, as the amount

of CD-APES-MNPs increases (curves 2 and 3), the secondary structural features of the

refolded CA become closer to that of the native CA (curve 1). The ellipticity values at

208 nm are different for curves 2, 3 and 4. These values are used to calculate the α-helix

content of proteins [158]. Based on the value of ellipticity obtained at different amount

of solid phase, this result also indicates that optimal refolding assistance was achieved

by using 40 mg/ml of CD-APES-MNPs. Only partial stripping of the detergent

molecules occurs in case of using too little CD-APES-MNPs which in turn leads to

partially stripped inactive intermediates. Under these circumstances, these partially

captured CA remain in the inactive detergent-protein complex states [187]. Sub-optimal

concentration of CD-APES-MNPs may allow the refolding intermediate complexes to

Chapter: 4

80

live long enough to form intermolecular interactions with each other which lead to

declined refolding yields [176].

Figure 4-13 Far-UV circular dichroism spectra of native (curve 1) and the refolded CA samples in the presence of 10 (curve 4), 30 (curve 3) and 40 (curve 2) mg/ml of the CD-APES-MNPs added in the solution. Each spectrum was obtained at 25C with a 1mm path length cell and is the average of two scans. All necessary corrections were made for background absorption.

4.4 Conclusions

In this work, we have described a new method to synthesize β-CD bonded magnetic

nanoparticles (CD-APES-MNPs) having combined effects of chaperoning behavior of CDs

and magnetic properties of iron oxide. The results from FTIR, XRD, TEM and VSM have

confirmed the formation of nanosized superparamagnetic particles functionalized with

β-CD. From the results of elemental analyses, the amount of grafted β-CD on the

surface of magnetic nanoparticles was calculated as 0.042 mmolg-1 solid. These as-

synthesized CD-APES-MNPs were effectively used in as solid phase artificial

chaperone in carbonic anhydrase (CA) refolding in vitro. Our result indicates that the

maximum refolding yields obtained were 85% for thermally denatured CA which was

-20

-16

-12

-8

-4

0

4

200 210 220 230 240 250

Elli

pti

city

, m

deg

Wavelength, nm

4

1

3

2

Chapter: 4

81

as the same level as that using liquid-phase artificial chaperone-assisted refolding and

the structures of the refolded CA were found as same as those of native protein. These

results suggest that CD-APES-MNPs could be used as a suitable and efficient stripping

agent in solid-phase artificial chaperone-assisted refolding due to easier and faster

separation of these stripping agents from the refolded samples and the recycling

potentials of these nanoparticles. This new approach will have a considerable impact on

reducing the production cost for the respective refolding processes by eliminating

further purification steps associated with the liquid-phase artificial chaperone assisted

refolding.

Chapter: 5

82

Chapter 5 Selective recognition and separation of nucleosides using carboxymethyl-β-cyclodextrin functionalized hybrid magnetic nanoparticles2

5.1 Introduction

As the constructing units of nucleic acids, nucleosides are involved in performing

significant functions in various biological processes. Analysis of the nucleosides

provides important information for the diagnosis of certain severe diseases and

metabolic disorders [192]. However, mixing of different nucleosides occurs frequently

when they perform various biological and chemical functions in biological raw

materials [193]. Extensive studies have been carried out to separate and quantify

nucleosides due to their important biomedical and pharmaceutical applications. The

traditional way to quantify nucleosides is ion-exchange method but unfortunately this

technique is unable to separate the nucleosides from their mixtures [194]. In the recent

years, chromatographic methods have been widely used to quantify and separate the

nucleosides in the mixing conditions, among which most of them are reverse phase high

performance liquid chromatography (RP-HPLC) with gradient elution or ion-pair

[192,195]. RP-HPLC requires organic solvents such as acetonitrile or methanol as

mobile phase to achieve satisfactory separation of the nucleosides within the reasonable

retention time. Umemura et al. utilized amphoteric surfactant to modify the

octadecylsilica (ODS) column of RH-HPLC so that a non-organic aqueous solvent can

be used as mobile phase instead [196]. However, the intrinsic disadvantages of HPLC

2. A.Z.M. Badruddoza, L. Junwen, K. Hidajat, M.S. Uddin, Selective recognition and separation of

nucleosides using carboxymethyl-β-cyclodextrin functionalized hybrid magnetic nanoparticles,

Colloids and Surfaces B: Biointerfaces, 92 (2012) 223-231.

Chapter: 5

83

are its cost and stringent requirements for sample purity. The nucleosides mixtures

usually contain small amounts of other large molecules and impurities from cell broth,

which may clog the column, bind to the mobile phase or precipitate out during the

separation process [197]. Another powerful alternative to determine nucleosides is

electrochemical detection. Recent literature reveals that several studies have been

carried out to use different electrodes such as mercury electrode [198], boron doped

diamond electrode [199] and fullerene-C60-modified glassy carbon electrode [198] to

improve the sensitivity and selectivity in the quantification and separation of adenosine

and guanosine. However, tedious sample preparation and high cost are the most

significant limitations of this method. In a recent study by Lan Jin et al, β-

cyclodextrin/layered double hydroxide intercalation compound was used for selective

adsorption of adenosine and guanosine [193]. The method is very effective and simple

but the subsequent removal of clay compound from nucleoside solution requires a very

long period of centrifugation and complete removal is difficult. Thus, development of a

simple, fast and accurate method to separate and detect nucleoside derivatives is

essential for accurate biomedical analysis. Although various magnetic beads or

magnetic latex particles were reported for molecular diagnosis consisting in the

extraction, purification, concentration, and amplification of nucleic acids [191,200],

cyclodextrin modified MNPs combining magnetic and molecular binding/recognition

properties would be useful as separators and receptors in biological research.

Recently, the functionalization of magnetic nanoparticle surfaces with macrocyclic host

molecules in well-defined host–guest interactions has gained significant attention

[201,202]. The combination of the physical properties of magnetic materials and the

molecular recognition ability of CD molecules adsorbed on the nanoparticles surface

makes them to exhibit promising applications in different fields such as catalysis [201],

Chapter: 5

84

drug delivery [16,202,203], environment [201,204,205], chemical sensing [206], and so

on. Recent work showed that based on differences of intermolecular weak interactions

i.g., inner interactions (van der waals force and hydrophobic interactions) and external

interactions (hydrogen bonding), β-CD could selectively separate several alcohols from

aldehyde solutions [207]. However, no work has been done so far utilizing the

combined properties of MNPs and CDs for the selective capture of biomolecules like

nucleosides.

Adenosine (A) Guanosine (G)

Figure 5-1 Chemical structure of adenosine (A) and guanosine (G)

Here, we developed a novel magnetic nanoadsorbent (CMCD-APTS-MNPs) by grafting

CM-β-CD onto the surface of APTS modified Fe3O4 MNPs and evaluated their

capacities to adsorb nucleosides. Adenosine (A) and guanosine (G) were selected as

model target guest molecules as shown in Figure 5-1. The individual and selective

adsorption behaviors of guanosine and adenosine with CMCD-APTS-MNPs were

studied. Effects of pH on adsorption were investigated. For better understanding to gain

insights into the molecular recognition mechanism of nucleosides and β-CD, the

inclusion relation between the immobilized β-CD and the guest substrates were also

investigated.

NH

N

N

O

NH2N

O

HOH

HH

HH

HO

1

2

3

4

567

8

9

1

24

567

8

9

N

NN

N

NH2

O

HOH

HH

HH

HO 3

Chapter: 5

85

5.2 Experimental

5.2.1 Materials

Iron (II) chloride tetrahydrate (99%), Iron (III) chloride hexahydrate (98%), N-

hydroxysuccinimide (NHS), 3-aminopropyltriethoxysilane (APTS, 98%), guanosine

(99%), adenosine (99%), chloroacetic acid (98+%) and N-(3-dimethylaminopropyl)-N’-

ethylcarbodiimide hydrochloride (EDC) was purchased from Alfa Aesar. Ammonium

hydroxide (25%) and ammonium phosphate, monobasic (99.99%) was purchased from

Merck (USA). β-CD (99%) was obtained from Tokyo Kasie Kogyo (Japan). All other

chemicals were used as received without further purification. The water in this work

was Milli-Q ultrapure water.

5.2.2 Preparation of carboxymethyl-β-cyclodextrin (CM-β-CD)

CM-β-CD was prepared following the procedure of literature [208], and the detail

synthesis of CM-β-CD was as following: a mixture of β-CD (10 g) and NaOH (9.3 g) in

water (37 ml) was treated with a 16.3% monochloroacetic acid solution (27ml) at 50ºC

for 5 h. Then the reaction mixture was cooled to room temperature, and pH was

adjusted (6~7) using HCl. The obtained neutral solution was then poured to superfluous

methanol solvent and white precipitates were produced. The solid precipitation was

then filtered and dried under vacuum to give carboxymethylated β-CD (CM-β-CD, 6 g).

1H NMR (300 MHz, D2O), δ (TMS, ppm): 5.08, 5.30 (s, 7H, H-1), 4.39 (s, 2.5H, H-7),

3.30-4.20 (m, 45.7H, H-2, H-3, H-4, H-5, H-6, H-7’). (The number of C is shown in

Figure 5-2). IR (KBr): ν (cm−1): 3140–3680 (–OH), 2923 (–CH), 1741 (C=O), 960–

1200 (C–O–C, C-C/C-O) (Figure 5-3d). The average number of carboxylate groups

(3.1) per CM-β-CD was calculated using 1H NMR [209].

Chapter: 5

86

5.2.3 Fabrication of CM-β-CD modified APES-MNPs (CMCD-APES-MNPs)

CM-β-CD was grafted onto APES-MNPs in the presence of EDC and NHS. Detailed

synthetic procedure for APES modified magnetic nanoparticles (APES-MNPs) are

given in Chapter 4 (Section 4.2.4). A solution of CM-β-CD (1.3 g, 1 mmol) in 40 ml of

distilled water was first activated with 0.192 g (1 mmol) of EDC and 0.115 g (1 mmol)

of NHS for 30cmin. With this mixture, 0.7 g of APES-MNPs dispersed in 10 ml of

distilled water was added and the pH of the reaction mixture was adjusted to 7.0. The

reaction was carried out at room temperature under constant stirring. After 24 h, the

product formed was separated by magnetic decantation and dried by freeze-drying.

5.2.4 Adsorption of nucleosides

Nucleosides adsorption experiments were carried out using batch equilibrium technique

in aqueous solutions at pH 4–9 at 25oC. In general, 110 mg of wet magnetic nano-

adsorbent was added to 5 ml of either guanosine or adenosine solution of various

concentrations and shaken in a thermostatic water-bath shaker operated at 230 rpm for a

specific period of time to reach adsorption equilibrium. After equilibrium was reached,

the magnetic nano-adsorbents were removed using a strong permanent magnet made of

Nd-Fe-B and the supernatant was collected. The amount of nucleosides in the

supernatant was then quantified by UV-vis spectrophotometer. The solution pH was

adjusted by NaOH or HCl.

To measure the adsorption selectivity of CMCD-APES-MNPS, adsorption experiments

were also carried out by putting same amount of adsorbent in a mixture of A and G

(containing equal amounts) with different concentrations. After adsorption equilibrium,

the supernatant were separated by magnetic decantation and the concentrations of A and

G were analyzed by HPLC.

Chapter: 5

87

For single nucleoside samples, UV-Vis spectrophotometer (Model 1601) was used to

determine the concentration of nucleosides. The detection wavelength of both

guanosine and adenosine was set at 260 nm. For mixed nucleoside samples, the amount

of residual nucleosides in solution was quantified by HPLC. HPLC experiments were

carried out with Agilent Technologies 1200 Series chromatography system equipped

with a 4.6 mm × 250 mm C18 column (Agilent). The mobile phase used was 0.05 M

(NH4)H2PO4 in methanol-water (10:90) at pH 5.0. The flow rate was 1.0 ml/min and the

UV detector was set 254 nm at 30oC.

5.3 Results and discussions

5.3.1 Synthesis and characterization of magnetic nanoparticles The reaction scheme for grafting CM-β-CD onto Fe3O4 nanoparticles is shown in

Figure 5-2. First, 3–aminopropyltriethoxysilane (APES) modified MNPs were

synthesized which provides free –NH2 groups on the surface. Then CM-β-CD moiety

was coupled on the magnetic surface through amide bond (–NH–CO–) formation.

When CMCD-APTS-MNPs were added into solution containing either A or G or both,

immobilized CM-β-CD adsorbed the nucleoside through inclusion and hydrophobic

interactions. The nucleoside adsorbed particles were then separated from solution by

applying an external magnetic field (Figure 5-2).

Chapter: 5

88

Figure 5-2 Schematic illustration of the fabrication of the carboxymethyl-β-cyclodextrin modified magnetic nanoadsorbents and mechanism for selective separation of nucleosides.

The successful grafting of carboxymethylated β-CD on the surface of APES-MNPs was

verified by FT-IR studies. Figure 5-3 shows IR spectra of bare MNPs, APES-MNPs,

CMCD-APES-MNPs, and CM-β-CD in the range of 400-4000 cm-1 wave number. It is

shown that the characteristics adsorption band of Fe–O bonds in the tetrahedral sites of

bare MNPs is 586 cm-1. Strong absorption band at 3422 cm-1 is due to O-H stretching

vibration. The spectrum of APES-MNPs shows peak at 997 and 1110 cm-1 which can be

Chapter: 5

89

attributed to siloxane bonds (Si-O-H) and Si-O-Si groups, respectively. The peaks at

2854 and 2953 cm-1 are assigned to C-H stretching vibration in aminopropyl groups.

This suggests that Fe3O4 surface is successfully coated with APES. In spectra for

CMCD-APES-MNPs, the characteristics peaks at 941, 1030 and 1153 cm-1 can be

observed due to immobilized CM-β-CD on the surface. The peak at 941 cm−1 was due

to the R-1,4-bond skeleton vibration of β-CD, and the peaks at 1030 and 1153 cm−1

corresponded to the antisymmetric glycosidic νas(C–O–C) vibrations and the coupled

ν(C–C/C–O) stretch vibration, respectively [16]. Thus, the comparison between FTIR

spectra of APES and CM-β-CD coated magnetite nanoparticles gives evidence for

successful grafting of CM-β-CD on the magnetite surface which is further supported by

XPS (Figure A-2 in Appendix A).

Chapter: 5

90

Figure 5-3 FTIR spectra of as-synthesized magnetic nanoparticles-(a) uncoated MNPs, (b) APES-MNPs, (c) CMCD-APES-MNPs and (d) CM-β-CD.

High resolution TEM was applied to characterize the size and morphology of the nano-

magnetic particles. Typical TEM image of CMCD–APES–MNPs is shown in Figure 5-

4(a). No significant layer due to CM-β-CD grafting can be observed on the surface of

magnetic nanoparticles. It shows that magnetic particles bonded with CM-β-CD were

spherical or ellipsoidal particles with nano-size range. The mean diameter calculated

from the TEM image was estimated to be 11.3 nm. These as-synthesized CM-β-CD

modified magnetic nanoparticles exhibit superparamagnetism as evidenced by VSM

(Figure A-1 in Appendix A). Figure 5-4(b) also shows the hydrodynamic diameter

distribution of CMCD-APES-MNPs in water determined by the dynamic light

scattering (DLS) technique. The mean hydrodynamic diameter was found to be 56.5

nm. The particle size from TEM measurements in a dried state cannot be compared

400 1400 2400 3400

% T

ran

smis

sion

Wavenumber, cm-1

1030

1155

997

997

948

1080

582

1741

1030

1155

9452923

1624

10803396

2924

2854

586

3398

3404

586

2923

1325

1217

1110

(a)

(b)

(c)

(d)

Chapter: 5

91

with the values from DLS measurements which are water-swollen particles and DLS

gives higher values compared to TEM.

Figure 5-4 (a) TEM micrograph and size distribution of CMCD-MNPs measured by TEM and (b) hydrodynamic size distribution by DLS.

The amount of CM-β-CD grafted on the surface of APES-MNPs was estimated from

the thermogravimetric analyses of uncoated, APES coated and CM-β-CD coated

magnetic nanoparticles. As shown in Figure 5-5, the TGA curve of uncoated MNPs

shows first weight loss of 1.6 % below 130oC which might be due to the loss of

adsorbed water in the sample. After this brief event of weight loss, an unexpected

weight gain takes place within the temperature range from 130 to 205oC. This weight

gain can be attributed to the oxidation of Fe3O4 to Fe2O3 [210]. The total weight loss

over the full temperature range is estimated to be 2.3% due to the loss of the adsorbed

water as well as dehydration of the surface –OH groups. The TGA curve for APES-

MNPs exhibits two steps of weight loss, contributed from the loss of residual water in

the sample in 30–200oC and the loss of APES (~2.7%) in the range of 200–450 oC.

From the TGA curve for CMCD-APES-MNPs, a drastic drop of 8.5% can be seen in

(a) (b)

Chapter: 5

92

the range 190–430oC and it is contributed from the thermal decomposition of APES and

CM-β-CD moieties. The TGA curves also confirm the successful grafting of CD

molecules onto the magnetic surface. From these data, the amount of CM-β-CD grafted

onto the surface of APES-MNPs could be estimated to be 5.8 wt% or 0.044 mmol/g.

Figure 5-5 TGA curves for (a) uncoated MNPs, (b) APES-MNPs and (c) CMCD-APES-MNPs.

Zeta potentials of uncoated and CM-β-CD coated magnetic nanoparticles (0.1 mg/ml)

were measured in 10-3 M NaCl aqueous solution at different pH. The solution pH was

adjusted by NaOH or HCl. As shown in Figure 5-6, the pH values of point of zero

charge (pHpzc) of uncoated Fe3O4 nanoparticles and APES coated magnetic particles

(APES-MNPs) are about 6.8 and 7.7, respectively. After being grafted with CM-β-CD,

the pHpzc has been shifted to 4.25, indicating that the grafting of CM-β-CD onto

uncoated MNPs is successful. Moreover, magnetic nanoparticles modified with CM-β-

CD yields acidic surface since pHpzc is lower than that of uncoated MNPs and this

85

88

91

94

97

100

0 200 400 600 800

%w

eigh

t los

s

Temperature, oC

(a)

(b)

(c)

Chapter: 5

93

surface acidity is due to the introduction of several oxygen-containing functional groups

[211].

Figure 5-6 Zeta potentials of uncoated, APES and CM-β-CD modified magnetic

nanoparticles at different pH.

5.3.2 Adsorption of nucleosides onto CMCD-APES-MNPs 5.3.2.1. Effects of pH It has been reported that the solution pH values are important controlling parameters on

the analysis and separation of nucleosides [192,212]. The effects of initial solution pH

on the adsorption of nucleosides onto CMCD-APES-MNPs were investigated from pH

4 to 9 with an initial A and G concentration of 1.0 g/l. The solution pH after adsorption

didn’t change significantly in the experiments. As shown in Figure 5-7, sorption

capacity increases from pH 4 to pH 5 for guanosine and pH 4 to pH 6 for adenosine.

Comparatively less adsorption takes place for both A and G at low pH (pH 4). At low

pH, A and G are positively charged to some extent [see Figure A-3 in Appendix A

where the distribution coefficient (δ) of A and G are calculated from the pH and pKa

-60

-40

-20

0

20

40

60

2 3 4 5 6 7 8 9 10 11 12

Zet

a p

oten

tial

, m

V

pH

Uncoated MNPs

APES-MNPS

CMCD-APES-MNPs

Chapter: 5

94

values and the dissociation equilibrium process of A and G are also shown]. The zeta-

potential analysis of the CMCD-APES-MNPs (Figure 5-6) shows that the magnetic

nanoparticles are also positively charged at pH 4. Therefore, the interactions between

the adsorption sites on CMCD-APES-MNPs and the G or A cations are electrostatically

repulsive. The optimum pH appears to be in the range of 5~7 and 7~9 for guanosine and

adenosine, respectively. Above pH 7, the sorption capacity of magnetic nanoparticles

decreases slightly for G. However, the largest difference in the adsorption capacity

between G and A is obtained at pH 5. Therefore, the selective adsorption of nucleosides

can be achieved at pH 5 when they are in a competitive environment. At pH 5, 3.1% of

adenosine molecule is positively charged and the mean charge is approximately +0.03V

[193]. Guanosine is almost a neutral molecule (δ= 100%) at this pH. It is well known

that CDs show stronger inclusion with neutral guest molecules than cations, accounting

for a larger adsorption capacity for G than that of A at pH 5.

Figure 5-7 Effect of pH on the adsorption of A and G by CMCD-APES-MNPs: Conditions: Initial A and G concentration: 1.0 g/l, temperature: 25oC.

Chapter: 5

95

5.3.2.2 Kinetic studies Figure 5-8 shows the effects of contact time on adsorption of nucleosides onto CMCD-

APES-MNPs under the standard conditions. The initial nucleoside concentration

provides the necessary driving force to overcome the mass transfer resistance A and G

between aqueous and solid phases. It can be seen that the most rapid adsorption of A

and G occurred at initial few minutes and then occurred slowly. The time taken to reach

adsorption equilibrium for both nucleosides adsorption is 80 min.

Figure 5-8 Effect of contact time on nucleoside adsorption by CMCD-APES-MNPs. Conditions: Initial A or G concentration: 1.0 g/l, pH: 5, temperature: 25oC, adsorbent: 110 mg.

5.3.2.3 Equilibrium Studies The adsorption isotherm for A and G at pH 5 and 25oC are shown in Figure 5-9. It can

be seen that the adsorption of A and G increased with increasing initial concentration.

The adsorption of G was consistently higher than that of A and the difference in

adsorption between A and G increased clearly at higher initial concentration. The

higher adsorption of G onto CMCD-APES-MNPs can be attributed to its stronger

0

4

8

12

16

20

0 20 40 60 80 100 120 140 160 180 200

q e(m

g/g)

Time (min)

G

A

Chapter: 5

96

affinity towards CM-β-CD. The evidence of adsorption of guanosine and adenosine

onto the magnetic surface is shown in Figure A-4 (Appendix A).

Figure 5-9 Effects of initial concentrations on the adsorption of A and G onto CMCD-APES-MNPs. Conditions: Initial A and G concentration: 0.1,0.2,0.4,0.6,0.8,1.0g/l, pH: 5, temperature: 25oC, adsorbent: 110 mg.

The adsorption equilibrium data were fitted by Langmuir isotherm. The Langmuir

equation can be expressed as:

1e e

e m m L

C C

q q q K (5-1)

where qe is the equilibrium adsorption capacity of A and G onto the adsorbents (mg/g),

Ce is the equilibrium concentration of A and G (mg/ml), qm is the maximum capacity of

adsorbent (mg/g) and KL is the Langmuir adsorption constant (ml/mg). The values of qm

and KL can be determined from the slope and y-intercept of the linear plot of Ce/qe

against Ce as shown in Figure 5-10. For predicting the favorability of an adsorption

0

4

8

12

16

20

0 0.2 0.4 0.6 0.8 1

q e(m

g/g)

Final concentration (g/l)

AGLangmuir fit

Chapter: 5

97

system, the Langmuir equation can also be expressed in terms of a dimensionless

separation factor (RL) defined as follows [213]:

0

1

1LL

RK C

(5-2)

where Co is the initial concentration of adsorbate. The RL indicates the favorability and

the capacity of adsorption system. When 0 < RL < 1.0, it represents good adsorption.

The adsorption parameters are shown in Table 5-1. The adsorption isotherms of A and

G are both fitted well by Langmuir isotherm with R2 values of 0.983 and 0.982,

respectively. The maximum capacity of adsorbent on A and G are 12.5 mg/g and 31.3

mg/g respectively, indicating that the adsorption of G is more than 2 times of that of A

under the same conditions. The higher values of RL indicate the high affinity of these

nano-adsorbents to the nucleosides.

Figure 5-10 Langmuir plot illustrating the linear dependence of Ce/qe on qe for A and G.

y = 0.0807x + 0.0255R² = 0.9832

y = 0.0335x + 0.0195R² = 0.9874

0.02

0.04

0.06

0.08

0.1

0.12

0 0.2 0.4 0.6 0.8 1

Ce/

q e

Ce

A

G

Chapter: 5

98

Table 5-1 Adsorption isotherm parameters for A and G at pH 5 and 25oC

Isotherm Model Parameters A G Langmuir qm (mg/g) 12.5 31.3

KL (ml/mg) 3.2 1.68 R2 0.983 0.982 RL 0.238-0.758 0.373-0.856

5.3.3 Selective adsorption of A and G

The mixture nucleoside solution was prepared by dissolving equal amount of A and G

in the same buffer (pH 5). CMCD-APTS-MNPs were added to the mixture solution and

subject to mechanical shaking for 12 h. HPLC was used to quantify the concentration of

remaining A and G in the supernatant. The adsorption capacities of the CMCD-APTS-

MNPs at different total concentration of nucleosides were plotted in Figure 5-11(a). The

adsorption of both A and G increased proportionally with the initial total concentration.

It can be seen that G was adsorbed preferentially over A, especially at higher initial

concentration. The selective adsorption of nucleoside molecules onto the CMCD-

APTS-MNPs could be attributed to a stronger recognition capability of CM-β-CD

toward G, while the recognition for A is relatively weak. Binding constant or inclusion

formation constant (K) which represents the inclusion interactions is measured between

nucleoside and CM-β-CD (see Section 5.2.3.5). It can be seen that the binding constant

of CM-β-CD towards G is larger than that towards A. Moreover, the shifting of

maximum absorption peak for G/CM-β-CD system is more obvious in compared to that

for A/CM-β-CD system indicating a stronger interaction between G and CM-β-CD.

From Figure 5-1, it can be seen, A and G have similar structures except for an extra

C=O present in G. Hence, the results for the selective adsorption of G over A can be

attributed to the stronger hydrophobic interactions and additional weak intermolecular

hydrogen bonding contributed by C=O group in G which is further evidenced through

Chapter: 5

99

the studies of interactions between nucleosides and CM-β-CD by UV-vis spectra and

FTIR analysis, respectively.

Thus, the structure and charge of the guest plays important role in host-guest

interactions. In order to further verify that the selective adsorption properties of the

adsorbents were due to the hydrophobic cavity of CM-β-CD, a similar experiment was

conducted using APTS-MNPs instead of CMCD-APTS-MNPs. The adsorption of A or

G onto APTS-MNPs is mainly governed by electrostatic attractive interactions since

APTS-MNPs are positively charged at pH 5 whereas G or A is almost neutral at the

same condition (Figure A-2, Appendix A). Previous studies on adsorption behavior of

oligonucleotides on cationic or anionic latex particles also showed that electrostatic

forces play a major role in the adsorption process [214]. The result in Figure 5-11(b)

indicated that the adsorption of A and G was approximately the same and hence it

proved that the selective adsorption was indeed directly related to the hydrophobic

cavity of CM-β-CD. However, nonspecific adsorption might occur due to residual

amine (NH2) groups on the surface of magnetic nanoparticles due to electrostatic

interactions [191]. But this nonspecific adsorption may be negligible in compared to the

inclusive adsorption due to CM-β-CD molecules which has more than 80% of the

surface coverage and act as effective binding sites for nucleosides adsorption.

Chapter: 5

100

Figure 5-11 Selective adsorption of A and G mixture solutions using (a) CMCD-APES-MNPs and (b) APES-MNPs. Conditions: pH: 5, temperature: 25oC, adsorbent: 110mg.

5.3.4 UV-vis spectra analysis and determination of binding constants The inclusion capability of CM-β-CD towards nucleosides was investigated by UV-vis

absorption measurements. In this study, the guest molecules G or A is assumed to be

included by CM-β-CD moiety grafted on CMCD-APTS-MNPs with the same

mechanism as parent CM-β-CD; while Fe3O4 or other molecule do not affect the

inclusion complexation. As the binding sites in the CMCD-APTS-MNPs are essentially

0

2

4

6

8

10

12

14

16

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

q e(m

g/g)

Initial concentration (g/l)

G

A

(a)

1

2

3

4

5

6

0.1 0.3 0.5 0.7 0.9 1.1

q e(m

g/g)

Initial concentration (g/l)

G

A

(b)

Chapter: 5

101

the CM-β-CD apolar cavities and that the global properties of these sites are not

significantly different from the properties of the isolated CM-β-CD molecule, the

measurement of binding constant between CM-β-CD host and nucleoside guest

molecules could explain the reasons for selective inclusion of G to some extent. Figure

5-12 shows the UV-vis spectra in the absorption of nucleosides (1x10–5 mol l–1) in

phosphate buffer solution (pH 5) containing various concentrations of CM-β-CD. It is

clear that absorption peaks of A or G were enhanced, with the addition of CM-β-CD.

This increase is not just from additional CM-β-CD hosts added, but from the inclusion

of G or A within the relatively non-polar cyclodextrin cavity. A significant shift in the

absorption maximum (a blue shift from 253 nm to 248.3 nm) was observed in the case

of G upon addition of CM-β-CD (Figure 5-12a), whereas the absorption peak was

slightly shifted (from 259.9 nm to 259.1 nm) for A (Figure 5-12b). Compared with the

pristine G, the absorption bands of the products CMCD–APTS-MNPs/G (see Fig. S5)

shifted to a lower wavelength for about 4 nm. The reason may be that the strong

interaction due to additional hydrogen bonding between the host and the G was formed,

thus including the G guest much more tightly. This increase in absorption maxima and

shift in peak position upon CM-β-CD addition could be attributed to the fact that G or

A was becoming included in the hydrophobic cavity of CM-β-CD.

The inclusion equilibrium constant or binding constant (K) is an important parameter,

which represents the strength of the inclusion interaction. In other words, the stability

mainly relies on the host–guest interactions. The stability of the inclusion complex is

mainly determined by the geometric capability and the polarity of guest molecules

[215]. Geometric factors are crucial in determining the guest molecules that can

penetrate into the relatively hydrophobic cavity. On the other hand, only those

substrates with less polarity than water can form inclusion complexes with cyclodextrin

Chapter: 5

102

moieties. If CMCD: guest complex was formed with 1:1 stoichiometry, the inclusion of

CMCD and guest could be expressed as:

(5-3)

The inclusion constants of complexes are estimated according to the equation of Benesi

and Hildebrand (double-reciprocal method) [216]:

∆ (5-4)

where ∆A denotes the difference of absorption of guest molecule in the presence and

absence of CDs. α is a constant, and [CMCD]o denotes the concentration of CM-β-CD.

K can be calculated from a plot of 1/∆A vs. 1/[CMCD]o. Insets of Figure 5-12 shows the

double reciprocal plots of 1/∆A versus 1/[CMCD]0 for A and G with CM-β-CD at pH 5.

The plot exhibits good linearity (the linear regression coefficient R2 ~ 0.99). This

verifies the formation of inclusion complexes with a stoichiometry of 1:1 between

nucleosides and CM-β-CD. The relative inclusion constant of CM-β-CD with G and A

is estimated to be 47 and 18.6 M-1, respectively. From this result, the complex of G with

CM-β-CD should be more stable than that of A, and this is the driving force for the

inclusive separation. The 1:1 stoichiometry of complexes of parent β-CD with

nucleosides (both A and G) was also determined by the circular dichroism spectral

changes (Figure A-5, Appendix A). It can be seen that G has higher binding capacities

with β-CD in compared to A as the binding constant of β-CD with G (30.3 M-1) was

higher than that with A (17.9 M-1). This result also indicates that incorporation of

carboxylic groups in β-CD moieties can enhance the strength of host-guest interactions

with G molecules.

Chapter: 5

103

Figure 5-12 Absorption spectra of (a) adenosine and (b) guanosine (1x10–5 mol l–1) in pH 5.0 buffers containing various concentrations of CM-β-CD. Concentration of CM-β-CD: (1) 0, (2) 1.0x10–3, (3) 2.0x10–3, (4) 2.5x10–3 and (5) 3.5x10–3 mol l–1. Insets: Double reciprocal plot for nucleoside complexes with CM-β-CD.

5.3.5 Interactions of the nucleosides with cyclodextrins

In order to investigate the vibrational changes upon host-guest interaction between

nucleosides and CM-β-CD, FTIR spectroscopy was used. FTIR spectroscopy is a useful

technique used to confirm the formation of an inclusion complex. Figure 5-13 shows

the infrared spectra of adenosine, guanosine, β-CD and their inclusion complexes. The

Chapter: 5

104

inclusion complexes were prepared following the procedure described in the Section A-

6 (Appendix A) and the formation of inclusion complexes are also confirmed by

Differential Scanning Calorimetry (DSC) analysis (Figure A-6, Appendix A). In the IR

spectrum of adenosine, the bands at 1666, 1605 and 1572 cm-1 are mainly assigned to

the bending mode of NH2, C=N stretching and the conjugated vinylic group (C=C) in

the aromatic ring, respectively. The two strong bands at 1734 and 1695 cm-1 in the

spectra of free guanosine are assigned to the cyclic carbonyl (C6=O) stretching [217].

The bands at 1624 and 1609 cm-1 corresponds to NH2 bending mode and the aromatic

C=C vibrations, respectively. The other bands in the region of 1600-1400 cm-1 in

guanosine are assigned to C-C, C=N and C-N vibrations.

In the IR spectra of inclusion complexes, there is a significant change in the

characteristic bands absorption of adenosine and guanosine, particularly in the range of

1400-1800 cm-1. Some bands disappeared and some bands became less intense after

forming the inclusion complexes. The strong peaks of adenosine at 1572 and 1605 cm-1

reduced its intensity significantly and shifted to 1574 and 1609 cm-1, respectively in its

inclusion complexes, The aromatic C=C strong band of guanosine at 1609 cm-1

disappeared in the spectrum of its inclusion complex. These results indicate that the

aromatic rings of adenosine and guanosine might be included into the cavity of β-CD.

The vibrational band for the C=O groups (1742 cm-1) of CM-β-CD showed a strong

band at 1634 cm-1 upon complexation with G. Moreover, the cyclic carbonyl (C=O)

stretching band of guanosine at 1695 cm-1 reduced its intensity and shifted to 1693 cm-1

in its inclusion complex suggesting the strong interactions through intermolecular

hydrogen bonds between guanosine and CM-β-CD moiety [215]. The peak at 1539 cm-1

of guanosine is also shifted to 1537 cm-1 and its intensity is reduced. The band located

at 1643 cm-1 in the IR spectra of CM-β-CD which is correlated to δ-HOH bending of

Chapter: 5

105

water molecules attached to cyclodextrins shifted to 1637 cm-1 in that of the inclusion

complex. σC–O–H peaks at 1336 cm-1 and 1250 cm-1 in the G/ CM-β-CD inclusion

complexes have much higher intensity compared to that of CM-β-CD. These are the

evidences of formation of hydrogen bonds between the hydroxyl groups of CM-β-CD

and the carbonyl groups of G [215]. The electron cloud density of the oxygen atom of

carbonyl in G might lead to the formation of the hydrogen bond between CM-β-CD and

G. Recent study showed that hydrogen bond interactions (e.g., O-H…O and C-H…O

etc.) play an important role in the complexes of alcohol/ β-CD and aldehyde/ β-CD and

based on the strength of the interactions, β-CD could selectively separate the alcohols

from the mixed solution [207]. Yang et al. [218] utilized insoluble β-CD polymer for

selective separation of cinnamaldehyde and benzaldehyde based on hydrogen bond

interactions between carbonyl (aldehyde) and secondary hydroxyl groups present in β-

CD moieties. It can also be concluded preliminarily from the FTIR results that the

adenine and guanine part of the nucleosides was probably enclosed in the β-CD

hydrophobic cavity. Several studies were done using different techniques to investigate

the interactions between the base (purine) moieties and CDs. For example, Seno et al.

proposed that the adenine group of nicotinamide adenine dinucleotide phosphate

(NADP) is incorporated into the CD cavity from the measurements of high performance

liquid chromatography [219]. Hao et al. also reported the inclusion of adenine molecule

in the more apolar environment inside the β-CD cavity based on enhancement of the

fluorescence intensity of adenine [220]. Kondo and Nishikawa suggested that adenine

and β-CD forms an inclusion complex based on the results of an ultrasonic relaxation

study [221]. However, from our studies it can be seen that the CMCD-APTS-MNPs

could selectively recognize and adsorb G over A as β-CD cavity primarily provides

Chapter: 5

106

hydrogen bonds between β-CD and polar groups of G in addition to hydrophobic

interactions.

Figure 5-13 FTIR spectra of adenosine and guanosine and their inclusion complexes with β-CD.

5.4 Conclusions

In summary, a simple and straightforward synthetic method was developed for the

preparation CM-β-CD modified Fe3O4 nanoparticles that combine magnetic and

molecular recognition properties. The properties of this novel nano-adsorbent were

Chapter: 5

107

evaluated for selective adsorption of guanosine and adenosine. The grafting of CM-β-

CD onto the surface of the magnetic particles was confirmed by FTIR, TGA and XPS.

The adsorption of nucleosides was found to be pH dependent and the adsorption of A

and G onto CMCD-APES-MNPs were well-described by Langmuir isotherm model.

Under the competitive environment, selective adsorption of G over A occurred at pH 5

onto these nano-adsorbents. This selectivity was due to the multiple recognition of the

CM-β-CD which might give strong hydrophobic and extra interaction due to hydrogen

bonding towards G. The stronger interactions between G and CM-β-CD were confirmed

by measuring the binding constants which represent the inclusion interactions and also

by FTIR studies. The results showed that this adsorbent would have wide applications

in selective separation, concentration, and analysis of nucleotides/nucleosides in

biological sample.

Indeed, this study provides a practical and promising technique to separate organic

compounds with similar structures based on the difference of inclusional binding

property by forming host–guest interactions. Combining the unique properties of MNPs

(superparamagnetism and electrocatalytic) and cyclodextrins (excellent supramolecular

recognition and host–guest inclusion capability), it is also possible to obtain the new

materials which will provide good opportunities for applications in the fields of sensors,

electrocatalysis etc.

Chapter: 6

108

Chapter 6 Adsorptive removal of dyes from aqueous water using carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbent3

6.1 Introduction

This chapter reports on synthesis and characterization of novel nanoadsorbents-

carboxymethyl-β-cyclodextrin conjugated magnetic nanocomposites and their use in the

removal of organic dyes from aqueous streams. The adsorption behaviors of a cationic

dye with theses nano-adsorbents have been discussed from the equilibrium and kinetic

viewpoints. Methylene blue (MB) was used in this study as the model dye compound

because of their extensive use in various industries.

Dye removal from industrial wastewater has become an important issue because of

environmental concern. Dye and dye stuffs are extensively used in many industries,

such as cosmetics, leather, paper, printing textile finishing industries. It has been

estimated that 10-15% of the dyes was lost during the dyeing process and released as

effluent. Since most of the used dyes are stable, recalcitrant, colorant, and even

potentially carcinogenic and toxic, their discharge into the environment without proper

treatment poses serious environmental, aesthetical and health problems [109]. The

removal of dyes from aqueous environment has been widely studied and used including

biological processes, coagulation, membrane filtration, adsorption, photocatalysis,

advance oxidation etc. Among these methods, adsorption has increasingly received

more attention because of its high efficiency, simplicity of design, and ease of operation

[114,115,141].

3. A.Z.M. Badruddoza, Goh Si Si Hazel, K. Hidajat, M.S. Uddin, Synthesis of carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbent for removal of methylene blue, Colloids Surf. A: Physicochem Eng. Aspects 367 (2010) 85-95.

Chapter: 6

109

One of the most widely used method for the removal of organic pollutants is their

adsorption on activated carbon, arranged in different arrays such as filters, dispersed

powder or fluidized beds. Activated carbon has been efficiently used to adsorb many

kinds of pollutants as it is characterized by a high porosity and a very large surface area.

In spite of its high adsorption capacity, the use of activated carbon on a large scale is

limited by process engineering difficulties such as the dispersion of the activated carbon

powder and the cost of its regeneration. Even if activated carbon can be used in granular

form, a lack of selectivity in adsorption is observed with activated carbon. Thus,

continuous and great efforts are being made by many researchers on optimizing

adsorption and developing novel alternative adsorbents with high adsorptive capacity

and low cost.

Recently, natural polymers such as polysaccharide and their derivatives have gained

significant interest as low-cost adsorbents of choice for wastewater treatment because

of their particular structure, physico-chemical characteristics, chemical stability, high

reactivity and excellent selectivity towards organic compounds and metals [141]. An

extensive list of low-cost polysaccharide based adsorbents for dye removal has been

reported by Crini [107]. An important class of polysaccharide derivatives are

cyclodextrins (CD) which can form inclusion complexes with a wide variety of organic

compounds in its hydrophobic cavity through host-guest interactions and also can

complex various metal ions [9]. Therefore, CD complexation is a procedure of choice

for decontaminating techniques. Several types of water insoluble β-CD-based polymers

which have been used for removal of contaminants from wastewaters are summarized

in Table 2-7. This kind of polymer is usually synthesized by reacting β-CD with

crosslinking agents such as epichlorohydrin, diisocyanates, polycarboxylic acids and

anhydrides. In particular, the β-CD-based polymers are very efficient in removing acid,

Chapter: 6

110

direct, disperse and reactive dyes from aqueous solutions [114]. However, they have

low affinity for basic (cationic) dyes. Recently it has been reported that incorporation of

suitable groups e.g. carboxyl groups onto β-CD can enhance the capability to adsorb

basic dyes on the polymer beads [11,113,141]. However, the application of these

polymeric materials for treating wastewater requires an additional separation step to

remove the adsorbent from the aqueous system. A step in developing adsorbents with

superior properties would be the inclusion of magnetic particles into natural polymers,

thus combining the advantages of both materials.

This work aims at exploring the possibility to fabricate carboxymethyl-β-cyclodextrin

(CM-β-CD) conjugated Fe3O4 nanoparticles with exploitable characteristics as nano-

adsorbent for removal of dyes from aqueous solution. Here, two adsorbents CMCD-

MNP(C) and CMCD-MNP(P) were synthesized via carbodiimide and precipitation

method, respectively. The size, structure and surface properties of the CM-β-CD

modified magnetic nanoparticles were characterized using different analytical tools.

The adsorption capability was demonstrated using methylene blue (MB) as model dye.

MB is a cationic dye which is toxic and carcinogenic. Its characteristics are given in

Table 6-1. The adsorption behaviors of CMCD-MNP(C) and CMCD-MNP(P) towards

MB adsorption were studied using both equilibrium and kinetic viewpoints. The effects

of several parameters such as effect of pH, initial MB concentration etc. and mechanism

of MB adsorption onto CM-β-CD modified magnetic nanoparticles were investigated in

this report.

Chapter: 6

111

6.2 Experimental

6.2.1 Materials

In this work, the following chemicals were used for fabrication of CM-β-CD modified

magnetic nanoparticles: Iron (II) chloride tetrahydrate (99%) [Alfa Aesar], iron (III)

chloride hexahydrate (98%) [Alfa Aesar], ammonium hydroxide (25%) [Merck (USA)],

β-cyclodextrin (β-CD, 99%) [Kasie Kogyo Co. (Tokyo)], chloroacetic acid (99%) [Alfa

Aesar], carbodiimide (cyanamide, CH2N2, 98%) [Alfa Aesar], methylene blue (Basic

blue 9: CI-52015) [Sigma]. All other chemicals were of analytical grade and were used

as received without further purification. Milli-Q water was used throughout this study.

Table 6-1 Characteristics of Methylene Blue (MB) dye

C.I. name Methylene Blue

C.I. number 52015

Abbreviation MB

Dye Type Basic

Dye content (%) 99

Molecular Weight (gmol-1) 319.86

Molecular formulae C16H18ClN3S

Wavelength (nm) 664

Chemical structure

6.2.2 Fabrication of CM-β-CD modified Fe3O4 nanoparticles [CMCD-MNP(C) & CMCD-MNP(P)]

To synthesize CMCD-MNP(C), the binding of CM-β-CD onto the Fe3O4 magnetite

surface was conducted following the work of Liao and Chen [17] on the preparation of

polyacrylic acid (PAA) bound magnetic nanoparticles with slight modification. Detailed

Chapter: 6

112

procedures to synthesize CM-β-CD are given in Chapter 5 (Section 5.2.5). For the

binding of CM-β-CD, 100 mg of magnetic nanoparticles were first added to 2 ml of

buffer A (0.003M phosphate, pH 6, 0.1 M NaCl) and sonicated for 5 min. Then, the

reaction mixture was further sonicated for 15 min after adding 0.5 ml of carbodiimide

solution (0.025 g/ml in buffer A). Finally, 2.5 ml of CM-β-CD (50 mg/ml in buffer A)

was added and the reaction mixture was sonicated for another 90 min. The CM-β-CD

bound magnetic nanoparticles were recovered from the reaction mixture using a

permanent magnet and washed 2-3 times with buffer A and then dried in a vacuum

oven.

CMCD-MNP(P) was fabricated by one step co-precipitation method. Briefly, 0.86 g of

FeCl2.4H2O, 2.36 g FeCl3.6H2O and 1.5 g CM-β-CD were dissolved in 40 ml of de-

aerated Milli-Q water with vigorous stirring at a speed of 1,200 rpm. 5 ml of NH4OH

(25%) was added after the solution was heated to 90ºC. The reaction was continued for

1 hr at 90ºC under constant stirring and nitrogen environment. The resulting

nanoparticles were then washed with Milli-Q water few times to remove any unreacted

chemicals and dried in a vacuum oven.

6.2.3 Adsorption/desorption of methylene blue

The adsorption of methylene blue (MB) onto the CM-β-CD-bound iron oxide magnetic

nanoparticles was carried out in 0.03 M phosphate buffer at desired MB concentrations,

pH and temperatures. In general, 120-130 mg wet nanoparticles were added to 5 ml of

MB solution. The initial concentrations of MB solution were 0.1 to 3 mg/ml. The effect

of pH on the amount of dye removal was analyzed in the pH range from 2 to 12. The

pH was adjusted using HCl or NaOH solutions. After equilibrium was reached,

nanoparticles were removed magnetically from MB solution. The adsorption amounts

Chapter: 6

113

of MB were estimated from the concentration change of MB in solution after adsorption

by the spectrophotometric method at 664 nm (Shimadzu UV-1601 UV/vis

spectrophotometer). Calibration curves were plotted between absorbance and

concentration of the MB solution. The amount of MB adsorbed onto CMCD-MNP(C)

and CMCD-MNP(P) was calculated by a mass balance relationship,

( )e t e

Vq C C

w (6-1)

where V (ml) is the volume, Ct and Ce (mg/ml) are the initial and final solution

concentration of MB, respectively, and w (mg) is the dry mass of the solid.

For desorption experiment, the MB-sorbed nano-adsorbents were washed thoroughly

with deionized water. Desorption of MB was then performed by putting the MB-sorbed

nanoparticles into methanol solution containing 0–5% acetic acid [40]. After

equilibrium was reached, nanoparticles were removed by magnetic decantation from

MB solution and the concentration of MB in liquid solution was measured to estimate

the amount of MB desorbed.

6.3. Results and Discussion

6.3.1 Synthesis and characterization of magnetic nanoparticles

CMCD-MNP(C) (BET surface area~110.9 m2/g) was synthesized using two-steps

(carbodiimide method). The first step involves the formation of carboxyl group onto β-

CD by reacting chloroacetic acid with β-CD in the alkaline condition. The average

number of carboxylate groups (3.1) per molecule of CM-β-CD is determined using 1H

NMR [209] as well as by titrating the carboxylate groups with NaOH solution [222].

Before titrating CM-β-CD with NaOH, the CM-β-CD sodium salt was converted to its

acidic form by passing 50 ml of a 100 g/l CM-β-CD solution twice through a column

Chapter: 6

114

containing an ion exchange resin (H+). Using the degree of substitution of 3.1

carboxymethyl substituents (each with a molecular weight of 58 amu) per CM-β-CD

molecule, we calculated an average atomic mass for CM-β-CD of 1312 amu. This

molecular weight was used to calculate the grafted amount of CM-β-CD on the surface

of Fe3O4 nanoparticles. In the second step, CM-β-CD was covalently bonded onto the

surface of magnetic nanoparticles via carbodiimide activation. Here, carbodiimide

(cyanamide) was added in CM-β-CD solution to pre-activate the carboxyl (-COOH)

groups which in turn form covalent bonding with the surface OH groups on magnetite.

On the other hand, CMCD-MNP(P) (BET surface area~77 m2/g) was synthesized by

simple one-step co-precipitation where iron precursors (Fe2+ and Fe3+) and CM-β-CD

were mixed together in the reaction medium. In one-step method, the carboxyl groups

of CM-β-CD directly react with the surface OH groups on the magnetite to form Fe-

carboxylate. The step-by-step reaction procedures to synthesize CM-β-CD modified

magnetic nanoparticles are shown in Figure 6-1.

Chapter: 6

115

Figure 6-1 An illustration for the carboxymethylation and binding of cyclodextrin onto Fe3O4 nanoparticles by two different methods.

The binding of CM-β-CD on surfaces of magnetic nanoparticles was confirmed by

FTIR spectroscopy. Figure 6-2 shows the FTIR spectra of bare MNP, CMCD-MNP(C)

and CMCD-MNP(P) and CM-β-CD in the 400-4000 cm-1 wave number range. The

broad band at 330-3500 cm-1 is due to –OH stretching vibrations. The spectrum of CM-

β-CD shows the characteristic peaks at 947, 1030, 1157 and 1741 cm−1. The peak at 947

cm−1 is due to the R-1,4-bond skeleton vibration of β-CD, and the peaks at 1030 and

1157 cm−1 corresponded to the antisymmetric glycosidic νa(C-O-C) vibrations and

coupled ν(C-C/C-O) stretch vibration [11,16]. The peak at 1741 cm-1 corresponds to

carbonyl group (=CO) stretching which confirms the incorporation of the

carboxymethyl group (-COOCH3) into β-CD molecule. All the significant peaks of

Chapter: 6

116

CM-β-CD in the range of 900–1200 cm−1 are present in the spectrum of CMCD-

MNP(C) and CMCD-MNP(P) with a small shift. Moreover, as shown in Figure 6-2(b),

two main characteristic peaks appeared at 1623 and 1401 cm−1 due to bands of COOM

(M represents metal ions) groups, which indicates that the COOH groups of CM-β-CD

reacted with the surface OH groups of Fe3O4 particles resulting in the formation of the

iron carboxylate [223,224]. So it can be concluded that CM-β-CD has been grafted

successfully on the surface of both magnetic nano-adsorbents.

Figure 6-2 FTIR spectra of (a) uncoated MNPs, (b) CMCD-MNP(P), (c) CMCD-MNP(C) and (d) CM-β-CD.

Typical TEM images and particle size distributions of CMCD-MNP(P) and CMCD-

MNP(C) are shown in Figure 6-3(a) and 6-3(c) and their insets. Well-shaped spherical

or ellipsoidal magnetic nanoparticles are observed. The mean diameter of both CMCD-

MNP(P) and CMCD-MNP(C) is about 12 nm. This indicates that binding process did

400 800 1200 1600 2000 2400 2800 3200 3600 4000

% T

Wavenumber, cm-1

945

582

3396

3386

29242854

582

58614001028

947

1030

3394

1030

947

10781153

1155

1080

1618

1635

(a)

(b)

(c)

(d)

17412923

10801157

2924

Chapter: 6

117

not significantly result in the agglomeration and more changes in their sizes. Similar

results have been observed for the magnetic nanoparticles binding with carboxymethyl-

chitosan by one-step and two-steps method [224]. No significant layer of CM-β-CD can

be observed on these nano-adsorbents. Figure 6-3(b) and 6-3(d) also show the

hydrodynamic diameter distribution of CMCD-MNP(C) and CMCD-MNP(P) in water

determined by the DLS technique. The mean hydrodynamic diameters were found to be

28.2 and 43.6 nm, respectively. The particle size from TEM measurements in a dried

state cannot be compared with the values from DLS measurements which are water-

swollen particles and DLS gives higher values compared to TEM. This can be attributed

to the reason that polymer coating increases the hydrodynamic diameter and modified

iron oxide particles are slightly aggregated in water. The difference in sizes obtained

from DLS and TEM has also been observed with other nano-materials [16,202].

Chapter: 6

118

Figure 6-3 TEM micrographs and hydrodynamic diameter distributions for CMCD-MNP(C) (a) & (b); and CMCD-MNP(P) (c) & (d) respectively.

The amount of CM-β-CD grafted on the surface of MNP was estimated from the

thermogravimetric analyses of uncoated and CM-β-CD coated magnetic nanoparticles.

As shown in Figure 6-4, the TGA curves for CMCD-MNP(C) and CMCD-MNP(P)

exhibits two steps of weight loss, contributed from the loss of residual water in the

sample in 30–200oC and the loss of CM-β-CD in the range of 200–400oC. From the

TGA curve for CMCD-MNP(C) and CMCD-MNP(P), a drastic drop of 4.7% and 12%,

respectively can be seen in the range 190–400oC and it is contributed from the thermal

decomposition of CM-β-CD moieties. Below 200oC, the rate of weight loss is relatively

slow owing to the loss of residual water adhering to the sample surface and adsorbed in

the CD cavities. Thus, the TGA curves also confirm the successful grafting of CM-β-

CD molecules onto the magnetic surface.

0

2

4

6

8

10

12

14

16

18

7 8 9 10 11 12 13 14 15 16 17 18


Fre

quen

cy (

%)

(a)

(b)

(d)

(c)

Chapter: 6

119

Figure 6-4 TGA curves for (a) bare Fe3O4 MNP, (b) CMCD-MNP(C) and (c) CMCD-MNP(P).

The XRD patterns of bare MNP, CMCD-MNP(C) and CMCD-MNP(P) indicates six

characteristic peaks at 2θ = 30.2o, 35.6o, 43.2o, 53.7o, 57.2o, and 62.9o as shown in

Figure 6-5(i). They relate to their corresponding indices (2 2 0), (3 1 1), (4 0 0), (4 2 2),

(5 1 1), and (4 4 0), respectively. This reveals that the resultant nanoparticles are pure

Fe3O4 with a spinel structure and the modification of the surface of magnetic

nanoparticles with CM-β-CD did not result in the phase change of Fe3O4. The crystal

sizes of CMCD-MNP(C) and CMCD-MNP(P) determined from the XRD pattern by

using Scherrer’s equation [Dhkl = 0.9λ/(βcos θ), where Dhkl is the average crystalline

diameter, λ is X-ray wavelength, β is the half width of XRD diffraction lines and θ is

Bragg’s angle in degree] are found to be 10.2 and 10.7 nm, respectively, slightly less

than that observed from the TEM image. Here, the (3 1 1) peak of the highest intensity

was picked out to evaluate the particle diameter of the MNPs.

Chapter: 6

120

Figure 6-5 (i) XRD patterns and (ii) magnetization curves (a) uncoated Fe3O4 MNPs, (b) CMCD-MNP(C) and (c) CMCD-MNP(P).

The magnetic properties of the bare and CM-β-CD coated Fe3O4 nanoparticles were

measured by VSM at room temperature. In Figure 6-5(ii), the hysteresis loops that are

characteristic of superparamagnetic behavior can be clearly observed for both bare and

CM-β-CD coated magnetic nanoparticles. Superparamagnetism is the responsiveness to

an applied magnetic field without retaining any magnetism after removal of the applied

magnetic field. From M vs. H curve, the saturation magnetization values (Ms) obtained

at same field for CMCD-MNP(C) and CMCD-MNP(P) were 68 and 54 emu g-1,

respectively, lower than that of bare Fe3O4 (75 emu g-1). This is mainly attributed to the

existence of non-magnetic materials (CM-β-CD layer) on the surface of nanoparticles.

However, the magnetization magnitude of sample synthesized by one-steps method is

lower than that by two-steps method [224]. This is because the adsorption quantity of

CM-β-CD onto Fe3O4 nanoparticles is higher; the magnetic particles amount per unit

quantity is lower. As a result, the saturation magnetization of the prepared by one-step

Chapter: 6

121

method is lower. These values of saturation magnetization are comparable to those of

other functionalized nanoadsorbents [108,225]. This feature allows the as-synthesized

nanoparticles for highly efficient magnetic manipulation when used as nano-adsorbents

for removal of dyes from aqueous solution under relatively low external magnetic field.

Zeta potentials of CMCD-MNP(P) and CMCD-MNP(C) (0.1 mg/ml of each adsorbent)


adjusted by NaOH or HCl. As shown in Figure 6-6, the isoelectric point (pI) of bare

Fe3O4 nanoparticles was about 6.8, consistent with the values reported in literature [40].

After being grafted with CM-β-CD, the pI for CMCD-MNP(C) and CMCD-MNP(P)

were shifted to 4.7 and 4.2, respectively indicating that the grafting of CM-β-CD onto

both adsorbents was successful. Thus, the CMCD-MNP(C) was positively charged at

pH<4.7 whereas CMCD-MNP(P) was positively charged at pH < 4.7.

Figure 6-6 Zeta potentials of bare Fe3O4 MNP, CMCD-MNP(C) and CMCD-MNP(P).

-60

-40

-20

0

20

40

60

1 2 3 4 5 6 7 8 9 10 11 12

Zet

a p

oten

tial

, m

V

pH

Bare Fe3O4 MNPCMCD-MNP(C)CMCD-MNP(P)

Chapter: 6

122

6.3.2 Adsorption of MB onto CM-β-CD modified MNPs

The CD molecules with proper size, shape, and polarity could fit the cavity to form

inclusion complex through host–guest interactions, which contribute to the application

of CDs in environmental pollution cleanup. CDs can remove typical organic pollutants,

such as dyes and phenolic compounds, from large volumes of water. In our work,

methylene blue (MB) was used as a model compound representing organic

contaminants. The use of MB as a model compound has been reported in earlier studies

[11,114,118,141,226,227]. Qualitative adsorption behavior of the MB on CM-β-CD-

functionalized nanoadsorbent is shown in Figure 6-7. Nanoadsorbents were shaken for

15-50 min in an alkaline aqueous solution of MB (blue colored). During the adsorption

process, the decrease in free MB could be qualitatively shown by the clear change of

the color in the aqueous phase, which indeed turned lighter and lighter with time. After

adsorption, the supernatant MB solution was separated by using strong magnet and

quantified by UV-vis spectrophotometer.

Chapter: 6

123

Figure 6-7 The schematic representation of the selective adsorption and separation of MB by CM-β-CD modified magnetic nanoparticles.

Figure 6-8 is the UV-vis spectra of MB before and after adsorption with different

nanoadsorbents at pH~12. It was found that CM-β-CD modified magnetic nanoparticles

had higher adsorption capabilities than uncoated magnetic particles, indicating surface

modification with CM-β-CD could enhance the adsorption capacities. Moreover,

CMCD-MNP(P) adsorbed more MB than CMCD-MNP(C) due to higher amount of

grafted CM-β-CD on the surface acting as the adsorption sites.

Chapter: 6

124

Figure 6-8 UV-vis spectra of methylene blue (0.5 mg/ml) at pH~12 upon addition of (a) 0 mg MNPs; (b) 50 mg uncoated MNPs; (c) 50 mg CMCD-MNP(C); (d) 50 mg CMCD-MNP(P).

6.3.2.1 Effect of pH

The effects of initial solution pH on the adsorption of methylene blue (MB) onto both

CMCD-MNP(P) and CMCD-MNP(C) were investigated at pH 2–12, 25ºC, and an

initial MB concentration of 2 mg/ml. As shown in Figure 6-10, very less adsorption of

MB took place on both adsorbents, when pH was less than 4. At lower pH values, the

dimethylamine group in MB molecule is changed, as shown in Figure 6-9 and the

strong hydrophilic MB may have weak interaction with hydrophobic cavity of CM-β-

CD. Weaker interaction of CM-β-CD with MB molecules was reported in acetic media

(pH 2) [228]. Moreover, at low pH the surface of adsorbents would be surrounded by

the hydrogen ions which compete with MB ions binding the sites of the sorbent.

Between the initial pH values of 4 and 10 (equilibrium pH 5.4 to 9.65), the solution pH

hardly affects the adsorption of MB onto both CMCD-MNP(P) and CMCD-MNP(C).

Here, the hydrophilic and hydrophobic groups in each MB molecule simultaneously

exist, which have the same interaction with the CMCD-MNPs. At pH > 10, the

0

0.2

0.4

0.6

0.8

1

1.2

400 450 500 550 600 650 700 750 800

Ab

sorb

ance

Wavenumber (nm)

(a)

(b)

(c)

(d)

(a)

(b)

(c)

(d)

(a)

(b)

(c)

(d)

(a)

(b)

(c)

(d)

Chapter: 6

125

adsorption capacities increase with increase of pH since MB molecules with ammonium

salt are neutralized and the interaction between neutral MB and the cavity of CM-β-CD

is strengthened. Similar observation was found on the binding of MB with self-

assembled multilayer thin-film fabricated with CM-β-CD/diazoresin [229]. As the

adsorption amount of MB remained almost same at pH 4–10, the following studies were

conducted at a fixed pH of 8 and also at optimum pH 12.

Figure 6-9 The molecular structure of MB.

Figure 6-10 Effect of pH on the adsorption of MB by CM-β-CD modified magnetic nanoparticles. Conditions: (Initial MB concentration: 2 mg/ml, temperature: 25ºC, adsorbents: 120 mg).

0

50

100

150

200

250

300

1 3 5 7 9 11 13

Ad

sorb

ed a

mou

nt

(mg/

g)

Equilibrium pH

CMCD-MNP(P)

CMCD-MNP(C)

Chapter: 6

126

6.3.2.2 Adsorption kinetics

Figure 6-11 shows the effects of contact time on adsorption of MB onto CM-β-CD

modified magnetic nanoparticles under various initial concentrations of MB and at

25ºC. The initial dye concentration provides the necessary driving force to overcome

the mass transfer resistance of MB between the aqueous and solid phases [230]. It can

be seen that a rapid adsorption of MB by both adsorbents occurred, with equilibrium

reached in approximately 20 and 50 min by CMCD-MNP(C) and CMCD-MNP(P),

respectively. Also the amount of dye adsorbed at equilibrium increased with the

increase in initial MB concentrations. Within almost 5-10 minutes, 85-90% MB of that

at equilibrium was achieved using both CMCD-MNP(P) and CMCD-MNP(C). Such a

fast adsorption rate could be attributed to the absence of internal diffusion resistance.

This suggests that both adsorbents could rapidly and effectively adsorb MB from

aqueous solution.

Chapter: 6

127

Figure 6-11 Effect of contact time on the adsorption of MB onto CMCD-MNP(P) (a) and CMCD-MNP(C) (b) and pseudo-second order kinetics of MB adsorption onto CMCD-MNP(P) (c) and CMCD-MNP(C) (d). Conditions: (Initial MB concentration: 0.25, 0.50 and 2 mg/ml, pH: 8, temperature: 25ºC).

The adsorption kinetics of MB onto CM-β-CD modified magnetic nanoparticles was

investigated with the help two kinetic models, namely the Lagergren pseudo-first order

and pseudo-second-order model. The Lagergren rate equation is one of the most widely

used adsorption rate equations for the adsorption of solute from a liquid solution. The

pseudo-first-order kinetic model is expressed by the following equation [150]:

1( )te t

dqk q q

dt (6-2)

Chapter: 6

128


gives:

ln(qe - qt) = lnqe – k1t (6-3)

where qe and qt refer to the amount of dye adsorbed (mg/g) at equilibrium and at any

time, t (min), respectively, and k1 is the equilibrium rate constant of pseudo-first-order

sorption (1/min). The slope and intercept of the plot of ln(qe - qt) versus t are used to

determine the first-order rate constant, k1. The corresponding kinetic parameters are

shown in Table 6-1. It was found that the correlation coefficient (R2) has low value

(<89%) for both adsorbents for all initial MB concentrations studied and a very large

difference exists between qe,cal and qe,exp, indicating a poor pseudo-first-order fit to the

experimental data. The inapplicability of the pseudo-first-order model to describe the

kinetics of basic blue 3 and Malachite Green (a basic dye) by adsorption using

cyclodextrin based polymer was also observed in some previous work [10,113].

Another kinetic model is pseudo-second order model, which is expressed by [149]:

21( )t

e t

dqk q q

dt (6-4)

Rearranging the variables in Eq. 6-4 gives,

12( )t

e t

dqk dt

q q

(6-5)


gives:

22

1 1

t e e

tt

q k q q (6-6)

Chapter: 6

129

where k2 is the equilibrium rate constant of pseudo second- order adsorption (g/mg

min). The slope and intercept of the plot of t/qt versus t were used to calculate the

second-order rate constant, k2 (Figures 6-11c and 6-11d). The corresponding kinetic

parameters from this model are listed in Table 6-2. The correlation coefficient (R2) for

the pseudo-second-order adsorption model has high value (> 99%) for both adsorbents.

The calculated equilibrium adsorption capacities, (qe,cal) by CMCD-MNP(P) and

CMCD-MNP(C) were 129.87 and 69.44 mg/g, respectively which were consistent with

the experimental data. These facts suggest that the pseudo-second-order adsorption

mechanism is predominant, and that the overall rate of the dye adsorption process

appears to be controlled by the chemisorption process [115]. Similar phenomena have

been observed for MB adsorption onto many adsorbents, for example- titanate

nanotubes [226], tea waste [231], β-CD/carboxymethyl cellulose polymer [141] etc.

Table 6-2 Adsorption kinetic parameters of MB onto the CM-β-CD modified magnetic nano-adsorbents at pH 8 and 25ºC

Adsorbent Initial conc. Co (mg/ml)

pseudo-first order pseudo-second order

k1, min-1 qeb, mg/g R2 qe

a, mg/g k2,g/(mg min) qeb, mg/g R2

CMCD-MNP(P)

0.25 0.005 5.93 0.719 47.68 0.032 44.70 0.999

0.50 0.015 7.77 0.798 74.60 0.020 73.53 0.999

2.00 0.023 25.20 0.785 130.96 0.004 129.87 0.999

CMCD-MNP(C)

0.25 0.013 3.84 0.662 27.23 0.026 26.46 0.999

0.50 0.016 5.00 0.658 42.53 0.020 41.33 0.998

2.00 0.016 4.10 0.883 68.75 0.016 69.44 0.999 a experimental b calculated

6.3.2.3 Adsorption isotherms of MB

The equilibrium isotherms for the adsorption of MB by CMCD-MNP(P) and CMCD-

MNP(C) at pH 8 and 12 and 25ºC are shown in Figure 6-12. The equilibrium data were

fitted by Langmuir and Freundlich isotherm equations. The Langmuir equation can be

expressed as:

Chapter: 6

130

1e e

e m m L

C C

q q q K (6-7)

where qe is the equilibrium adsorption capacity of MB on the adsorbent (mg/g), Ce the

equilibrium MB concentration in solution (mg/ml), qm the maximum capacity of

adsorbent (mg/g), and KL is the Langmuir adsorption constant (ml/mg). The linear form

of Freundlich equation, which is an empirical equation used to describe heterogeneous

adsorption systems, can be represented as follows:


where qe and Ce are defined as above, KF is Freundlich constant (ml/mg), and n is the

heterogeneity factor. The values of qm and KL were determined from the slope and

intercept of the linear plots of Ce/qe versus Ce (Figure 6-13a) and of values of KF and

1/n were determined from the slope and intercept of the linear plot of lnqe versus lnCe

(Figure 6-13b). The isotherm parameters are shown in Table 6-3. The high values of

correlation coefficients, R2 (> 0.99) for both CMCD-MNP(P) and CMCD-MNP(C),

indicated that the equilibrium data were fitted better by Langmuir equation than by

Freundlich equation. Thus, the adsorption of MB on both adsorbents obeyed the

Langmuir adsorption isotherm. From Table 6-3, the qm values at optimum pH (~12) for

CMCD-MNP(P) and CMCD-MNP(C) were estimated to be 277.8 and 140.8 mg/g,

respectively, and the KL values for MNPs and CMCD-MNPs were calculated as 9.0 and

4.44 ml/mg, respectively. CMCD-MNP(P) could adsorb MB molecule almost twice

than that by CMCD-MNP(C) at both pH 8 and 12. This could be explained by the

amount of CM-β-CD grafted on the surface of both adsorbents. CMCD-MNP(P)

adsorbent contained more CM-β-CD (12 wt%) than CMCD-MNP(C) (4.7 wt%)

providing more adsorption sites for adsorption. The maximum amount of MB adsorbed

by both adsorbents in this work is compared to other adsorbents recently reported in

Chapter: 6

131

literature shown in Table 6-4. The adsorption capacities of these as-synthesized nano-

adsorbents for MB are comparatively higher than some of other cyclodextrin polymer

based adsorbents. For example, the adsorption capacities of MB on β-CD polymer (β-

CDP) crosslinked by citric acid and β-CD/carboxymethyl cellulose polymer were 105

mg/g [11] and 56.5 mg/g [141], respectively. Our results reveal the potential of these

materials to be an effective nano-adsorbent for removing basic dyes.

Figure 6-12 Equilibrium isotherm for the adsorption of MB on CMCD-MNP(P) and CMCD-MNP(C) at 25ºC.

0

50

100

150

200

250

300

0 0.5 1 1.5 2 2.5 3

q e(m

g/g)

Ce (mg/ml)

CMCD-MNP(P) at pH12CMCD-MNP(P) at pH 8CMCD-MNP(C) at pH12CMCD-MNP(C) at pH 8Langmuir fitting

Chapter: 6

132

Figure 6-13 Langmuir plot illustrating the linear dependences of Ce/qe on Ce (a) and Freundlich plot illustrating the linear dependences of lnqe on lnCe (b).

(a)

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0 0.5 1 1.5 2 2.5 3

Ce/

q e

Ce

CMCD-MNP(P) at pH 12CMCD-MNP(P) at pH 8CMCD-MNP(C) at pH12CMCD-MNP(C) at pH 8

(b)

2

2.5

3

3.5

4

4.5

5

5.5

6

-5 -3 -1 1

lnq e

lnCe

CMCD-MNP(P) at pH 12

CMCD-MNP(P) at pH 8

CMCD-MNP(C) at pH 12

CMCD-MNP(C) at pH 8

Chapter: 6

133

Table 6-3 Adsorption isotherm parameters for MB adsorption onto CM-β-CD modified magnetic nanoparticles at 25ºC

Isotherm Models

Parameters CMCD-MNP(P) CMCD-MNP(C)

pH 12 pH 8 pH 12 pH 8

Langmuir

qm (mg/g) 277.8 138.9 140.8 77.5

KL (ml/mg) 9.0 10.28 4.44 8.60

R2 0.999 0.998 0.993 0.996

RL 0.31-0.036 0.49-0.031 0.69-0.07 0.54-0.037

Freundlich n 2.913 2.97 2.20 2.84

KF (ml/g) 256.1 120.60 112.26 66.14

R2 0.921 0.957 0.975 0.945

Table 6-4 Maximum adsorption capacities (qm in mg/g) for MB by some other adsorbents reported in literature

Adsorbents Maximum adsorbed amount, qm (mg/g)

pH References

Activated carbon from steam activated bituminous coal

580.0 11 [232]

Fe3O4-activated carbon nanocomposite

321.0 NA* [137]

M-β-CD modified magnetic nanoparticles [CMCD-MNP(P)]

277.8 12 This study

Polyacrylic acid-bound iron oxide magnetic nanoparticles

199.0 9 [40]

Activated carbon from groundnut shell (GNSC)

164.9 7.4 [233]

CM-β-CD modified magnetic nanoparticles [CMCD-MNP(C)]

140.8 12 This study

β -CD polymer (β-CDP) crosslinked by citric acid

105.0 NA* [11]

Spent activated clay 91.23 5.5 [234] Tea waste 85.16 8 [231] β -CD/carboxymethyl cellulose polymer

56.5 8 [141]

Silica 56.2 11 [235] Yellow passion fruit waste 44.7 8 [227] Fe(III)/Cr(III) hydroxide 22.8 NA* [236] Activated carbon from waste biomass

16.43 6 [230]

*NA: Not available

Chapter: 6

134

The essential features of the Langmuir isotherm can be expressed in terms of a

dimensionless constant called separation factor or equilibrium parameter (RL) which is

given by the following equation [213]:

0

1

1LL

RK C

(6-9)

where C0 is the initial MB concentration (mg/ml) and KL is the Langmuir constant

related to the energy of adsorption (ml/mg). The value of RL indicates the shape of the

isotherms to be either unfavorable (RL > 1), linear (RL = 1), favorable (0 < RL < 1) or

irreversible (RL = 0). It was observed that the value of RL in the range of 0-1 (Table 6-2)

confirmed the favorable uptake of MB by both adsorbents. The calculated RL values at

different initial MB concentration are presented in Figure 6-14. Also higher RL values at

lower MB concentrations indicate that adsorption was more favorable at lower

concentration. In addition, the low RL values (< 0.05) implied that the interaction of MB

molecules with CM-β-CD modified magnetic nanoparticles might be relatively strong

[226].

Figure 6-14 Separation factor for MB onto CM-β-CD modified magnetic nanoparticles at pH 8 and 25ºC.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 1 2 3

RL

Co (mg/ml)

CMCD-MNP(P)

CMCD-MNP(C)

Chapter: 6

135

The free energy change (∆G) for adsorption at 25oC was calculated using the following

equation:

∆G = −RT lnKa = −RT lnKc = −RT ln(Cads/Ce ) (6-10)

where T is the temperature (K), R the gas constant (Jmol-1K-1), Ce the dye concentration

in solution at equilibrium and Cads the concentration of dye adsorbed at equilibrium.

The above equation implies that only at very low concentration of solute, the

distribution constant (Kc) is equal to the thermodynamic equilibrium constant (Ka) and

only in this case it can be used for calculation of ∆G [237]. Kc can be estimated from the

slope of the linear plot of Cads vs. Ce and the linear relationship holds only for dilute

solutions. The calculated ∆G value was found to be −3.61 kJ/mol and -3.21 kJ/mol at

pH~8 and -4.62 KJ/mol and -2.98 KJ/mol at pH~12 for CMCD-MNP(P) and CMCD-

MNP(C), respectively. The negative values of free energy change indicate the

spontaneous nature of sorption and confirm affinity of sorbents for the dye.

6.3.3 Adsorption interactions

For understanding of the MB/CMCD-MNP(P) interaction, FTIR data were introduced

to gain insight into the adsorption mechanism. Figure 6-15 shows the spectra of

methylene blue (MB) and CMCD-MNP(P) nano-adsorbent before and after MB

adsorption. Figure 6-15(a) shows the broad peak at 3386 cm−1 corresponded to the

stretching vibration of highly -OH group on the CMCD-MNP(P). The other peaks at

1028 and 1153 cm−1 are due to C-OH and O-C-O groups of cyclodextrin moieties,

respectively. After being adsorbed by methylene blue, the peaks at 900-1100 cm-1 in the

spectrum for CMCD-MNP(P) have shifted which indicates that these functional groups

may be involved into interaction with MB. The absorption band at 3383 cm−1 also

shifts to higher wave number (3389 cm−1) which can be explained by the disappearance

Chapter: 6

136

of water molecules from the cyclodextrin cavity, similarly to the case of true inclusion

complexes [238]. In the spectrum for MB shown in Figure 6-15(c), the peak

corresponding to the vibration of the aromatic ring at 1600 cm−1 was very prominent.

Two peaks at 1490 and 1396 cm−1 are assigned to the C–N stretching vibrations [226].

The vibrations of the CH3 group are found at 1357 and 1342 cm−1. Several weak peaks

between 1252 and 669 cm−1 were ascribed to the C–H in plane and out of plane bending

vibrations [232]. In the spectrum of CMCD-MNP(P) after adsorption of MB (Figure 6-

15b), several peaks corresponding to MB with the aromatic ring vibration at 1606 cm−1,

the C–N stretching vibrations at 1492 and 1394 cm−1 and the CH3 group vibrations at

1354 and 1337 cm−1, can be observed which reflects the evidence for the strong

interaction between MB and CMCD-MNP(P). Similar MB-adsorbent interaction was

observed after adsorption of MB onto titanate nanotube [226].

Figure 6-15 FTIR spectra of CMCD-MNP(P) of before (a) and after (b) adsorption of MB, and only MB (c).

400 900 1400 1900 2400 2900 3400 3900

%T

Wavenumber, cm-1

10281623

3386

1606

10321337

1600

1396

1357

3389

1083

2923

586

586

13421028

1081

(b)

(a)

(c)

1490

1492

Chapter: 6

137

It is reported that due to its specific characteristics (the presence of cyclodextrin

moieties and complexing chemical groups), the sorption mechanism of cyclodextrin

based materials is different from those of other materials [10]. The main interactions in

the complexation process between cyclodextrin and other organic molecule are dipole-

dipole, hydrogen bonding, electrostatic, van der Waals, hydrophobic and charge transfer

interaction [9]. Among these forces, hydrophobic interactions have been mostly

recognized as the main factors involved in the inclusion complex with cyclodextrin. In

this study, the adsorption of MB is found to be pH dependent and the

hydrophilic/hydrophobic nature of MB depending on various pHs plays an important

role in the formation of inclusion complex with CM-β-CD (Figure 6-10). It has been

found that the adsorption process follows the pseudo-second-order kinetics and this

suggests that chemical sorption might be the rate-limiting step in controlling the

adsorption process. The negative values of free energy change (∆G) confirmed the

affinity of adsorbents for the MB dye.

6.3.4 Desorption and regeneration experiments

Desorption study was conducted to explore the possibility of recyclability of the

magnetic nano-adsorbent. Desorption of MB from MB loaded-CMCD-MNP(P) was

demonstrated using a methanol solution containing acetic acid (0-5%) as eluent [40]. It

can be seen from Figure 6-16(a) that pure methanol solution could desorb 57.2% of MB

from the nano-adsorbent surface. The percentage of MB desorbed could be increased by

using acetic acid in methanol solution. About 99.0% of desorption efficiency was

achieved when 5% (v/v) of acetic acid was used in methanol solution, respectively.

Desorption of MB from MB loaded-CMCD-MNP(P) was also carried out using acidic

buffer (pH< 3). Since adsorption of MB was very less at low pH as shown in Figure 6-

Chapter: 6

138

10, it was hypothesized that acidic buffer might aid in desorption of MB from the CM-

β-CD modified magnetic nanoparticles. The desorption efficiency was achieved also as

high as 79.7% when acidic buffer (pH = 2) was used as desorption eluent.

Figure 6-16 (a) Desorption of MB from CMCD-MNP(P) as a function of acetic acid concentration in methanol, and (b) performance of CMCD-MNP(P) by multiple cycles of regenaration. Adsorption conditions: (CMCD-MNP(P): 120 mg; MB concentration: 2 mg/ml; temperature: 25ºC; pH: 8; contact time: 2 hr). Desorption conditions: (CMCD-MNP(P): 120 mg; temperature: 25ºC; contact time: 2 hr; desorption eluent: methanol with 5 % acetic acid)

0

20

40

60

80

100

0 1 2.5 5

% R

emov

al

Acetic acid in methanol (vol%)

(a)

0

20

40

60

80

100

120

140

1 2 3 4

q e(m

g/g)

No. of cycles

(b)

Chapter: 6

139

As shown as Figure 6-16(b), CMCD-MNP(P) could be recycled four times for MB

adsorption with more than 90% of adsorption capacity being retained. This result

indicates the stability and reusability of the adsorbents in wastewater treatment.

6.4 Cost analysis

Since its first introduction for heavy metals removal, activated carbon has undoubtedly

been the most popular and widely used adsorbent for environmental pollution control.

Activated carbons are the most effective adsorbents as they can efficiently adsorb many

pollutants. Although the commercial activated carbon is relatively cheap, but further

modification of these adsorbents for some specific applications could be quite

expensive. The regeneration of saturated carbon by thermal and chemical procedure is

also expensive, and results in loss of the adsorbent. This had led many researchers to

search for more efficient adsorbents with high adsorption capacities and low cost.

Cost is actually an important parameter for comparing the adsorbent materials. But,

which low-cost adsorbent is better? There is no direct answer to this question because

each low-cost adsorbent has its specific physical and chemical characteristics such as

porosity, surface area, physical strength and chemical stability, as well as inherent

advantages and disadvantages in wastewater treatment. A sorbent can be considered

low-cost if it requires little processing, is abundant in nature, or is a by-product or waste

material from another industry [107,116]. This adsorbent should also possess high

capacity and adsorption rate, high selectivity for different concentrations and rapid

kinetics. β-CDs are synthetic polysaccharides, formed by the action of an enzyme on

starch. Starches are unique raw materials in that they are very abundant natural

polymers, inexpensive and widely available in many countries. β-CD molecules are

available commercially at a low-cost. Both soluble and insoluble CD-based materials

Chapter: 6

140

are also easy to prepare with relatively inexpensive chemical reagents and are available

in a variety of structures with a variety of properties. Anchoring the β-CD derivatives

on the surfaces of Fe3O4 nanoparticles in one step precipitation method is also a simple

and inexpensive method. The particles synthesized in this way, shows high adsorption

capacities and fast kinetics and they could be regenerated and recycled using simple

magnetic separation technology which is obviously a great advantage. Due to scarcity

of consistent cost information in the literature data, cost comparisons are difficult to

make. However, the relative cost of the material used in the present study can be

comparable with modified activated carbons.

6.5 Conclusions

In this work, CM-β-CD modified Fe3O4 nanoparticles with combined properties of

superparamagnetism and inclusion functionalities were successfully synthesized. The

binding of CM-β-CD on the magnetic surface was confirmed by FTIR, TGA and zeta

potential analysis. MB was recovered fast and efficiently from the aqueous solution

using the as-synthesized magnetic nanoadsorbents. The adsorption of MB on these

adsorbents was found to be pH dependant. The kinetics of MB adsorption onto both

adsorbents followed the pseudo-second-order model. The adsorption of MB reached

equilibrium within 50 min and 85-90% of MB being adsorbed within 20 min. The

equilibrium data for both CMCD-MNP(P) and CMCD-MNP(C) were fitted well by the

Langmuir isotherm model of adsorption, showing monolayer coverage of MB

molecules at the outer surface of these nanoparticles. The maximum sorption capacities

of MB for CMCD-MNP(P) and CMCD-MNP(C) were 277.8 and 140.8 mg/g,

respectively at pH~12 and 25ºC and the negative values of free energy change indicated

the spontaneity of adsorption process. The magnetic nano-adsorbents (CMCD-MNP(P))

Chapter: 6

141

prepared by one-step method showed better adsorption performance than CMCD-

MNP(C) prepared by two-steps method. This is because, CMCD-MNP(P) has higher

content of CM-β-CD which in turn provides more available active sites for adsorption.

The adsorbed MB could be desorbed from the surface by using methanol solution

containing acetic acid and these nano-adsorbents could be recycled and handled easily

by using magnetic field gradient. The experimental results suggest that the CM-β-CD

modified magnetic nanoparticles could be employed as efficient and suitable adsorbents

for the removal of methylene blue dye from wastewater.

Chapter: 7

142

Chapter 7 Carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbents for removal of copper ions from aqueous water4

7.1 Introduction

This Chapter presents adsorption studies of heavy metal [Cu2+] ions onto CM-β-CD

bonded magnetic nanoparticles. Considered the special advantages of the adsorption

capacity of β-CD through complexation with multiple functional groups and the

separation convenience of magnetic Fe3O4 nanoparticles, grafting of β-CD on the

magnetic nanoparticles is considered as a promising alternative to develop magnetic

materials for the efficient removal of heavy metal pollutants.

Heavy metal pollution in industrial wastewater has attracted global attention because of

its adverse effects on the environment and human health [108,239]. The effluents of

industrial or municipal wastewaters contain considerable amount of toxic and polluting

heavy metals. One of those metals in focus is copper (Cu2+) which is an abundant and

naturally occurring element present in the wastewaters [108]. Copper ingestion might

cause vomit, cramps, liver damage, kidney damage and even death. It is also toxic to

aquatic organisms even at very small concentrations in natural water [152]. It is,

therefore, crucial to remove these heavy metals from wastewaters effectively before

their discharge into the environment.

4. A.Z.M. Badruddoza, A.S.H. Tay, P.Y. Tan, K. Hidajat, M.S. Uddin, Carboxymethyl-β-cyclodextrin

conjugated magnetic nanoparticles as nano-adsorbents for removal of copper ions: Synthesis and

adsorption studies, J. Hazard. Mater. 185(2-3) (2011) 1177-1186.

Chapter: 7

143

Currently heavy metal removal methods include chemical precipitation, ion exchange,

liquid-liquid extraction, membrane filtration, adsorption and biosorption [111]. Among

these methods, adsorption has increasingly received more attention as it is simple and

cost effective [41,112]. Recently, adsorption using magnetic nano-sized adsorbents has

attracted significant attentions in environmental applications due to the specific

characteristics [8,37,41,108,125,240]. Their properties- extremely small size, high

surface-area-to volume ratio and the absence of internal diffusion resistance provide

better kinetics for adsorption of metal ions from aqueous solution. Magnetic nano-

adsorbents have the advantages of both magnetic separation techniques and nano-sized

materials, which can be easily recovered or manipulated with an external magnetic

field. This technique overcomes many issues present in other physicochemical process

such as fouling problems and operation cost, and requires much less energy to achieve a

given level of separation [128]. The incorporation of magnetic particles with other

functionalized materials such as multiwalled carbon nanotubes (MWCNT)

[155,241,242], zeolites [243], activated carbon [244] etc. which are effective for the

removal of both organic and inorganic pollutants is potentially a promising method to

facilitate the separation and recovery of the adsorbents. Various types of magnetic

nano-adsorbents with tailored surface reactivity by using natural or synthetic polymers

such as, chitosan, gum arabic, alginate, poly(acrylic acid) etc. have been used recently

for heavy metal removal from wastewater [8,37,41,44,108,124,125,241,245]. However,

the effectiveness of magnetic nano-adsorbents modified with cyclodextrin–an important

class of polysaccharides has not been attempted.

Although there is less report on environmental applications of cyclodextrins for heavy

metal removal, there is evidence that metal can be complexed by cyclodextrins through

hydroxyl groups. Metal ion complexes with cyclodextrins could have a wide range of

Chapter: 7

144

applications in catalysis and molecular recognition [246]. Some properties such as

aqueous solubility and metal complexation potential can be altered by substituting

functional groups to the outside of the cyclodextrin [222]. For example, carboxymethyl-

β-cyclodextrin (CM-β-CD) has the ability to complex heavy metals such as cadmium,

nickel, strontium and mercury through the interactions between the metal ions and -

COOH functional groups [247,248]. Insoluble cyclodextrin polymers and cyclodextrin

immobilized on various supports including inorganic carriers are currently being

investigated for adsorptive specificity towards heavy metal ions [179,249]. Very

recently, Hu et al. showed that the grafted β-CD on the MWCNT/iron oxides

contributes to an enhancement of the adsorption capacity because of the strong

complexation abilities of the multiple hydroxyl groups in β-CD with the lead ions

[242]. However, to our knowledge, no adsorptive study of heavy metals on magnetic

nanoparticles covalently bonded with CM-β-CD has been reported so far.

In this study, a novel magnetic nano-adsorbent was synthesized for the adsorption of

metal ions by surface modification of Fe3O4 nanoparticles with CM-β-CD. The

adsorption behavior of these sorbents was investigated using Cu2+ ions as the target

metal contaminant because of its extensive environmental impacts. The effects of

several factors such as, pH, initial Cu2+ concentration and temperature were studied.

The adsorption interactions of Cu2+ onto CMCD-MNP(C) were investigated through

FTIR and XPS analyses. In addition, the regeneration and reusability of the adsorbents

were also evaluated.

Chapter: 7

145

7.2 Experimental

7.2.1 Materials

In this work, the following chemicals were used for fabricating CMCD-MNP(C): Iron

(II) chloride tetrahydrate (99%) [Alfa Aesar], iron (III) chloride hexahydrate (98%)

[Alfa Aesar], ammonium hydroxide (25%) [Merck], β-cyclodextrin (β-CD, 99%)

[Tokyo Kasie Kogyo (Japan)], carbodiimide (cyanamide, CH2N2, 98%) [Sigma],

copper(II) nitrate [Sigma], chloroacetic acid (99%) [Alfa Aesar]. All other chemicals

were of analytical grade and used as received without further purification.

7.2.2 Adsorption/ desorption of Cu2+ ions

The adsorption of Cu2+ by the CMCD-MNP(C) was investigated using batch

equilibrium technique in aqueous solutions at pH 2–6 at 25–55 ◦C. In general, 120 mg

of wet magnetic nano-adsorbent was added to 10 ml of copper nitrate solution of

various concentrations and shaken in a thermostatic water-bath shaker operated at 230

rpm. After equilibrium was reached, the magnetic nano-adsorbents were removed using

a strong permanent magnet made of Nd–Fe–B and the supernatant was collected. The

solution pH was adjusted by NaOH or HCl. The concentrations of copper ions were

measured using Inductive Couple Plasma Mass Spectrometry (Agilent ICP-MS 7700

series).

For the kinetic experiments, the initial copper ion concentrations were 50, 100 and 200

mg/l and the initial solution pH value was 6.0 (the solution pH was not controlled

during the experiments). These copper solutions containing the magnetic particles were

agitated for a contact time varied in the range of 0–240 min at a speed of 230 rpm at

25◦C. At various time intervals, samples were collected after separation of magnetic

adsorbents by magnetic decantation process and the copper concentration was

Chapter: 7

146

determined as mentioned previously. In the investigation of the effect of solution pH on

equilibrium copper adsorption, the initial Cu2+ concentrations in the solution were 50

and 200 mg/l, but the solution pH values were changed from 2 to 6. In the adsorption

isotherm experiments, the initial solution pH was 6.0 while the initial Cu2+

concentrations in the solutions were changed from 50 to 400 mg/l and temperatures

were varied from 25◦C to 55◦C. The samples were shaken for 4 h (which was enough to

achieve equilibrium) at 230 rpm. Duplicate experiments were carried out in each case

and the reproducibility was found to be fairly good. The error percentage was within

5%. Desorption study was conducted using 0.1 M acetic acid, 0.1 M citric acid and 0.1

M Na2EDTA as desorption eluents. Adsorption was first conducted using 120 mg of

wet CMCD-MNPs in 10 ml of 200 mg/l Cu2+ solution for 4 h at pH 6. Desorption was

then studied by adding 10 ml of either acid solution to the Cu2+-sorbed CMCD-

MNP(C). After shaking at 200 rpm for 3 h, the CMCD-MNPs were removed and the

concentration of copper ions was measured.

7.3 Results and Discussion

7.3.1 Synthesis and characterization of magnetic nanoparticles

The detailed synthesis procedures of CMCD-MNP(C) are shown step-by-step in

Chapter 6 (section 6.2.1). In Chapter 6, the characterization results of these nano-sized

magnetic particles performed by FTIR, TEM and VSM are also discussed. In this

section, only the results of characterization by TGA and zeta potential measurement

have been discussed.

The amount of CM-β-CD grafted on Fe3O4 MNPs is estimated from TGA analyses of

uncoated and CM-β-CD coated magnetic nanoparticles. As shown in Figure 7-1(a), the

TGA curve for CMCD-MNPs exhibits two steps of weight loss, contributed from the

Chapter: 7

147

loss of residual water in the sample in 30–190oC and the loss of CM-β-CD in the range

of 200–450oC. A drastic drop of weight loss can be seen in the range 190–400oC with

the occurrence of a weak heat absorption at 282.5oC and it is contributed from the

thermal decomposition of CM-β-CD. Thus, the TGA curves also confirm the successful

grafting of CM-β-CD onto the magnetic surface.

Figure 7-1 (a) TGA curves for uncoated Fe3O4 MNPs and CMCD-MNP(C); (b) Effect of the amount of CM-β-CD in solution on the grafted CM-β-CD contents onto magnetic nanoparticles.

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

92

93

94

95

96

97

98

99

100

101

0 200 400 600

% w

eigh

t lo

ss

Temperature, oC

Fe3O4 MNPs

CMCD-

Der

ivat

ive

of w

eigh

t lo

ss

121.9 oC

282.5 oC

(a)

0

1

2

3

4

5

6

0 50 100 150 200 250 300 350

% C

M-β

-CD

gra

fted

on

to M

NP

s

CM-β-CD added in solution (mg)

(b)

Chapter: 7

148

The effect of the amount of CM-β-CD used in solution on the amount of grafted CM-β-

CD onto the surface of magnetic nanoparticles was also demonstrated in this study. The

experiment was performed in 5 ml of CM-β-CD (5-60 mg/ml) solution containing a

constant Fe3O4 amount of 100 mg. The percentage amount of CM-β-CD grafted on

Fe3O4 MNPs was estimated from the weight loss measured by TGA analysis. As shown

in Figure 7-1(b), with increasing amounts of CM-β-CD in solution, the amount of

grafted CM-β-CD on the nanoparticles increases first and then remains almost constant

when the amount of CM-β-CD in solution is above 150 mg. This may be due to the fact

that as the amount of Fe3O4 is fixed which means the number of surface hydroxyl (-OH)

groups are fixed, no more -OH groups are available which can react with the –COOH

groups on CM-β-CD to form metal carboxylate. Similar finding was reported in

covalent binding of poly(acrylic acid) (PAA) to Fe3O4 nanoparticles via carbodiimide

activation [5]. However, the maximum amount of CM-β-CD bound to Fe3O4

nanoparticles could be determined to be 4.8 wt% i.e. 0.0366 mmol/g.

Figure 7-2 Zeta potentials of uncoated and CM-β-CD coated magnetic nanoparticles and copper(II) nitrate solution at different pH.

-60

-40

-20

0

20

40

60

2 4 6 8 10 12

Zet

a p

oten

tial

, m

V

pH

Uncoated Fe3O4 MNPs

CMCD-MNPs

copper(II) nitrate

Chapter: 7

149

Zeta potentials of uncoated and CM-β-CD coated magnetic nanoparticles (0.1 mg/ml)


adjusted by NaOH or HCl. As shown in Figure 7-2, the pH value of point of zero

charge (pHpzc) of uncoated Fe3O4 nanoparticles is about 6.8, consistent with the values

reported in literature. After being grafted with CM-β-CD, the pHpzc has been shifted to

4.7, indicating that the grafting of CM-β-CD onto uncoated MNPs is successful.

Moreover, magnetic nanoparticles modified with CM-β-CD yields acidic surface since

pHpzc is lower than that of uncoated MNPs and this surface acidity is due to the

introduction of several oxygen-containing functional groups [211].

7.3.2 Adsorption of Cu2+ ions on CMCD-MNP(C) 7.3.2.1 Effect of pH

The pH of the aqueous solution is an important controlling parameter in the adsorption

process [108,112,250]. The effects of initial solution pH on Cu2+ adsorption onto

CMCD-MNP(C) were investigated at pH 2–6, 25ºC, and an initial Cu2+ ion

concentration of 50 and 200 mg/l. As shown in Figure 7-3, it is noteworthy that the

adsorption capacity of Cu2+ ions increases with the solution pH at an initial Cu2+

concentration of 200 mg/l. This might be due to the less insignificant competitive

adsorption of hydrogen ions [8,37,251]. At a lower initial Cu2+ concentration (50 mg/l),

the adsorption capacity increases with increasing the solution pH and then remains

almost unchanged at pH 4–6. This might be due to the fact that almost all Cu2+ ions are

adsorbed. It is well known that Cu(II) species can be present in aqueous solution in the

forms of Cu2+, Cu(OH)+, Cu(OH)2, Cu(OH)3− and Cu(OH)4

2− and the predominant

copper species at pH < 6.0 is Cu2+ [250,252]. Therefore, less adsorption of Cu2+ that

takes place on CMCD-MNP(C) at lower pH, can be explained by the fact that at these

Chapter: 7

150

pH values the H+ concentration is high, which can compete with copper cations for the

same adsorption sites [250]. The adsorption studies at pH> 6 were not conducted

because of the precipitation of Cu(OH)2 from the solution.

Figure 7-3 Effect of pH on the adsorption of Cu2+ ions by CMCD-MNP(C) at 25ºC. Open points: qe vs. initial pH values; solid points: equilibrium pH values vs. initial pH values.

The effect of pH can be also explained by considering the surface charge on the

adsorbent material. The zeta potential of Cu2+ solution is presented in Figure 7-2 and it

shows that copper solution has positive zeta potential at pH< 7. Since the zeta potentials

of the CMCD-MNP(C) are positive at pH< 4.7 (point of zero charge, pHpzc), the

interactions between the adsorption sites on CMCD-MNP(C) and the copper ions are

electrostatically repulsive. As the solution pH increases, the repulsion between

adsorbent surface and metal ions becomes weaker, thus enhancing the adsorption

capacity [253]. On contrary, at pH above pHpzc, the negatively charged CMCD-

MNP(C) nano-adsorbents attract the positively charged metal ions. Moreover, the

1

2

3

4

5

6

0

10

20

30

40

1 2 3 4 5 6

q e(m

g/g)

Initial pH

Eq

uili

bri

um

pH

Chapter: 7

151

adsorption of Cu2+ ions depends on the adsorption sites available for copper attachment

on the surface of CMCD-MNP(C), possibly through the complexation with mainly –OH

and –COOH functional groups (see the discussions in the section 7.2.2.5). The

equilibrium pH values are also plotted as a function of initial pH values for each

experimental data (Figure 7-3). One can see that the final pH value is higher than the

initial pH value for lower pH range. When the initial solution pH exceeds to 4.0, the

final pH value is lower than the initial one and this difference becomes higher for

higher adsorption capacity of CMCD-MNP(C). The pH decreasing after Cu2+ ions

adsorption suggests that part of H+ is released from the CMCD-MNP(C) surface to

solution. Due to the increase of pH, more surface functional groups are deprotonated to

provide more metal binding sites, which results in a high metal ion adsorption and more

H+ are released into the solution resulting in the decrease in the pH values after metal

adsorption [250].

7.3.2.2 Adsorption kinetics

Figure 7-4(a) shows the effects of contact time on adsorption of Cu2+ onto CMCD-

MNP(C) at different Cu2+ concentrations ranging from 50 to 200 mg/l and at 25ºC. It

can be seen that a rapid adsorption of Cu2+ by CMCD-MNP(C) occurs, with equilibrium

reached in approximately 30 min. The adsorption capacity increases as initial metal ion

concentration increases. Within 15 min, 90% of Cu2+ adsorption equilibrium is

achieved using CMCD-MNP(C). This suggests that CMCD-MNP(C) could easily and

rapidly adsorb Cu2+ from aqueous solution due to high specific surface area and the

absence of internal diffusion resistance. Rapid adsorption rate for copper removal has

also been observed using several magnetic nanoparticles based nano-adsorbents

[37,41,124]. However, a shaking time of 4 h was used in all further adsorption

experiments to ensure equilibrium.

Chapter: 7

152

The adsorption kinetics of Cu2+ ions onto CMCD-MNP(C) is investigated with the help

two kinetic models, namely the Lagergren pseudo-first-order and pseudo-second-order

model. The pseudo-first-order kinetic model is expressed by the following equation

[150]:

ln(qe – qt) = lnqe – k1t (7-1)

where qe and qt refer to the adsorption capacity of Cu2+ (mg/g) at equilibrium and at any

time, t (min), respectively, and k1 is the rate constant of pseudo-first-order adsorption

(min-1). The slope of the linear plot of ln(qe – qt) versus t is used to determine k1 (figure

not shown).

The pseudo-second-order kinetic rate equation is expressed as follows [149]:

22

1 1

t e e

tt

q k q q (7-2)

where k2 is the rate constant of pseudo-second-order adsorption (g mg-1min-1). The

slope and intercept of the plot of t/qt versus t are used to calculate k2 and qe,cal (Figure 7-

4b). The corresponding kinetic parameters from both models are listed in Table 7-1. It

is found that the correlation coefficients (R2) for the pseudo-first-order kinetic model

are less than 90% indicating a poor pseudo-first-order fit to the experimental data. On

the other hand, the correlation coefficient (R2) for the pseudo-second-order adsorption

model has high value (> 99%), and the q values (qe,cal) calculated from pseudo-second-

order model are more consistent with the experimental q values (qe,exp) than those

calculated from pseudo-first-order model. These facts suggest that the adsorption data

are well represented by pseudo-second-order kinetic model. The pseudo-second-order

adsorption has also been reported for many adsorbents such as natural kaolinite clay

Chapter: 7

153

[254], H2SO4 modified chitosan [154], chitosan-coated MNPs modified with alpha-

ketoglutaric acid [245].

Figure 7-4 (a) Effect of contact time on Cu2+ adsorption by CMCD-MNP(C); (b) pseudo-second-order kinetics for adsorption of Cu2+. (Initial pH: 6, temperature: 25ºC).

0

10

20

30

40

0 50 100 150 200 250

q t(m

g/g)

t (min)

200 mg/mL100 mg/mL50 mg/mLpseudo-second-order kinetics

(a)

0

4

8

12

16

0 40 80 120 160 200 240 280

t/qt

t (min)

200 mg/mL

100 mg/mL

50 mg/mL

(b)

Chapter: 7

154

Table 7-1 Adsorption kinetic parameters of Cu2+ onto CMCD-MNP(C).

Initial conc. Co(mg/ml)

pseudo-first-order pseudo-second-order k1 (min-1) qe,cal

(mg/g)R2 qe,exp

(mg/g)k2

g/(mg min)qe,cal (mg/g) R2

50 0.0053 10.92 0.688 14.88 0.038 14.47 0.999 100 0.0082 20.45 0.641 23.48 0.027 23.25 0.999 200 0.0116 34.75 0.896 36.77 0.009 36.90 0.999

7.3.2.3 Adsorption isotherm of Cu2+ ions The equilibrium isotherms for the adsorption of copper ions by uncoated MNPs at 25oC

and by CMCD-MNP(C) at different temperatures (25oC, 40oC and 55oC) in pH 6 are

shown in Figure 7-5. The equilibrium data are fitted by Langmuir and Freundlich

isotherm equations [255]. The Langmuir equation can be expressed as:

1e e

e m m L

C C

q q q K (7-3)

where qe is the amount of adsorbed material at equilibrium (mg/g), Ce the equilibrium

concentration in solution (mg/l), qm the maximum capacity of adsorbent (mg/g), and KL

is the “affinity parameter” or Langmuir constant (l/mg). The linear form of Freundlich

equation, which is an empirical equation derived to model the multilayer adsorption,

can be represented as follows:


where qe and Ce are defined as above, KF is Freundlich constant (l/mg), and n is the

heterogeneity factor.

Chapter: 7

155

Figure 7-5 Equilibrium isotherms for Cu2+ adsorption by CMCD-MNP(C) at different temperature and by uncoated Fe3O4 nanoparticles (inset) at 25ºC and initial pH 6.

The values of qm and KL are determined from the slope and intercept of the linear plots

of Ce/qe versus Ce (Figure 7-6a) and the values of KF and 1/n are determined from the

slope and intercept of the linear plot of lnqe versus lnCe (Figure 7-6b). The isotherm

parameters are shown in Table 7-2. Namely, in all temperature studied, the Langmuir

isotherm shows a better fit to experimental data in compared to the Freundlich isotherm.

Moreover, Table 7-2 shows that the Langmuir constant, KL, related to the affinity of

binding sites decreases with increasing temperature indicating that the adsorption is

favorable at lower temperatures. The maximum adsorption capacities (qm) for uncoated

MNPs and CMCD-MNP(C) are found to be 20.8 and 47.2 mg/g, respectively, at 25oC.

The qm for uncoated MNPs is found quite similar as done by Banerjee and Chen (19.3

mg/g) [41]. CMCD-MNP(C), on the other hand, could adsorb copper ions almost or

more than twice that by uncoated MNPs indicating that the modification of magnetite

surface by CM-β-CD which has multiple hydroxyl and carboxyl functional groups

could enhance the adsorption capabilities. Hu et al. [242] also found that introducing

0

5

10

15

20

25

30

35

40

45

0 100 200 300 400

q e (

mg/

g)

Ce (mg/ml)

25oC40oC55oCLangmuir fitting

0

5

10

15

20

25

0 100 200 300 400 500

q e

Ce

Chapter: 7

156

cyclodextrin onto the surface of MWCNT/iron oxide composites enhances the

adsorption capacity with Pb(II). Chen et al. [241] studied the adsorption of Eu(III) onto

CNT-iron oxide composite in presence of poly(acrylic acid) (PAA) as a surrogate for

natural organic matter and found that the presence of PAA could strongly enhance the

Eu(III) adsorption and adsorbed PAA anions can be considered as a ‘bridge’’ between

the magnetic composite and Eu(III). A positive effect of PAA on Ni2+ adsorption at

pH< 8 using MWCNT is also reported by Yang et al. [256]. The high complexation

ability of pre-adsorbed PAA onto oxidized MWCNT with Ni2+ contributes to enhanced

adsorption on the surfaces. The maximum uptake of Cu2+ by CMCD-MNP(C) obtained

in this work is compared with other results reported in literature shown in Table 7-3.

However, the adsorption capacity of CMCD-MNP(C) for copper ions is comparatively

higher than some of other magnetic particles based adsorbents.

Table 7-2 Adsorption isotherm parameters for Cu2+ ions adsorption on uncoated and CM-β-CD coated magnetic nanoparticles at pH 6.

Adsorbents T (oC)

Langmuir isotherm constants Freundlich isotherm constants

KL (l/mg)

qm (mg/g)

R2 KF (mg/g)(l/mg)

n R2

CMCD-MNP(C)

25 0.0237 47.20 0.995 7.064 3.15 0.973 40 0.0221 42.01 0.993 5.044 2.78 0.961 55 0.0202 38.75 0.992 4.002 2.57 0.993

Uncoated MNPs

25 0.0514 20.80 0.984 4.635 3.86 0.853

Chapter: 7

157

Table 7-3 Comparison of maximum adsorption capacity of CMCD-MNP(C) with those of some other magnetic adsorbents reported in literature for Cu2+ ions adsorption.

Adsorbents Maximum adsorbed amount, qm (mg/g)

References

Chitosan-coated Fe3O4 NPs modified with alpha-ketoglutaric acid

96.15 [108]

Glutaraldehyde modified Fe3O4 NPs (GA-APTES-NPs)

61.07 [124]

Calcium alginate modified Fe3O4 NPs 60.00 [125] CM-β-CD modified MNPs 47.20 This study Gum Arabic modified MNPs 38.50 [41] Amino functionalized Fe3O4@SiO2 core-shell NPs

29.85 [126]

Chitosan bound MNPs 21.50 [37] Uncoated Fe3O4 nanoparticles 19.30 [41] Amino-functionalized MNPs 12.43 [8] Carbon encapsulated MNPs 3.21 [127]

Figure 7-6 The Langmuir (a) and Freundlich (b) isotherm plots for Cu2+ ions adsorption by CMCD-MNP(C) at pH 6.

7.3.2.4 Effect of temperature and thermodynamic parameters

The effect of temperature on the adsorption isotherm was investigated under isothermal

conditions in the temperature range of 25-55ºC and at pH 6. As shown in Figure 7-5,

the adsorption capacities for CMCD-MNP(C) decrease with the increase in temperature

Chapter: 7

158

from 25oC to 55oC. Thermodynamic parameters such as change in free energy (∆Go),

enthalpy (∆Ho), and entropy (∆So) are calculated using the following thermodynamic

functions:

∆Go = -RT lnKp (7-5)

∆ ° ∆ ° (7-6)

KP is the thermodynamic equilibrium constant, i.e., the ratio of the equilibrium

concentration of Cu2+ on CMCD-MNP(C) to that in solution. This constant KP for the

sorption process is determined by plotting ln(qe/Ce) versus qe and extrapolating to zero

qe [257,258]. Figure 7-7 shows the van’t Hoff plots of lnKP versus 1/T (R2=0.99) and

the values of ∆Ho and ∆So for CMCD-MNP(C) are determined from the slopes and

intercepts, respectively. The calculated thermodynamic parameters are listed in Table 7-

4. The negative value of all ∆Go indicates the feasibility of the process and spontaneous

nature of metal ion adsorption onto magnetic nano-adsorbents. Moreover, the

magnitude of ∆G◦ decreases with increasing temperature indicating that adsorption is

not favorable at higher temperatures. The changes of enthalpy (∆Ho) and entropy (∆So)

at 25-55ºC could be determined to be -19.26 kJmol-1 and -0.0546 kJmol-1K-1,

respectively for CMCD-MNP(C). The negative value of ∆Ho confirms the exothermic

nature of adsorption which is also supported by the decrease in value of Cu2+ uptake of

the adsorbent with the rise in temperature. Similar results have been obtained for Cu2+

adsorption by cellulosic fibers modified by β-CD [259]. The negative entropy change

(∆So) for the process is caused by the decrease in degree of freedom by the adsorbed

species and indicates the stability of adsorption process with no structural change at

solid–liquid interface [154,260].

Chapter: 7

159

Figure 7-7 van’t Hoff plot for the adsorption of Cu2+ by CMCD-MNP(C).

Table 7-4 Thermodynamic parameters for the adsorption of Cu2+ ions onto CMCD-MNP(C).

∆Go (kJmol-1) ∆Ho (kJmol-1) ∆So (kJmol-1K-1)

298.15K 313.15K 328.15K -2.93 -1.99 -1.29 -19.26 -0.0546

7.3.3 Adsorption interactions

For understanding of the copper/ CMCD-MNP(C) interactions, FTIR data are analyzed.

Figure 7-8 shows the spectra of CMCD-MNP(C) nano-adsorbent before and after Cu2+

adsorption and the possible IR frequencies of CMCD-MNP(C) are listed in Table 7-5.

FTIR spectrum of metal (Cu2+)-sorbed CMCD-MNP(C) shows that the peaks expected

at 1030, 1155, 1635 and 3423 cm-1 have shifted, respectively to 1028, 1153, 1628 and

3406cm-1. The shifting of the peaks indicates that their corresponding functional groups

(mainly hydroxyl and carbonyl groups) may directly be involved in interaction with

metal to form surface complexes. Since Cu2+ adsorption on the magnetic nano-

adsorbent affected all the chemical bonds with oxygen atoms, it is reasonable to assume

that the oxygen atoms would be the main adsorption sites for Cu attachment. After Cu2+

0.2

0.4

0.6

0.8

1

1.2

1.4

0.003 0.0031 0.0032 0.0033 0.0034

lnK

p

1/T (K-1)

Chapter: 7

160

adsorption, a new sharp adsorption band at 1385cm−1 can also be observed which is

assigned to nitrate (NO3-) anion [261,262]. Hence, NO3

- ions are on the surface of

CMCD-MNP(C) to balance the electrical charges of the adsorbed copper ions [262]. In

addition, the peak of Fe-O has a slight shift from 582 to 586 cm-1 as some of metal ions

are adsorbed onto the iron oxide [263].

Figure 7-8 FTIR spectra of CMCD-MNP(C) before (a) and after (b) adsorption of Cu2+ ions.

Table 7-5 FTIR absorption frequencies (cm-1) of CMCD-MNP(C) before and after adsorption and their possible assignments

CMCD-MNP(C) (before adsorption)

CMCD-MNP(C) (after adsorption)

Assignment

582 586 υFe–O 947 946 Skeletal vibration involving α-1,4 linkage

1030 1028 υasC-O-C α-glycoside bridge 1080 1080 υC-C 1155 1153 υC-OH

- 1385 NO3-

1635 1628 υC=O (COO-) 2924 2924 υasC-H 3423 3406 υO-H

υ: stretching vibration, υas: asymmetric stretching vibration

400 800 1200 1600 2000 2400 2800 3200 3600 4000

% T

ran

smis

sion

Wavenumber, cm-1

10301080

1155947

1385582 10801028

11532924

2924

586

3423

3406

946

(a)

(b)

1635

1628

Chapter: 7

161

To further verify the findings from the FTIR results, XPS studies of the CMCD-

MNP(C) before and after Cu2+ adsorption are conducted. Figure 7-9 shows the high

resolution spectra of the C1s and O1s regions. Deconvolution of the C1s peak produces

four peaks of binding energies 284.6, 286.2, 287.9 and 288.7eV, respectively (before

and after Cu2+ adsorption) (Figure 7-9a and 7-9b). These peaks can be assigned to the

carbon atoms in the forms of C–C (aromatic), C–O (alcoholic hydroxyl and ether), C=O

(carbonyl) and COO- (carboxyl and ester) [264]. However, C1s XPS spectra do not

clearly show any significant changes of the CMCD-MNP(C) before and after Cu2+

adsorption which indicates that C atoms are not involved in chemical adsorption of

Cu2+ ions.

Deconvolution of the O1s peak yields three individual component peaks which come

from different groups and overlap on each other (Figure 7-9c and 7-9d). Peaks at 529.8

and 530.6eV (before and after Cu2+ adsorption) are attributable to the C=O functional

groups [264]. Peaks at 530.7 and 532.5eV (observed before Cu2+ adsorption) and at

532.0 and 533.7eV (observed after Cu2+ adsorption) are attributable to the C-O groups

(alcoholic hydroxyl and ether) and COO- groups, respectively. The shift in the binding

energies after Cu2+ adsorption is likely to be caused by the binding of copper ions onto

the oxygen atoms and thus reduces its electron density. Similar observation has been

reported by Lim et al. [263] and Zheng et al. [264]. The relative content of oxygen in

C–O (alcoholic hydroxyl and ether) is reduced (from 39.2% to 33.3%) and that in form

of C=O increases (from 32.7% to 46.5%) after copper adsorption. These changes,

together with binding energy shift, suggest that oxygen atoms are the main adsorption

sites and the Cu2+–O coordinate bonds are formed in Cu2+ adsorption by CMCD-

MNP(C), which is in good agreement with FTIR results.

Chapter: 7

162

Figure 7-9 XPS spectra of CMCD-MNP(C): (a) C1s before adsorption; (b) C1s after adsorption; (c) O1s before adsorption; and (d) O1s after adsorption; (e) Cu(2p3/2) core-level spectrum of Cu-loaded CMCD-MNPs.

Figure 7-9e shows the Cu(2p3/2) core-level spectrum of the CMCD-MNP(C) with

adsorbed copper ions at pH 6.0. The binding energy of the copper at 935.1eV and the

presence of a satellite band nearby is representative of the oxidation state (+2) of copper

Chapter: 7

163

for the Cu(2p3/2) orbital [265,266]. This indicates the existence of Cu (i.e., Cu2+) being

adsorbed on the surface of the magnetic nano-adsorbents.

7.3.4 Desorption and regeneration studies

From practical point of view, repeated availability is a crucial factor for an advanced

adsorbent [155,255,267]. Such adsorbent has higher adsorption capability as well as

better desorption property which will reduce the overall cost for the adsorbent. To

evaluate the possibility of regeneration and reusability of CMCD-MNP(C) as an

adsorbent, we performed the desorption experiments. Desorption of Cu2+ from CMCD-

MNP(C) were demonstrated using three different eluent buffers, namely 0.1M citric

acid, 0.1M Na2EDTA and 0.1M acetic acid. Below pH 2, CMCD-MNP(C) tends to be

unstable and have a tendency to disintegrate. It is found that the quantitative desorption

efficiencies using citric acid, Na2EDTA and acetic acid were 96.2, 89.4 and 66.8%,

respectively. It is also noted that no obvious Fe ions were observed in the desorbent

solutions after desorption, an indication that there was no dissolution of Fe3O4

nanoadsorbents during the desorption process.

The reusability was checked by following the adsorption–desorption process for three

cycles and the adsorption efficiency in each cycle was analyzed. In each cycle, 120 mg

of CMCD-MNP(C) and 10 ml of an initial Cu2+ concentration of 200 mg/l was used for

the adsorption process, and 10 ml of 0.1M citric acid was used as desorption eluent. The

desorption kinetics of Cu2+ from Cu-loaded nanoparticles and the adsorption and

desorption capacity of CMCD-MNP(C) is presented in Figure 7-10. It is observed that

the desorption reaches equilibrium within 1 h using 0.1 M citric acid solution as

desorption eluent. Cu2+ adsorption capacity of CMCD-MNP(C) remains almost

constant for the three cycles, indicating that there are no irreversible sites on the surface

of CMCD-MNP(C). Recently Hu et al. [242] reported that β-cyclodextrin grafted

Chapter: 7

164

multiwalled carbon nanotubes/iron oxides (MWCNTs/iron oxides/CD) can be recycled

five times for Pb(II) adsorption with less than 5% of decline in efficiency, indicating

good reusability of the adsorbents. Our recyclability studies suggest that theses nano-

adsorbents can be repeatedly used as efficient adsorbents in wastewater treatment.

Figure 7-10 (a) Kinetics of desorption of Cu2+ from Cu-loaded CMCD-MNP(C) in 0.1M citric acid solution; (b) performance of CMCD-MNPs by multiple cycles of regeneration.

7.4 Conclusions

In this study, we have successfully synthesized a magnetic novel nano-adsorbent

comprising Fe3O4 nanoparticles modified with CM-β-CD having magnetic properties

0

20

40

60

80

100

0 20 40 60 80 100 120 140 160

% d

esor

pti

on

Time (min)

(a)

0

10

20

30

40

1 2 3

q e(m

g/g)

No. of Cycles

Adsorption Desorption(b)

Chapter: 7

165

exhibited by magnetite and adsorption properties of CM-β-CD. Grafting of CM-β-CD

onto the magnetic nano-adsorbents is confirmed by FTIR, TGA, and XPS analyses.

These magnetic nano-adsorbents were used to effectively remove Cu2+ from aqueous

solution. The solution pH greatly affects the adsorption of Cu2+ onto CMCD-MNP(C).

Adsorption of Cu2+ reaches equilibrium within 30 min and 90% of Cu2+ is being

adsorbed at 15 min. The kinetics of Cu2+ adsorption follows the pseudo-second-order

model. The equilibrium data are fitted well by the Langmuir model of adsorption. The

maximum adsorption capacity of CMCD-MNP(C), determined to be 47.2 mg/g, is

considerably higher than that of some other reported magnetic adsorbents. Furthermore,

the adsorption process is spontaneous and exothermic in nature. Both FTIR and XPS

analyses clearly reveal that the oxygen atoms on CMCD-MNP(C) are the main binding

sites for Cu2+ to form surface-complexes. In addition, the copper ions were desorbed

effectively from CMCD-MNP(C) by treatment with citric acid and Na2EDTA solution.

Results of this work suggest that the CMCD-MNP(C) may be promising adsorbents for

the removal of heavy metal ions from wastewater using the technology of magnetic

separation.

Chapter: 8

166

Chapter 8 Multifunctional core-shell silica nanoparticles with magnetic, fluorescence, specific cell targeting and drug-inclusion functionalities

8.1 Introduction

In recent years, the advent of multifunctional nanomaterials attracted enormous interest

of biomedical research community [268]. Rational combination of diverse building

blocks such as metallic or semiconductor nanomotifs, organic molecules/polymers,

biomolecules give rise to composite nanomaterials which exhibit a wide range of

diagnostic and therapeutic capabilities; bioimaging, specific cell targeting, drug storage

and controlled drug release etc [269,270]. Magnetic nanoparticles evidently are

becoming prominent inorganic building blocks of such multifunctional nanocomposites.

Because, colloidal magnetic nanoparticles (MNPs), typically iron oxide (Fe3O4), exhibit

superparamagnetism which can respond to external magnetic field. The fact that these

superparamagnetic materials do not retain any magnetization after removal of the

external magnetic field, and thus avoid aggregation, is a clear advantage for in vivo

applications [271]. MNPs can be magnetically guided inside a biological system, e.g,

artery or tumor by an external magnetic field [272] and tracked by magnetic resonance

imaging (MRI) [273]. Moreover, a metal/semiconductor/polymer coating on the

magnetic particles enhances their stability, biocompatibility, as well as expands

applicability by integrating more functionalities via physical and/or chemical methods.

A shell of silica around MNPs, known as Fe3O4@SiO2 core-shell, constitute the basic

architecture of most multifunctional nanocomposites. Because silica has low-toxicity,

less prone to degradation in a biological environment, plus numerous chemical methods

exist to functionalize silica shell with molecular, biomolecular and polymeric ligands

for biomedical purposes [274,275]. To date most of the magnetic core-shell

Chapter: 8

167

nanocomposites exhibit typically two or a maximum of three functionalities [275,276]

by combining the magnetic core a fluorescent label [277,278] or quantum dot

[279,280], cell targeting ligand [278,280,281] or drugs [282], however little attempt has

been made to incorporate four functionalities: magnetism, fluorescence, selective cell-

targeting and drug storage-delivery in one single platform was made [283,284]. We

present a novel core-shell nanocomposite which has such quadruple of functionalities

[36].

We rationally design the bottom-up synthesis of a core-shell nanocomposite by

stepwise coating of Fe3O4 magnetic crystal (~10 nm) with layers of silica shells for

colloidal protection, dye-doping and molecular fictionalization (Figure 8-1).

Microemulsion sol-gel chemistry was opted to construct the first layer of silica gel to

obtain Fe3O4@SiO2 core-shell structure. We then overlay a Fluorescent dye-conjugated

(FITC) silica layer to optically track the nanocomposite for any cellular uptake by

fluorescence imaging. FITC is chemically encapsulated into the silica shell to enhance

photochemical stability [270] and fluorescence intensity due to “caging effects” [285].

Being both magnetic and fluorescently labeled these materials have the potential for

bimodal imaging by optical and magnetic resonance means [284,286,287].

For a nanoparticulate delivery device for selective drug delivery to specific sites, the

challenge lies in successfully combining therapeutic and targeting functionalities along

with imaging capability into one single nano-system. To this end, the outermost layer of

silica is furnished with a cancer cell targeting ligand and an interesting ‘molecular cage’

for drug storage. We covalently conjugated folic acid to the amino-termini on the outer

silica shell. Folic acid or folate (FA) has emerged as an attractive cancer-targeting

ligand because cancerous cells overexpress high-affinity FA receptors (FARs) [288].

Chapter: 8

168

Thus FA-conjugated nanomaterials show preference towards malignant cells than the

healthy ones.

In order to armor our nanocomposite with a drug storage and delivery vehicle we finally

conjugated β-cyclodextrin molecules onto the shell. In the magnetic core-shell

literature, mesopores in the silica shell are used as the physical reservoir for storing

drug molecules [36,284]. However, in most cases these magnetic-mesoporous silica

nanocomposites can carry only hydrophilic drugs, because the mesopores of silica can

accommodate only water-soluble drug molecules [289-291]. Since the majority of

potent anticancer drugs are water-insoluble, applications of silica nanoparticles for

anticancer drugs delivery have been largely restricted. Unlike physically entrapping

drug molecules into the mesopores of silica, we relied on the supramolecular inclusion

of drug molecules into the doughnut-shaped β-cyclodextrin (β-CD) cavity. β-CD

molecule has a hydrophilic exterior while the internal cavity is sufficiently hydrophobic

to host water-insoluble drug molecules [292,293]. As a drug carrier, β-CD not only

facilitates solubilization, stabilization, and transport of hydrophobic drugs but also

minimizes unwanted side effects associated with the corresponding drugs

[60,117,294,295]. Several studies have been directed towards constructing MNP-

cyclodextrin nanocomposites as magnetically guided drug carrier [202,296]. However,

the agglomeration of these as-synthesized magnetic nanocarriers as evidenced by TEM

may limit their applications in biomedical research fields.

The feasibility of our magnetic nanocomposite as an optically trackable, cancer-cell

targeting and delivery vehicle for anticancer drugs is evaluated by investigating their

capability in discriminating between cancerous and healthy cells, cellular uptake,

fluorescent imaging, formation of hydrophobic drug-inclusion complexes and the in

vitro release profile using all-trans-retinoic acid (RA) as a model hydrophobic drug.

Chapter: 8

169

After combining superparamagnetic, luminescent, specific cell-targeting and

hydrophobic drug-encapsulation properties, these multi-functional nanoparticles pose

great potentials as a smart nano ‘Swiss army knife’ in diverse theranostic applications

[297,298].

8.2 Experimental 8.2.1 Materials

Iron (II) chloride tetrahydrate (99%), Iron (III) chloride hexahydrate (98%), 3-

aminopropyltriethoxysilane (APTS) (98+%), all-trans-retinoic acid (RA) (98%),

chloroacetic acid (99%) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

hydrochloride (EDC) was purchased from Alfa Aesar. Triton X-100 and N-

hydroxysuccinimide (NHS) (98%) were purchased from Sigma-Aldrich. Ammonium

hydroxide (25%) was purchased from Merck (USA). 1-hexanol (>98%), oleic acid

(99%) and tetraethyl orthosilicate (TEOS) (99%) was purchased from Fluka. β-

cyclodextrin hydrate (99%) was obtained from Sinopharm Chemicals. Folate free

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and 3-[4,5-

dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide were purchased from Sigma-

Aldrich. All other chemicals were used purchased from either Sigma-Aldrich or Fisher

Scientific and received without further purification. All the experiments were

performed in deionized Milli-Q water.

8.2.2 Synthesis of multifunctional core-shell silica magnetic nanoparticles

8.2.2.1 Preparation of oleic acid coated Fe3O4 MNPs (OA-MNPs)

Oleic acid stabilized Fe3O4 MNPs were prepared according to the method described by

Lopez-Lopez et al. [299]. Briefly, 3.04 g FeCl3.6H2O and 1.26 g FeCl2.4H2O were

dissolved in 50 ml DI water under nitrogen and vigorous stirring with ultrasonification

Chapter: 8

170

for 10 min. 0.8 ml of oleic acid was then added immediately after 8 ml ammonium

hydroxide (NH4OH, 25 wt%) had been added to the solution, and the resulting emulsion

was stirred vigorously for 1 h at 25ºC. Then the solution was heated up to 95 ºC to

convert iron hydroxides into magnetite. As soon as that temperature was reached, the

suspension was cooled to room temperature. The mixture was then acidified to pH 5–6

with HNO3. The resulting particles were collected by magnetic decantation, washed 4-5

times with DI water to remove the unreacted reactants, then washed with ethanol 3

times to remove water and residual oleic acid. Finally the particles were dried in

vacuum oven for 3 h.

8.2.2.2 Synthesis of Fe3O4@SiO2(FTIC) NPs

Typically, 12 mg of dried magnetic Fe3O4 powder was dissolved first in 77 ml

cyclohexane and then 20 g triton X-100, 16 ml hexanol, and 3.4 mL H2O were added

with intense stirring to generate the microemulsion system [34]. 0.4 ml of tetraethyl

orthosilicate (TEOS) was added to this mixture. After 6 h of stirring, 1.2 ml aqueous

ammonia (25 wt %) was introduced drop wisely to initiate the TEOS hydrolysis. The

microemulsion was kept stirring for another 24 h, and then N-1-(3-

triethoxysilylpropyl)-N’-fluoresceyl thiourea (FITC-APTS) ethanolic solution and

TEOS (0.3 ml) was added to the microemulsion. FITC-APTS conjugate was prepared

by adding 1.25 mg FITC with 0.15 ml APTS in 0.2 ml absolute alcohol and stirring in

dark for 24 h in room temperature. For FITC-APTS conjugate synthesis, an excess

amount of APTS (molar ratio of APTS to FITC =20) was used to ensure complete

conversion of FITC to FITC-APTS conjugates. After stirring in dark for 24 h at room

temperature, ethanol was added to destabilize the microemulsion system. The resulting

MNPs were collected by magnetic separation and washed 5 times by anhydrous ethanol

Chapter: 8

171

and then by deionized water to remove surfactant and unreacted reactants and dried by

air.

8.2.2.3 Synthesis of Fe3O4@SiO2(FTIC)-FA/NH2 NPs

To attach folic acid (FA) to the Fe3O4@SiO2(FTIC) MNPs surface, FA-APTS

conjugate was prepared first. In a flask, 2 mg of folic acid and 1 µL of APTS were

mixed in 20 ml of DMSO. Next, 0.53 mg of N-hydroxysuccinimide (NHS) and 2.35 mg

of N,N'-dicyclohexylcarbodiimide (DDC) (FA: NHS: DCC = 1: 1: 2.5) were added into

the mixture and stirred for 2 h. In a separate flask containing 80 ml of toluene and the

Fe3O4@SiO2(FTIC) MNPs (50 mg) in DMSO suspension, the FA-APTS conjugate and

free APTS (7 µL) (total APTS/FA molar ratio = 8) solution was added, and the mixture

was stirred for 20 h at room temperature. The materials were recovered by magnetic

separation, washed twice with toluene, then ethanol and dried under vacuum.

8.2.2.4 Synthesis of Fe3O4@SiO2(FTIC)-FA/CMCD NPs

CM-β-CD was grafted onto Fe3O4@SiO2(FITC)-FA/NH2 NPs in the presence of EDC

and NHS. Detailed synthetic procedure for CM-β-CD is given in Chapter 5 (Section

5.2.2). A solution of CM-β-CD (0.65 g, 0.5 mmol) in 20 ml of distilled water was first

activated with 0.96 g (0.5 mmol) of EDC and 0.575 g (0.5 mmol) of NHS for 30 min.

With this mixture, 100 mg of dried Fe3O4@SiO2(FITC)-FA/NH2 was added and the pH

of the reaction mixture was adjusted to 6~7. The reaction was carried out at room

temperature under constant stirring. After 24 h, the product formed was separated by

magnetic decantation and dried by vacuum oven.

Chapter: 8

172

8.2.3 Cell culture and intracellular uptake study The HeLa cells were grown in DMEM (Dulbecco’s modified Eagle’s medium)

supplemented with 10% FBS (fetal bovine serum) at 37oC and 5% CO2. The MCF-7

cells were grown in DMEM supplemented with 10% FBS and 1% insulin (10 ml: 400

U) at 37oC and 5% CO2. Cells (5 x108/ L) were plated on 14 mm glass cover slips and

allowed to adhere for 24 h. Subsequently, after washing with phosphate buffer solution

(PBS), the cells were incubated in PBS buffer containing 50 µg/ml Fe3O4@SiO2(FTIC)-

FA/CMCD MNPs at 37oC for 1 h under 5% CO2, and then washed with PBS

sufficiently to remove excess nanoparticles. Nanoparticle uptake by HeLa and MCF-7

cells was studied by confocal microscopy methods. Confocal microscopy images of the

cells were acquired with a 488 nm laser on a Leica SP2 confocal laser scanning

microscope.

8.2.4 Cytotoxicity assay

In vitro cytotoxicity was evaluated by performing methyl thiazolyl tetrazolium (MTT)

assays on the HeLa cells. Cells were seeded into a 96-well cell culture plate at 5x104

per well in DMEM with 10% FBS at 37oC and 5% CO2 for 24 h; then the cells were

incubated with Fe3O4@SiO2(FTIC)-FA/CMCD NPs with different concentrations (0, 1,

10, 20, 30, 50 µg/ml diluted in DMEM) for 6 h or 24 h at 37oC under 5% CO2.

Untreated wells were used as control. Thereafter, MTT (10 µl, 5 mg/ml) was added to

each well, and the plate was incubated for 4 h at 37oC. The assays were carried out

according to the manufacturer’s instructions. The OD570 value (Abs.) of each well,

with background subtraction at 690 nm was measured by microplate reader (GENios,

Tecan, Switzerland).

Chapter: 8

173

8.2.5 Inclusion/adsorption of retinoic acid The inclusion of retinoic acid was performed by immersing precisely weighed amount

of Fe3O4@SiO2(FTIC)-FA/CMCD MNPs (about 0.12 g) in 10 ml methanol solution of

retinoic acid (0.2 mg/ml concentrations), and stirred with a thermostatic water-bath

shaker (200 rpm) for 12 h at room temperature. The magnetic nanoparticles were then

removed magnetically from the solution. The concentration of retinoic acid solution

was determined by using a spectrophotometer at 341 nm. The amount of included

retinoic acid (A) by Fe3O4@SiO2(FTIC)-FA/CMCD MNPs was calculated as follows:

A = V (C0–C1) (8-1)

where V is the volume of RA solution (ml), C0 is the initial concentration of RA

(mg/ml), C1 is the concentration of RA solution after inclusion or absorption (mg/ml).

8.2.6 Release study The drug release experiment was performed in an Erlenmeyer flask at 37oC in PBS

buffer (pH 7.4). 120 mg of Fe3O4@SiO2(FITC)-FA/CMCD NPs containing a known

amount of retinoic acid were suspended in 250 ml of release medium, stirred at 150 rpm

in a thermostatic water-bath heater maintained at 37oC. 5 ml of samples were

periodically removed and assayed. The volume of each sample withdrawn was replaced

by the same volume of fresh medium. The amount of released retinoic acid was

determined at 341 nm using a UV-Visible spectrophotometer. The drug release study

was performed in duplicate for each of the samples. To prevent decomposition of RA

during the release experiment, the samples were kept in dark.

Chapter: 8

174

8.3 Results and discussions

8.3.1 Synthesis and characterization of as-synthesized magnetic nanoparticles

The synthetic route for multifunctional core-shell magnetic nanoparticles

[Fe3O4@SiO2(FITC)-FA/CMCD NPs] is outlined in Figure 8-1. Fluorescently-doped

core-shell structure Fe3O4@SiO2(FITC) was obtained by a one-pot two-step method. In

the first step, oleic-acid-stabilized Fe3O4 MNPs was coated with a layer of silica to

make the basis of the core-shell structure. Fe3O4 MNPs were dispersed in a water-in-oil

(cyclohexane-hexanol) microemulsion prior to the sol-gel formation of the silica layer

onto the magnetite surface. In the second step, this emulsion was further treated with a

mixture of FITC-APTS conjugate and free APTS overlay thin FITC-doped silica on the

primary shell. The purpose of the primary silica shell is to protect the core Fe3O4 MNP.

As the dye molecules are incorporated into the second silica layer, any possibility of

FITC degradation or fluorescence quenching by Fe3O4 is minimized. Secondly,

encasing FITC within a thin layer of condensed APTS-layer would provide

photostability and additional chemical protection in the cellular environment. The

outermost layer of Fe3O4@SiO2(FITC) was then decorated with a cancer targeting

ligand, folate by silanization with folic acid-APTS conjugate. A fair excess of APTS

(molar ratio of APTS to FA = 10) was used to ensures sufficient free amino groups

dangling out from the outer surface for further functionalization. CM-β-CD, the

molecular host from hydrophobic drugs was attached onto the outermost surface via

amide bond formation between the carboxylic groups of CM-β-CD with free amino

groups.

Chapter: 8

175

Figure 8-1 Synthesis of CM-β-CD conjugated fluorescein-doped magnetic mesoporous silica nanoparticles [Fe3O4@SiO2(FITC)-FA/CMCD NPs].

Physical and chemical features of the core-shell multifunctional particles are

investigated by several analytical methods. The XRD pattern (Figure B-1 in Appendix

B) confirmed the existence of Fe3O4, and a broad peak ranging from 20˚ to 30˚ was

assigned to the amorphous silica. This indicates that silica is successfully coated onto

the Fe3O4 MNP surfaces. However, silica shell did not result in the phase change of

Fe3O4 nanoparticles. Further investigation with the FTIR spectra confirms the presence

of the silica shell and also the incorporation of APES-FITC conjugate in the silica

matrix (Figure B-2 in Appendix B). The sample exhibits a strong broad band at around

1090 cm-1 corresponding to the Si-O-Si asymmetric stretching vibration and a weak

band at 956 cm-1 associated to Si–OH vibration [288]. The bands at 1630 cm-1 and 1560

cm-1 are the bendings of N–H, characteristic of the presence of NH2 group. The peak at

Chapter: 8

176

2938 cm-1 due to the C–H stretching vibrations of alkyl groups. The magnetization

curve of Fe3O4@SiO2(FITC)-FA/CMCD NPs measured at 300 K exhibits no hysteresis

loop, which means that these multifunctional nanocomposites retain the

superparamagnetic property (Figure B-3 in Appendix B).

The successful attachment of fluorescent probe molecule (FITC) and cell-targeting

ligand (folate) into/onto the silica shell was investigated by UV-Vis spectroscopic

analyses (Figure 8-2). UV-vis absorption spectra of all the samples were measured in

aqueous medium. Fe3O4@SiO2 nanocomposites do not show any prominent absorption

peaks. Characteristic absorption bands for FITC and folate, at 488 and 280 nm,

respectively, are clearly visible in the case of Fe3O4@SiO2(FITC)-FA/NH2 NPs and are

not blue/red shifted due to the integration inside/on a silica matrix (when compared

with UV absorptions for free FITC and FA). Since FITC-APES conjugate was

covalently linked to the silica matrix, no dye leaking was observed in an aqueous

suspension.

Figure 8-2 UV–vis spectra of (a) pure FITC, (b) pure folic acid, (c) Fe3O4@SiO2, (d) Fe3O4@SiO2(FITC), and (e) Fe3O4@SiO2(FITC)–FA/NH2 NPs in water.

Chapter: 8

177

The incorporation of FITC dye in Fe3O4@SiO2(FITC)-FA/CMCD NPs enabled the

nanoprobes with excellent fluorescent property in water (Figure 8-3). The emission

spectrum of the dye-doped multifunctional NPs shows a broad band ranging from 490

to 580 nm with a maxima at 515 nm. Fe3O4@SiO2(FITC)-FA/CMCD nanoprobes and

pure FITC dye in water have similar excitation and emission spectra (not shown here),

so the fluorescent characteristics of FITC dye did not alter after incorporation into a

silica-shell. The spectral properties (excitation and emission spectra) of

Fe3O4@SiO2(FITC)-FA/CMCD NPs were comparable with FITC dye-doped magnetic

silica nanoparticles in the literature [270,284]. Inset picture in Figure 8-3 shows

dispersions of these nanoparticles under room light and under the 365 nm UV

excitation. Clearly, these core-shell MNPs are highly dispersible in water and emit

strong green fluorescence under 365 nm UV excitation. Facile magnetic separation is

evident from its magnetic responsivity towards a permanent NdFeB magnet. Moreover,

after being harvested by an external magnet, absence of any fluorescence in the aqueous

part confirms that FITC is robustly embedded into the silica shell by chemical bonding

and does not leach out.

Chapter: 8

178

Figure 8-3 Excitation (dashed line, recorded at 515 nm emission) and emission (solid line, recorded at 480 nm excitation) spectra of Fe3O4@SiO2(FITC)-FA/CMCD NPs dispersed in water. Insets: visual photographs of (a) Fe3O4@SiO2(FITC) NPs dispersion in water under room light; (b) the green emission of these MNPs in water and (c) in response to a NdFeB magnet under the 365 nm excitation portable UV lamp.

High resolution TEM images of as-prepared multifunctional Fe3O4@SiO2(FITC)-

FA/CMCD NPs show that highly monodispersed particles with mean diameter 70 nm

(silica shell thickness = 30 nm) are formed (Figure 8-4). Moreover, a thin ring on the

outer surface can be observed which may be due to the surface organic group [34]. For

each core-shell particle a singular oleic acid coated Fe3O4 particle, rather than a cluster,

is encased within a thick layer of silica shell. Oleic acid modified bare Fe3O4 MNP

nanoparticles have an average diameter of 9.5 nm. The synthesized highly

monodispersed core-shell silica nanoparticles is achieved by microemulsion-based sol-

gel growth of silica in limited domain of water in the water-in-oil microemulsion. It can

be assumed that aqueous microdroplets in the microemulsion are monodisperse in size

and contain a single Fe3O4 MNP particle around which a well-defined shell of silica is

grown through hydrolysis of tetraethyl orthosilicate (TEOS) and subsequent

condensation of silica onto the surface of Fe3O4 cores. The silica layer can be tuned by

Chapter: 8

179

varying the ratio of water to surfactant (Figure B-4 in Appendix B). A thick silica shell

was preferred in this case since a thicker silica shell allowed high payloads of dye

molecules to be achieved. The uniformity of as-synthesized particles could be attributed

to the microemulsion method, which allows better control over the particle size and size

distribution.

Figure 8-4 TEM mages of (a) OA-MNPs, (b) Fe3O4@SiO2(FITC) NPs and (c) Fe3O4@SiO2(FITC)-FA/CMCD NPs.

The stepwise conjugation of functional groups on the silica surface was monitored by

measuring the surface charges and surface chemistry at different stages of synthesis

(Figure 8-1). As shown in Figure 8-5A, a negative zeta potential (ξ) value of -27 mV (at

pH 7.4) of dye-doped silica particles [Fe3O4@SiO2(FITC) NPs], can be attributed to the

presence of ionizable silanol groups of surface silica and acid functionalities (COOH

and phenolic-OH) of FITC molecules [300]. After modification with APTS/FA

conjugate, the ξ value of Fe3O4@SiO2(FITC)-FA/NH2 nanoparticles reverts to positive

value (ξ ~ +4.0 mV at pH 7.4) due to the presence of excess amount of amino groups

which exist mainly as NH3+. Folic acid (FA) is expected to furnish silica surface with

negative charge, however excess amount of NH2 groups presumable nullify it, making

the overall surface charge slightly positive. Further modification of the surface by

conjugating cyclodextrin groups is manifested in the reversal of surface charge back to

Chapter: 8

180

a negative value -20 mV attributable to the high ionizability of CM-β-CD (pKa< 4)

groups. As a result, Fe3O4@SiO2(FITC)-FA/CMCD NPs were observed to be well

dispersed in aqueous medium without any visible aggregation (insets of Figure 8-3).

Good aqueous dispersibility and colloidal stability of the nanoparticles are essential for

their biological applications. Dynamic light scattering (DLS) analyses suggest long-

term colloidal stability over a few day time and narrow size distribution (hydrodynamic

diameter = ca. 125 nm and polydispersity index = 0.20) (Figure B-5 in Appendix B).

Figure 8-5 (A) Zeta potentials of as-synthesized nanoparticles at pH 7.4; (B) TGA curves of (a) Fe3O4@SiO2(FITC)-FA/NH2 and (b) Fe3O4@SiO2(FITC)-FA/CMCD MNPs.

Chapter: 8

181

High density grafting of cyclodextrin molecules onto the outermost silica surface is

desirable as these are the host for hydrophobic drug molecules. TGA curves (25–800oC)

in Figure 8-5B shows the weight loss due to organics decomposition in both

Fe3O4@SiO2(FITC)-FA/NH2 and Fe3O4@SiO2(FITC)-FA/CMCD NPs; a comparison

of two TGA curves revealed an increased weight loss for Fe3O4@SiO2(FITC)-

FA/CMCD NPs, presumably due to the presence of substantial amount of CM-β-CD.

The amount of CM-β-CD grafted on the surface was calculated to be 0.034 mmol/g.

The average values of surface concentration obtained from XPS spectra of

Fe3O4@SiO2(FITC), Fe3O4@SiO2(FITC)-FA/NH2 and Fe3O4@SiO2(FITC)-FA/CMCD

are presented in Table 8-1. The elemental composition percentage of Si and N atom was

34.95% & 2.48% on the surface of Fe3O4@SiO2(FITC)-FA/NH2 and was 29.57% &

2.03% on the surface of Fe3O4@SiO2(FITC)-CMCD, respectively. The decreased

percentages may be attributed to the lack of Si and N in CM-β-CD moiety. In addition,

percentage of C atom was observed to increase from 16.8% to 23.3% after conjugation

of CM-β-CD. Therefore, XPS analysis together with the results of TGA confirmed the

successful grafting of CM-β-CD on the multifunctional magnetic surface.

Table 8-1 Surface composition of Fe3O4@SiO2(FITC), Fe3O4@SiO2(FITC)-FA/NH2 and Fe3O4@SiO2(FITC)-FA/CMCD nanoparticles obtained from XPS spectra.

Samples Surface elemental composition percentage (%)

C 1s O 1s Si 2p N 1s Fe3O4@SiO2(FITC) 15.62 46.90 36.46 1.02 Fe3O4@SiO2(FITC)-FA/NH2 16.80 45.76 34.95 2.48 Fe3O4@SiO2(FITC)-FA/CMCD 23.23 45.17 29.57 2.03

Chapter: 8

182

8.3.2 Cytotoxicity assay After successful synthesis and full physical and chemical characterizations biomedical

relevance of these multifunctional core-shell particles was investigated. The

cytotoxicity of the Fe3O4@SiO2(FITC)-FA/CMCD MNPs was evaluated using an MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with HeLa cells,

a human cervical carcinoma cell line. The concentration-dependent effect of

Fe3O4@SiO2(FITC)-FA/CMCD MNPs on the cell viability at 6 and 24 h was

determined (Figure 8-6). It is clear that the high cell viability can still be attained even

at a MNP concentration of 50 µg/ml. This indicates that the Fe3O4@SiO2(FITC)-

FA/CMCD MNPs is biocompatible at MNP concentrations up to 50 µg/ml.

Figure 8-6 In vitro cell viability of HeLa cells incubated with Fe3O4@SiO2(FITC)-FA/CMCD MNPs at different concentrations for 6 and 24 h at 37oC.

8.3.3 Confocal microscopy observations We carried out fluorescence imaging of Fe3O4@SiO2(FITC)-FA/CMCD NPs by for

their ability to discriminate between healthy and cancerous cells and cellular uptake.

After 1 hr incubation of HeLa and MCF-7 cells with MNPs at 37oC, cells were

Chapter: 8

183

observed using confocal laser scanning microscope (CLSM) (Figure 8-7).

Corresponding bright field images of the cells were also recorded. A clear preference

for the cancerous cells (HeLa) by these MNPs over the healthy cells (MCF-7) is

demonstrated in Figure 8-7. Cytoplasm of all the HeLa cells (Figure 8-7A) is highly

fluorescent due to the presence of FITC-tagged MNPs, while most of MCF-7 cells are

either less or non-invaded by MNPs (by comparing dark and bright field images) under

similar conditions. This suggests that the Fe3O4@SiO2(FITC)-FA/CMCD NPs were

preferentially targeted to the cancer cells and were internalized, presumably by folate

receptor mediated endocytosis [281,288]. These results suggest that

Fe3O4@SiO2(FITC)-FA/CMCD NPs could be used for simultaneous targeting and

imaging of cancerous cells.

Figure 8-7 CLSM images of cells incubated with Fe3O4@SiO2(FITC)-FA/CMCD NPs at 37oC: (A) HeLa cells (FA-positive) and (C) MCF-7 cells (FA-negative). Bright field images of HeLa cells (B) and MCF-7 cells (D) were also supplied. The nanoparticle concentration is 50 µg/ml and the incubation time is 1 h.

Chapter: 8

184

8.3.4 Drug inclusion/adsorption and release studies

Since Fe3O4@SiO2(FITC)-FA/CMCD NPs are capable of preferentially target and

invade cancerous cells, use of these particles as an anti-cancer drug carrier would be

ideal for tumor-specific drug delivery. In this direction, we investigated drug loading

capacity of these MNPs which are already decorated with well-known molecular drug

carrier: β-cyclodextrin. We chose Retinoic acid (RA) as a model hydrophobic anti-

cancer drug. In order to get an idea about supramolecular interactions between RA and

free CM-β-CD molecules in solution, host-guest type complex formation was

investigated by UV-vis spectroscopy. Figure 8-8 shows the UV-vis spectra of RA

(7.3x10–6 mol l–1) in 10 vol% methanol solutions (pH 7.4) containing various

concentrations of CM-β-CD. Previous studies showed that methanol even up to 20

vol% does not influence the inclusion of cyclodextrin with other guest molecules [301].

The UV absorption peak is shifted to shorter wavelengths with an increase in the CM-β-

CD concentrations, accompanied by the increase in absorbance. This indicates the

formation of the RA/CM-β-CD inclusion complexes. Similar inclusion behaviors of

cyclodextrin with various drugs by UV-Vis spectroscopy have been reported in

literature [302,303].

Chapter: 8

185

Figure 8-8 (a) Absorption spectra of RA (7.3x10–6 mol l–1) in pH 7.4 buffers containing various concentrations of CM-β-CD. Concentration of CM-β-CD: (1) 0, (2) 7.5x10–5, (3) 1.1x10–4, (4) 1.6x10–4, (5) 2.3x10–4, (6) 5.6x10–4 and (7) 3.75x10–3 mol l–1. Inset: Double reciprocal plot for RA complexes with CM-β-CD.

The inclusion constants of complexes are estimated according to the double-reciprocal

method as in Eq 8-2.

0][

111

CDKA

(8-2)

where ∆A denotes the difference of absorption of guest molecule in the presence and

absence of CDs. α is a constant, and [CD]0 denotes the concentration of CDs. K can be

calculated from a plot of 1/∆A vs. 1/[CD]. Inset of Figure 8-8 shows the double

reciprocal plots of 1/∆A versus 1/[CD] for RA with CM-β-CD at pH 7.4. The plot

exhibits good linearity (the linear regression coefficient R2=0.998). This verifies the

formation of inclusion complexes with a stoichiometry of 1:1 between RA and CM-β-

CD. The relative inclusion constant of CM-β-CD with RA is estimated to be 779 M-1.

The lipophilic character of RA provides the main driving force for their inclusion into

Chapter: 8

186

the apolar CDs cavities. Literature values for CD-drug inclusion constant varies from

100 to 10,000 M−1 [304]. For therapeutic applications, a moderate value for K is

desirable that ensures thermodynamic feasibility for drug inclusion in the drug loading

stage, while at the same time does not impede drug release when needed [305].

Figure 8-9 shows the capabilities of Fe3O4@SiO2(FITC)-FA/CMCD and

Fe3O4@SiO2(FITC)-FA/NH2 NPs for RA loading in methanol solution. The effects of

adsorbent dosage on RA adsorption/inclusion were studied. It was obvious that

Fe3O4@SiO2(FITC)-FA/CMCD NPs exhibited a considerable adsorption capability for

RA, but very less retinoic acid loading by Fe3O4@SiO2(FITC)-FA/NH2 MNPs was

observed. This revealed that the CM-β-CD grafted on the Fe3O4@SiO2(FITC)-FA/NH2

NPs could indeed enhance the adsorption of retinoic acid effectively due to its special

hydrophobic cavity structure which acts as a host–guest complex with RA. Maximum

adsorption capacity of these multifunctional nanoparticles with RA was estimated to be

3.0 mg/g which is comparable with previous studies. For example, Banerjee et al. found

in their magnetic nanoparticle assisted drug delivery experiments that the maximum

uptakes were 2.72 mg/g of ketoprofen using CD-citrate-GAMNPs [202], 7.53 mg/g of

RA using CD-GAMNPs [16], 1.68 mg/g of ketoprofen using HCD-GAMNP [203] and

7.53 mg/g of RA using HCD-HMDI-GAMNP [306].

Chapter: 8

187

Figure 8-9 Effects of nanoparticle dosage on adsorption/inclusion of retinoic acid.

We then focused on the drug release profile from RA complexed-Fe3O4@SiO2(FITC)-

FA/CMCD NPs in phosphate buffer saline (pH 7.4) at 37oC. Figure 8-10 shows the

release profiles of RA-loaded Fe3O4@SiO2(FITC)-FA/CMCD NPs (RA loading

capacity of 3.0 µg/mg) as a function of time. The typical release profile was

characterized by an initial rapid release phase in the first 60 min when almost 23% of

the total drug was released. These results suggest that a portion of the total drug–CD

complexes in the nanocomposite are quite labile, whereas the rest apparently more

stable. Apart from Van der Walls interaction between hydrophobic part of RA and CD

cavity, at certain conformational arrangements hydrogen bonding between the polar

functional groups’ of RA and CD and the release of high energy water molecules from

the cavity during the complex formation may contribute to further stabilization for some

RA-CD complexes, contributing to slow down the release process [50]. Thus the

reversible and predictive nature of complex formation with hydrophobic molecules

makes CD to serve as a dual agent of drug loading and unloading in our

nanocomposite. Apart from relying on passive drug release (vide supra), the magnetic

Chapter: 8

188

core can be exploited for magneto-responsive release of the drug from the MNP surface

[290,307] which we envisage as a potential research direction towards a remotely

controllable, sustained-release of drugs from CD-decorated nanocomposites.

Figure 8-10 Release profile of retinoic acid from Fe3O4@SiO2(FITC)-FA/CMCD NPs.

In the present work the release data were fitted to the Ritger–Peppas equation given as

follows [308]:

Mt/M∞ = ktn (8-3)

where Mt/M∞ is the amount of drug released, t is the time of release, k is a kinetic

constant, and n is a diffusion exponent which describes the drug release mechanism.

For spherical metrics, when n ≤0.43, diffusion is the major driving force, normally

termed ‘‘Fickian diffusion”. When n > 0.85 drug release is mainly controlled by

degradation, termed case II transport. When the value of n is between 0.43 and 0.85, the

drug release mechanism is anomalous. Our experimental data showed a fairly good fit

to the Ritger–Peppas equation for RA release as the correlation coefficient (R2) is 0.972.

Chapter: 8

189

The value of the exponent n is 0.26, indicating a Fickian diffusion controlled

mechanism.

8.4 Conclusions

We synthesized highly uniform and monodispersed multifunctional magnetic

nanocomposite that is capable of performing a quadruple of activities: magnetic

manipulation, bioimaging, cell-targeting, drug storing-and-releasing. The synthesis

involves several stages of covalent attachments of functional molecules and ligands

orchestrated in rational way to ensure the chemical robustness. All functional groups

were capable of performing independently being within one single nanosystem. Our

magnetically maneuverable nanocomposite shows to be highly dispersible in aqueous

medium and benign towards healthy cells. Folate ligand leads to preferential

internalization into the target cancerous cells. Fluorescence tag acts as a beacon to know

the whereabouts of the particles within a cell and the cyclodextrin moiety can carry and

release drug pay-load in a biologically relevant environment. All these features are ideal

for a nano Trozan horse for targeted drug delivery and imaging. Magnetic core material

has two other features which could further expand the potency of our nanocomposite:

magnetic resonance would facilitate in vivo imaging (MRI) and magneto-responsive

drug release should enable a remotely controllable and sustained drug-delivery system.

Chapter: 9

190

Chapter 9 Summary and Recommendations

9.1 Summary of findings

The main aim of this thesis work is to present a systematic and comprehensive study on

design, synthesis and characterization of β-cyclodextrin conjugated magnetic

nanoparticles and also their potential applications in the field of bio-/biomedical

applications as well as inorganic/organic pollutants removal from wastewater. To each

technique whether protein refolding, adsorption, it has been demonstrated that addition

of magnetic materials aides to improvisation in efficiency of separations.

Chapter 4 describes the method to synthesize β-CD bonded magnetic nanoparticles

(CD-APES-MNPs) having combined effects of chaperoning function of β-CD and

magnetic properties of iron oxide. These as-synthesized CD-APES-MNPs were

effectively used as solid phase artificial chaperone in carbonic anhydrase (CA)

refolding in vitro. The maximum refolding yields obtained were 85% for thermally

denatured CA which was as the same level as that using liquid-phase artificial

chaperone-assisted refolding and the structures of the refolded CA were obtained as

same as those of native protein. Moreover, these magnetic stripping agents could be

easily separated from the refolded samples using the technology of magnetic separation.

Taking into account the recycling potentials of these magnetic based stripping agents,

with significant cost-cutting capabilities by eliminating further purification steps

associated with the liquid-phase artificial chaperone assisted refolding, these stripping

agents would be very suitable materials for protein refolding.

In Chapter 5, a simple and straightforward synthetic method was developed for the

preparation of CM-β-CD modified APES-Fe3O4 nanoparticles that combine magnetic

Chapter: 9

191

and molecular recognition properties. The adsorption of both adenosine (A) and

guanosine (G) onto CMCD-APES-MNPs was found to be pH dependent and the

adsorption equilibrium data were well-described by Langmuir isotherm. Under the

competitive environment, selective adsorption of G over A occurred at pH 5 onto these

nano-adsorbents. This selectivity was due to the multiple recognition sites of the CM-β-

CD which might give strong hydrophobic interaction and hydrogen bonding towards G.

The results showed that this adsorbent would have wide applications in selective

separation, concentration, and analysis of nucleotides/nucleosides in biological samples.

In Chapter 6 and 7, two novel nanoadsorbents, CMCD-MNP(C) and CMCD-MNP(P)

were synthesized via carbodiimide and precipitation method, respectively. These

adsorbents could effectively be used in methylene blue (MB) and Cu2+ ions removal

from aqueous solution. Surface modification of Fe3O4 nanoparticles by CM-β-CD could

greatly enhance the adsorption capacity of MB and Cu2+ ions due to inclusion behavior

and surface complexation with multiple hydroxyl and carboxyl groups of CM-β-CD,

respectively. The adsorption of MB and Cu2+ ions on these adsorbents was found to be

dependent on pH dependant and initial concentration of these pollutants. Moreover,

these pollutants could be recovered fast and efficiently from the aqueous solution. The

adsorption kinetics and isotherms studies demonstrated that the adsorption process

obeyed the pseudo-second-order kinetics and Langmuir isotherm model, respectively.

The maximum uptakes for MB and Cu2+ ions were 277.8 and 47.2 mg/g, respectively

which are considerably higher than that of some other reported magnetic adsorbents.

The negative values of free energy change indicated the spontaneity of adsorption

process. Moreover, the Cu2+ adsorption process was exothermic in nature. In addition,

desorption and regeneration studies suggest that theses nano-adsorbents could be

Chapter: 9

192

repeatedly used as efficient and suitable adsorbents for the removal of both organic and

inorganic pollutants from wastewater using the technology of magnetic separation.

In Chapter 8, highly uniform and monodispersed multifunctional magnetic

nanocomposites were synthesized that are capable of performing a quadruple of

activities: magnetic manipulation, bioimaging, cell-targeting, drug storing and

releasing. The results of retinoic acid inclusion and release experiments indicate that

this system seems to be a very promising vehicle for the administration of hydrophobic

anticancer drugs. Moreover, these prepared nanoparticles possessing assortment of

functionalities would serve simultaneously in efficient bioimaging, specific cell

targeting, hydrophobic drug storage and controlled release systems.

This study has a lot of implications in the field of bio-/biomedical or environmental

applications. All the properties such as superparamagnetism, inclusion/ molecular

recognition properties found in one nano-system imply a wide variety of applications.

The bifunctional or multifunctional magnetic nanomaterials synthesized in this thesis

work can act as a promising candidate for bio-separation, environmental pollutants

cleanup and controlled drug delivery for biomedical applications.

9.2 Recommendations for future work

This thesis has successfully introduced methods for the development and synthesis of

magnetite/β-CD nanocomposites. Several bio- and environmental applications have

been demonstrated using these as-prepared nanoparticles combining magnetic

responsiveness and supramolecular host-guest behaviors. This thesis work is only a

preliminary study. There are several interesting directions for future work. Based on the

current study and above discussion further work should be carried out in the following

aspects:

Chapter: 9

193

9.2.1 Protein refolding using cyclodextrin conjugated magnetic nanoparticles

(a) In Chapter 4, β-CD conjugated magnetic nanoparticles as stripping agents was used

in only CA refolding. Further work might focus on refolding of other structurally

diverse non-homologous proteins such as, hen’s egg white lysozyme (containing

disulfide links in the native state), citrate synthase (a dimer with almost exclusively α-

helical secondary structure) etc. using β-CD conjugated magnetic nanoparticles to

evaluate their efficiencies as stripping agents.

(b) Though refolding experiment was carried out at low protein concentrations (0.029

mg/ml CA) in this study, for many applications, it is advantageous to conduct refolding

at higher protein concentrations. CA refolding at higher protein concentrations can be

made more efficient by increasing the concentrations of the artificial chaperones and by

adding Na2SO4 as suggested by Rozema and Gellman [65]. However, in our study using

solid phase chaperones, CD-APES-MNPs at above optimal concentration (40 mg/ml)

would lead to lower refolding yields. To solve this problem, one approach would be to

synthesize stripping agents with increased number of cyclodextrin moieties per volume

or per mass of solid matrix (magnetic nanoparticles).

(c) In this study, CD-APES-MNPs were used as stripping agents to strip away the

detergent molecules from protein-detergent complex. However, the use of cyclodextrin

as protein refolding aids was reported previously where it could prevent the formation

of protein aggregates during renaturation [70,82,83]. Natural CDs may interact with

proteins via hydrophobic forces, hydrogen bonding and Van der Waals interactions.

Charged chemically modified CDs can also bind to the folding polypeptide chain via

electrostatic interactions. Our as-prepared CD-APES-MNPs could be used as refolding

aids in ‘dilution additive approach’ described in Section 2.4. Moreover, it could be used

Chapter: 9

194

as a separation tool for separating the inclusion bodies (protein aggregates) from cell

debris.

(d) To evaluate the possibility of repeated use of CD-APES-MNPs as a solid-phase

artificial chaperone, regeneration of these stripping agents could be investigated after

recovery of refolded proteins. It could be regenerated by washing out adsorbed

detergent from solid phase with pure water [104].

9.2.2 Pollutants removal from aqueous water using cyclodextrin bonded magnetic

nanoparticles

(a) CM-β-CD conjugated magnetic nano-adsorbents were used as novel adsorbent for

the removal of methylene blue from aqueous streams. MB is a basic or cationic dye.

Adsorption behaviors of other dyes like acid, direct, disperse or reactive dye could be

studied with these magnetic nano-adsorbents to gain better understanding of adsorption

mechanism with different dyes. Similarly, these nano-adsorbents could be used for

adsorption of other heavy metals such as lead, cadmium etc. in non-competitive and

competitive adsorption environment. As CM-β-CD can complex various metals, its

complexation ability could be improved by increasing the number of carboxyl groups

i.g. the degree of carboxymethylation. Indeed, we have synthesized CM-β-CD polymer

(Mp = 13,000, 40wt% COOH groups) and grafted it on the magnetic nanoparticles.

These as-synthesized magnetic nanocomposites can be used for selective adsorption of

lead (II) from contaminated water. Currently, this work is in progress and some of

preliminary results are shown in Appendix C.

(b) Various ions exist in wastewater. The presence of other solutes may influence the

adsorption of any given adsorbate. Thus, K+, Na+, and Ca2+, as well as the anion of Cl−

Chapter: 9

195

and NO3−, which are the commonly coexisting ions with metal ions could be chosen for

further adsorption study. Natural organic matter (NOM) also exists ubiquitously in

natural aquatic environments, with two of its main fractions as humic acid (HA) and

fulvic acid (FA). The presence of natural organic matter (NOM) may also influence the

adsorption of heavy metal ions or organic pollutants on adsorbents in natural

environment. Therefore, competitive adsorption of metal ions and methylene blue could

be investigated in presence of natural organic matters.

(c) In this thesis, a small and limited recyclability study was done for the removal of

methylene blue and Cu2+ ions from aqueous water. For repeated use of these magnetic

nanoadsorbents, their chemical stability in various chemical and physical environments

including acid or base media needs to be investigated. The stability could be tested

through treatment of the particles with acids and several washing steps described in

literature [309]. Furthermore, the stability of the functional moieties (CM-β-CD) on the

adsorbent surface could be examined by FTIR after desorption. At very low pH (pH

<1), the magnetic phase was observed to be unstable and underwent leaching which

might limit the use of these nanoadsorbent in combination with complexing agents in

acidic media. However, magnetic nanoparticles can be coated with graphitic carbon

which can protect and isolate the encapsulated magnetic core and provide them

excellent chemical and thermal stability [309-311] .

(d) Organic and inorganic contaminant mixtures present at contaminated sites is one of

the major challenges in remediating water. Due to the fundamentally different

physiochemical properties of these contaminants, research has to be extended focusing

on remediating organic and inorganic contaminants separately rather than with a single

treatment. Some previous studies on soil remediation purposes showed that CM- β-CD

Chapter: 9

196

has the ability to simultaneously complex low-polarity organic molecules and heavy

metals. It was also found that the presence of organic molecules had a negligible effect

on the complexation of metal cations by CM-β-CD or vice versa [247,248]. Therefore,

CMCD-MNPs magnetic nanoparticles could be used for simultaneous removal of

organic and inorganic pollutants from wastewater as they can complex with both

pollutants as shown in Figure 9-1.

Figure 9-1 Schematic presentation of complexation of CMCD-MNPs magnetic nanoparticles with both organic and inorganic pollutants.

(e) The present work was conducted in batch lab-scale operation. A continuous

adsorption/desorption process more relevant for industrial processes could be developed

and studied. For this purposes, superconducting high gradient magnetic separator

(HGMS) suitable for a large-scale wastewater treatment in wastewater treatment plants

could be used. The availability of reasonably priced and efficient super-conducting

magnets and new methods for generating high magnetic field gradients enabled the use

of magnetic extractions on a large scale. Several research groups used HGMS for

contaminants removal from wastewater [123,312]. A multistage countercurrent

continuous contact process (Figure 9‐2) was proposed by Egashira and Hatton to

Chapter: 9

197

improve the treatment capacity [313]. Very recently, Rossier et al. demonstrated the use

of a true moving bed of chemically modified, carbon-coated metal magnetic

nanoparticles to adsorb small amounts of a solute (arsenate) out of a large volume, and

the desorption of the solute into a smaller volume, thereby enriching the solution [314].

The schematic diagram of their overall process is shown in Figure 9-3. However, for

large-scale wastewater treatment, a large quantity of magnetic nanoadsorbents would be

required unless they have extremely high adsorption capacity, which might limit their

practical applicability in treatment plants. Also for large-scale applications, the need of

producing high magnetic field generators and developing materials with high saturated

magnetization is a reality.

Figure 9-2 Schematic of the proposed multistage countercurrent continuous contact [313].

Chapter: 9

198

Figure 9-3 Schematic description of the overall process: in the first part (left), the target solute is extracted from a large volume using modified magnetic nanoparticles. In the second part (right), the loaded magnetic nanoparticles are washed (enrichment) and release the solute into a small volume. The moving solids (the true moving bed, i.e. the nanoparticles) links the two tank systems and transports the nanoparticles-bound solute against the concentration gradient between the large tank (left, low concentration) and the small tank (right, high concentration) [314].

9.2.3 Multifunctional silica core-shell magnetic nanoparticles for biomedical

applications

- Cellular Uptake of multifunctional nanoparticle in cancer cells could be evaluated and

measured quantitatively. Recent study by Yang et al. showed that the uptake of silica-

coated manganese oxide nanoparticles in targeted cells was quantitatively analyzed

through flow cytometry.

- Further experiments could be carried out on the specific delivery of hydrophobic drug

to the targeted cancer cells to assess their cytotoxicity effects on cells.

Chapter: 9

199

9.3 Research opportunities

In this thesis work, though various β-CD modified Fe3O4 nanoparticles has been

successfully synthesized and used in several potential applications, these

nanocomposites will also be useful in some other aspects as below:

Enantioseparation of amino acid enantiomers: The importance of amino acids in a

variety of bioscience, pharmaceutical areas and food analysis is indispensible. Amino

acids can be present as L- or D-isomers. Though they have identical physical and

chemical properties except for optical rotation, they can have very different

pharmacological properties and bioactive effects in stereo environments, specifically in

biological environments. Because of these similarities, chiral discrimination of

enantiomers is a very challenging task. Therefore chiral analysis of D/L-amino acids is

of increasing importance in pharmaceutical and biological fields. Though a number of

complicated approaches have been used for the chiral separation of organic compounds,

including gas chromatography (GC), high-performance liquid chromatography (HPLC),

capillary electrophoresis (CE) and the use of chiral reagents in chromatography [315],

most of these techniques are usually limited to the analytical scale, and only allow a

small amount of materials to be separated per run. However, β-CD modified Fe3O4

nanospheres synthesized in this thesis work as a chiral selecting system could be used in

efficient and economical enantioseparation of amino acid enantiomers [316]. The

combination of chiral recognition ability of amino acids enantiomers provided by β-CD

[169], and the magnetic separation ability of Fe3O4 nanoparticles, would provide an

effective tool to separate amino acid enantiomers.

Removal of pharmaceuticals, personal care products and endocrine disruptors

from wastewater: Over the last decade, environmental pollution by “emerging

Chapter: 9

200

contaminants” namely pharmaceuticals and personal care products (PPCPs) and also by

endocrine disrupting chemicals (EDCs) in different water sources and industrial

effluents has aroused the public concern [317,318]. Their occurrence is most often a

result of municipal wastewater discharge, as these compounds are not completely

removed in sewage treatment plants [319]. Wide range of PPCPs (naproxen, ibuprofen,

salicylic acid, carbamazepine, clofibric acid, diclofenac etc.) and EDCs (biphenol A, β-

Estradiol etc.) were found ubiquitously up to µg/l level in aqueous environment, soil,

wastewater, surface waters, sediments, groundwater, and even drinking water

[320,321]. Due to the adverse effects on living creatures and persistency of these PPCPs

and EDCs in environment, removal of these contaminants is of great importance. Based

on same principle used in Chapter 6 for the effective removal of dyes and in Chapter 8

for inclusion of hydrophobic drug (retinoic acid), β-CD modified Fe3O4

nanocomposites could also be used to remove PPCPs and EDCs from wastewater.

Chemical sensing. Selective recognition and separation of nucleosides using β-CD

bonded Fe3O4 nanocomposites has been demonstrated in Chapter 5. This method

provides a practical way to separate substrates based on the difference of binding

property by forming host–guest inclusion. Combining the unique properties of MNPs

(superparamagnetism and electrocatalytic) and CDs (excellent supramolecular

recognition and host–guest inclusion capability), it is possible to obtain the new

materials which will provide good opportunities for applications in the fields of sensors,

electrocatalysis etc. For detecting and monitoring chemicals, new electrodes modified

with β-CD bonded Fe3O4 nanocomposites could be designed. Recently, different β-CD

functionalized magnetic nanoparticles have been synthesized and electrochemical

response to dopamine, uric acid and tryptophan with the assistance of a magnet was

observed [206,322,323].

References

201

References

[1] A.K. Gupta, M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications, Biomaterials 26 (2005) 3995-4021.

[2] I.J. Bruce, T. Sen, Surface modification of magnetic nanoparticles with alkoxysilanes and their application in magnetic bioseparations, Langmuir 21 (2005) 7029-7035.

[3] E.K.U. Larsen, T. Nielsen, T. Wittenborn, H. Birkedal, T. Vorup-Jensen, M.H. Jakobsen, L. Ostergaard, M.R. Horsman, F. Besenbacher, K.A. Howard, J. Kjems, Size-dependent accumulation of PEGylated silane-coated magnetic iron oxide nanoparticles in murine tumors, ACS Nano 3 (2009) 1947-1951.

[4] D.L. Huber, Synthesis, properties, and applications of iron nanoparticles, Small 1 (2005) 482-501.

[5] M.H. Liao, D.H. Chen, Preparation and characterization of a novel magnetic nano-adsorbent, J. Mater. Chem. 12 (2002) 3654-3659.

[6] R. Hong, B. Feng, L. Chen, G. Liu, H. Li, Y. Zheng, D. Wei, Synthesis, characterization and MRI application of dextran-coated Fe3O4 magnetic nanoparticles, Biochem. Eng. J. 42 (2008) 290-300.

[7] A. Lopez-Cruz, C. Barrera, V.L. Calero-DdelC, C. Rinaldi, Water dispersible iron oxide nanoparticles coated with covalently linked chitosan, J. Mater. Chem. 19 (2009) 6870-6876.

[8] S.H. Huang, D.H. Chen, Rapid removal of heavy metal cations and anions from aqueous solutions by an amino-functionalized magnetic nano-adsorbent, J. Hazard. Mater. 163 (2009) 174-179.

[9] J. Szejtli, Introduction and general overview of cyclodextrin chemistry, Chem. Rev. 98 (1998) 1743-1754.

[10] G. Crini, H.N. Peindy, F. Gimbert, C. Robert, Removal of C.I. Basic Green 4 (Malachite Green) from aqueous solutions by adsorption using cyclodextrin-based adsorbent: Kinetic and equilibrium studies, Sep. Purif. Technol. 53 (2007) 97-110.

[11] D. Zhao, L. Zhao, C.S. Zhu, W.Q. Huang, J.L. Hu, Water insoluble ß-cyclodextrin polymer crosslinked by citric acid synthesis and adsorption properties toward phenol and methylene blue, J. Inclusion Phenom. Macrocyclic Chem. 63 (2009) 195-201.

References

202

[12] C.T. Yavuz, A. Prakash, J. Mayo, V.L. Colvin, Magnetic separations: From steel plants to biotechnology, Chem. Eng. Sci. 64 (2009) 2510-2521.

[13] Y. Sun, X. Ding, Z. Zheng, X. Cheng, X. Hu, Y. Peng, A Novel Approach to Magnetic Nanoadsorbents with High Binding Capacity for Bovine Serum Albumin, Macromol. Rapid Commun. 28 (2007) 346-351.

[14] Z.G. Peng, K. Hidajat, M.S. Uddin, Adsorption of bovine serum albumin on nanosized magnetic particles, J. Colloid Interface Sci. 271 (2004) 277-283.

[15] N. Shamim, L. Hong, K. Hidajat, M.S. Uddin, Thermosensitive-polymer-coated magnetic nanoparticles: adsorption and desorption of bovine serum albumin, J. Colloid Interface Sci. 304 (2006) 1-8.

[16] S.S. Banerjee, D.H. Chen, Cyclodextrin conjugated magnetic colloidal nanoparticles as a nanocarrier for targeted anticancer drug delivery, Nanotechnology 19 (2008) 265601-265607.

[17] W. Jiang, H.C. Yang, S.Y. Yang, H.E. Horng, J.C. Hung, Y.C. Chen, C.Y. Hong, Preparation and properties of superparamagnetic nanoparticles with narrow size distribution and biocompatible, J. Magn. Magn. Mater. 283 (2004) 210-214.

[18] G.M. da Costa, E.D. Grave, P.M.A. de Bakker, R.E. Vandenberghe, Synthesis and characterization of some iron oxides by sol-gel method, J. Solid State Chem. 113 (1994) 405-412.

[19] Y. Deng, L. Wang, W. Yang, S. Fu, A. Elaıssari, Preparation of magnetic polymeric particles via inverse microemulsion polymerization process, J. Magn. Magn. Mater. 257 (2003) 69-78.

[20] R. Abu Mukh-Qasem, A. Gedanken, Sonochemical synthesis of stable hydrosol of Fe3O4 nanoparticles, J. Colloid Interface Sci. 284 (2005) 489-494.

[21] D. Chen, R. Xu, Hydrothermal synthesis and characterization of nanocrystalline Fe3O4 powders, Mater. Res. Bull. 33 (1998) 1015-1021.

[22] S. Sun, H. Zeng, Size-controlled synthesis of magnetite nanoparticles, J. Am. Chem. Soc. 124 (2002) 8204-8205.

[23] S. Veintemillas-Verdaguer, M. Morales, C. Serna, Continuous production of γ-Fe2O3 ultrafine powders by laser pyrolysis, Mater. Lett. 35 (1998) 227-231.

[24] C.E. Sjagren, K. Briley-Saebra, M. Hanson, C. Johansson, Magnetic characterization of iron oxides for magnetic resonance imaging, Magn. Reson. Med. 272 (2005) 268-272.

References

203

[25] W. Wu, Q. He, C. Jiang, Magnetic iron oxide nanoparticles synthesis and surface functionalization strategies, Nanoscale Res. Lett. 3 (2008) 397-415.

[26] R. Cornell, U. Schertmann, Iron oxides in the laboratory; preparation and characterization, Weinheim: VCH, 1991.

[27] A. Figuerola, R. Di Corato, L. Manna, T. Pellegrino, From iron oxide nanoparticles towards advanced iron-based inorganic materials designed for biomedical applications, Pharmacol. Res. 62 (2010) 126-143.

[28] M. Mahmoudi, S. Sant, B. Wang, S. Laurent, T. Sen, Superparamagnetic iron oxide nanoparticles (SPIONs): Development, surface modification and applications in chemotherapy, Adv. Drug Deliv. Rev. 63 (2011) 24-46.

[29] J. Xie, J. Huang, X. Li, S. Sun, X. Chen, Iron oxide nanoparticle platform for biomedical applications, Curr. Med. Chem. 16 (2009) 1278-1294.

[30] R. De Palma, S. Peeters, M.J. Van Bael, H.V. den Rul, K. Bonroy, W. Laureyn, J. Mullens, G. Borghs, G. Maes, Silane ligand exchange to make hydrophobic superparamagnetic nanoparticles water-dispersible, Chem. Mater. 19 (2007) 1821-1831.

[31] R. Hao, R. Xing, Z. Xu, Y. Hou, S. Gao, S. Sun, Synthesis, functionalization, and biomedical applications of multifunctional magnetic nanoparticles, Adv. Mater. 22 (2010) 2729-2742.

[32] S.R. Dave, X. Gao, Monodisperse magnetic nanoparticles for biodetection, imaging, and drug delivery: a versatile and evolving technology, Nanomed. Nanobiotechnol. 1 (2009) 583-609.

[33] Y. Lu, Y. Yin, B.T. Mayers, Y. Xia, Modifying the surface properties of superparamagnetic iron oxide nanoparticles through A sol-gel approach, Nano Lett. 2 (2002) 183-186.

[34] C.W. Lu, Y. Hung, J.K. Hsiao, M. Yao, T.H. Chung, Y.S. Lin, S.H. Wu, S.C. Hsu, H.M. Liu, C.Y. Mou, C.S. Yang, D.M. Huang, Y.C. Chen, Bifunctional magnetic silica nanoparticles for highly efficient human stem cell labeling, Nano Lett. 7 (2007) 149-154.

[35] C. Zhang, B. Wängler, B. Morgenstern, H. Zentgraf, M. Eisenhut, H. Untenecker, R. Krüger, R. Huss, C. Seliger, W. Semmler, F. Kiessling, Silica- and alkoxysilane-coated ultrasmall superparamagnetic iron oxide particles: a promising tool to label cells for magnetic resonance imaging, Langmuir 23 (2007) 1427-1434.

References

204

[36] M. Liong, J. Lu, M. Kovochich, T. Xia, S.G. Ruehm, A.E. Nel, F. Tamanoi, J.I. Zink, Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery, ACS Nano 2 (2008) 889-896.

[37] Y.C. Chang, D.H. Chen, Preparation and adsorption properties of monodisperse chitosan-bound Fe3O4 magnetic nanoparticles for removal of Cu(II) ions, J. Colloid Interface Sci 283 (2005) 446-451.

[38] H.V. Tran, L.D. Tran, T.N. Nguyen, Preparation of chitosan/magnetite composite beads and their application for removal of Pb(II) and Ni(II) from aqueous solution, Mater. Sci. Eng. 30 (2010) 304-310.

[39] X. Xu, H. Shen, J. Xu, J. Xu, X. Li, X. Xiong, Core-shell structure and magnetic properties of magnetite magnetic fluids stabilized with dextran, Appl. Surf. Sci. 252 (2005) 494-500.

[40] S.Y. Mak, D.H. Chen, Fast adsorption of methylene blue on polyacrylic acid-bound iron oxide magnetic nanoparticles, Dyes and Pigments 61 (2004) 93-98.

[41] S.S. Banerjee, D.H. Chen, Fast removal of copper ions by gum arabic modified magnetic nano-adsorbent, J. Hazard. Mater. 147 (2007) 792-799.

[42] L. Zhang, F. Yu, A.J. Cole, B. Chertok, A.E. David, J. Wang, V.C. Yang, Gum arabic-coated magnetic nanoparticles for potential application in simultaneous magnetic targeting and tumor imaging, AAPS J. 11 (2009) 693-699.

[43] S. Shin, J. Jang, Thiol containing polymer encapsulated magnetic nanoparticles as reusable and efficiently separable adsorbent for heavy metal ions, Chem. Commun. (2007) 4230-4232.

[44] J.F. Liu, Z.S. Zhao, G.B. Jiang, Coating Fe3O4 magnetic nanoparticles with humic acid for high efficient removal of heavy metals in water, Environ. Sci. Technol. 42 (2008) 6949-6954.

[45] A. Aqil, S. Vasseur, E. Duguet, C. Passirani, J.P. Benoît, R. Jérôme, C. Jérôme, Magnetic nanoparticles coated by temperature responsive copolymers for hyperthermia, J. Mater. Chem. 18 (2008) 3352-3360.

[46] B. Du, A. Mei, P. Tao, B. Zhao, Z. Cao, J. Nie, J. Xu, Z. Fan, Poly[N-isopropylacrylamide-co-3-(trimethoxysilyl)-propylmethacrylate] Coated Aqueous Dispersed Thermosensitive Fe3O4 Nanoparticles, J. Phys. Chem. C 113 (2009) 10090-10096.

[47] W. Yantasee, C.L. Warner, T. Sangvanich, R.S. Addleman, T.G. Carter, R.J. Wiacek, G.E. Fryxell, C. Timchalk, M.G. Warner, Removal of heavy metals from

References

205

aqueous systems with thiol functionalized superparamagnetic nanoparticles, Environ. Sci. Technol. 41 (2007) 5114-5119.

[48] A.R. Khan, P. Forgo, K.J. Stine, V.T. D'Souza, Methods for selective modifications of cyclodextrins, Chem. Rev. 98 (1998) 1977-1996.

[49] E.V. Chernykh, S.B. Brichkin, Supramolecular complexes based on cyclodextrins, High Energy Chem. 44 (2010) 83-100.

[50] L. Liu, Q.X. Guo, The driving forces in the inclusion complexation of cyclodextrins, J. Inclusion Phenom. Macrocyclic Chem. 42 (2002) 1-14.

[51] E. Del Valle, Cyclodextrins and their uses: a review, Process Biochem. 39 (2004) 1033-1046.

[52] I.M. Trofymchuk, L.A. Belyakova, A.G. Grebenyuk, Study of complex formation between β-cyclodextrin and benzene, J. Inclusion Phenom. Macrocyclic Chem. 69 (2011) 371-375.

[53] R. Singh, N. Bharti, J. Madan, S. Hiremath, Characterization of Cyclodextrin Inclusion complexes- A Review, J. Pharm. Sci. Technol. 2 (2010) 171-183.

[54] F. Dragan, I. Bratu, G. Borodi, M. Toma, A. Hernanz, S. Simon, G. Cristea, R. Peschar, Spectroscopic investigation of β-cyclodextrin-metoprolol tartrate inclusion complexes, J. Inclusion Phenom. Macrocyclic Chem. 59 (2007) 125-130.

[55] B. Szafran, J. Pawlaczyk, Preparation and Characterization of the β-Cyclodextrin Inclusion Complex with Sulfafurazole, J. Inclusion Phenom. Mol. Recogn. Chem. 23 (1996) 277-288.

[56] Tian-xiang Xiang and Bradley D. Anderson, Inclusion complexes of purine nucleosides with cyclodextrins II. Investigation of inclusion complex geometry and cavity microenvironment, Int. J. Pharm. 59 (1990) 45-55.

[57] M. Spulber, M. Pinteala, A. Fifere, V. Harabagiu, B.C. Simionescu, Inclusion complexes of 5-flucytosine with β-cyclodextrin and hydroxypropyl-β-cyclodextrin: characterization in aqueous solution and in solid state, J. Inclusion Phenom. Macrocyclic Chem. 62 (2008) 117-125.

[58] T. Loftsson, D. Duchêne, Cyclodextrins and their pharmaceutical applications, Int. J. Pharm. 329 (2007) 1-11.

[59] K. Uekama, Design and evaluation of cyclodextrin-based drug formulation, Chem. Pharm. Bull. (Tokyo) 52 (2004) 900-915.

References

206

[60] K. Uekama, F. Hirayama, T. Irie, Cyclodextrin drug carrier systems, Chem. Rev. 98 (1998) 2045-2076.

[61] E.D.B. Clark, Refolding of recombinant proteins, Curr. Opin. Biotechnol. 9 (1998) 157-163.

[62] B.E. Fischer, Renaturation of recombinant proteins produced as inclusion bodies, Biotechnol. Adv. 12 (1994) 89-101.

[63] M.E. Goldberg, R. Rudolph, R. Jaenicke, A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme, Biochemistry 30 (1991) 2790-2797.

[64] B.M. Baynes, D.I.C. Wang, B.L. Trout, Role of arginine in the stabilization of proteins against aggregation, Biochemistry 44 (2005) 4919-4925.

[65] D. Rozema, S.H. Gellman, Artificial chaperone-assisted refolding of carbonic anhydrase B, J. Biol. Chem. 271 (1996) 3478-3487.

[66] D.L. Daugherty, D. Rozema, P.E. Hanson, S.H. Gellman, Artificial chaperone-assisted refolding of citrate synthase, J. Biol. Chem. 273 (1998) 33961-33971.

[67] D. Rozema, S.H. Gellman, Artificial chaperone-assisted refolding of denatured-reduced lysozyme: modulation of the competition between renaturation and aggregation, Biochemistry 35 (1996) 15760-15771.

[68] D. Rozema, S. Gellman, Artificial chaperones: protein refolding via sequential use of detergent and cyclodextin, J. Am. Chem. Soc. 117 (1995) 2373-2374.

[69] M. Winkler, J.Y. Chang, D.I. Wang, J.L. Cleland, S.E. Builder, J.R. Swartz, Polyethylene glycol enhanced protein refolding, Biotechnology 10 (1992) 1013-1019.

[70] A. Sharma, N. Karuppiah, cyclodextrin as protein folding aids, Biochem. Biophys. Res. Commun. 211 (1995) 60-66.

[71] Y. Xie, D.B. Wetlaufer, Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase II, Protein Sci. 4 (1995) 1535-1543.

[72] H.C. Chiu, S. Lin, S.C. Lin, K.L. Lin, Enhanced protein renaturation by temperature-responsive polymers, Biotechnol. Bioeng. 67 (2000) 505-512.

[73] P.M. Horowitz, G. Zardeneta, Protein refolding at high concentrations using detergent/phospholipid mixtures, Anal. Biochem. 218 (1994) 392-398.

References

207

[74] P.M. Horowitz, S. Tandon, Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent, J. Biol. Chem. 262 (1987) 4486-4491.

[75] P. Horowitz, S. Tandon, Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effect of lauryl maltoside, J. Biol. Chem. 261 (1986) 15615-15618.

[76] Y.L. Liu, H.M. Zhou, H.W. Xi, Y.X. Zhang, Y. Zhu, Refolding and reactivation of calf intestinal alkaline phosphatase with excess magnesium ions, Int. J. Biochem. Cell Biol. 34 (2002) 1241-1247.

[77] Y.X. Zhang, H.M. Zhou, S.L. Yan, X.J. Tian, X.H. Song, Study of refolding of calf intestinal alkaline phosphatase, J. Protein Chem. 22 (2003) 417-422.

[78] H. Zhou, Y. Park, F. Meng, Role of proline, glycerol, and heparin as protein folding aids during refolding of rabbit muscle creatine kinase, Int. J. Biochem. Cell Biol. 33 (2001) 701-709.

[79] H.M. Zhou, Y.D. Park, W.B. Ou, Effect of osmolytes as folding aids on creatine kinase refolding pathway, Int. J. Biochem. Cell Biol. 34 (2002) 136-147.

[80] S.L. Wang, V.D. Trivedi, K.C. Hwang, C. Yu, D.K. Chang, M.M. Chang, T.K. Kumar, D. Samuel, G. Ganesh, P.W. Yang, G. Jayaraman, Proline inhibits aggregation during protein refolding, Protein Sci. 9 (2000) 344-352.

[81] H. Ogino, H. Ishikawa, A. Sowa, M. Yasuda, Y. Murakami, Effect of additives on refolding of a denatured protein, Biotechnol. Prog. 14 (1998) 601-606.

[82] L. Sharma, A. Sharma, A. Desai, C. Lee, Lysozyme refolding with cyclodextrins: structure-activity relationship, Biochimie 88 (2006) 1435-1445.

[83] L. Sharma, A. Sharma, Influence of cyclodextrin ring substituents on folding-related aggregation of bovine carbonic anhydrase, Eur. J. Biochem. 268 (2001) 2456-2463.

[84] K. Shiraki, H. Hamada, L-argininamide improves the refolding more effectively than L-arginine, J. Biotechnol. 130 (2007) 153-160.

[85] K. Tsumoto, T. Arakawa, The effects of arginine on refolding of aggregated proteins: not facilitate refolding, but suppress aggregation, Biochem. Biophys. Res. Commun. 304 (2003) 148-152.

References

208

[86] M. Sola-Penna, P. Zancan, Trehalose and glycerol stabilize and renature yeast inorganic pyrophosphatase inactivated by very high temperatures, Arch. Biochem. Biophys. 444 (2005) 52-60.

[87] R. Yazdanparast, R. Khodarahmi, Refolding of chemically denatured alpha-amylase in dilution additive mode, Biochim. Biophys. Acta 1674 (2004) 175-181.

[88] R. Yazdanparast, R. Khodarahmi, Suppression effect of guanidine hydrochloride on a-cyclodextrin-assisted refolding of denatured a-amylase, Process Biochem. 40 (2005) 2973-2979.

[89] Y. Sun, Y. Huang, X.Y. Dong, Refolding kinetics of denatured-reduced lysozyme in the presence of folding aids, J. Biotechnol. 114 (2004) 135-142.

[90] Y. Lin, Z. Liu, J. Wang, D. Lu, How CTAB assists the refolding of native and recombinant lysozyme, Biochem. Eng. J. 24 (2005) 269-277.

[91] D.L. Hevehan, E. De Bernardez Clark, Oxidative renaturation of lysozyme at high concentrations, Biotechnol. Bioeng. 54 (1997) 221-230.

[92] C. Vial, E. Clottes, F. Couthon, Refolding of SDS- and thermally denatured MM-creatine kinase using cyclodextrins, Biochem. Biophys. Res. Commun. 227 (1996) 854-860.

[93] R. Yazdanparast, B. Eftekharzadeh, F. Khodagholi, A new artificial chaperone for protein refolding: sequential use of detergent and alginate, Protein J. 27 (2008) 123-129.

[94] Y. Aoyama, K. Akiyoshi, N. Yamaguchi, Y. Nomura, M. Ikeda, Protein refolding assisted by self-assembled nanogels as novel artificial molecular chaperone, FEBS Lett. 553 (2003) 271-276.

[95] T. Narita, Y. Aoyama, K. Akiyoshi, Y. Nomura, Y. Sasaki, M. Takagi, Thermoresponsive controlled association of protein with a dynamic nanogel of hydrophobized polysaccharide and cyclodextrin: heat shock protein-like activity of artificial molecular chaperone, Biomacromolecules 6 (2005) 447-452.

[96] M. Rao, D. Nath, Artificial chaperone mediated refolding of xylanase from an alkalophilic thermophilic Bacillus sp. Implications for in vitro protein renaturation via a folding intermediate, Eur. J. Biochem. 268 (2001) 5471-5478.

[97] D. Balasubramanian, B. Raman, C. Sivakama Sundari, Artificial chaperoning of insulin, human carbonic anhydrase and hen egg lysozyme using linear dextrin chains--a sweet route to the native state of globular proteins, FEBS Lett. 443 (1999) 215-219.

References

209

[98] R. Khodarahmi, F. Khodagholi, R. Yazdanparast, Artificial chaperone-assisted refolding of chemically denatured alpha-amylase, Int. J. Biol. Macromol. 35 (2005) 257-263.

[99] T. Takaha, K. Fujii, K. Hayashi, S. Machida, S. Ogawa, S. Xiaohua, Cycloamylose as an efficient artificial chaperone for protein refolding, FEBS Lett. 486 (2000) 131-135.

[100] Z. Liu, K. Zhang, D. Lu, Protein refolding assisted by an artificial chaperone using temperature stimuli responsive polymer as the stripper, Biochem. Eng. J. 25 (2005) 141-149.

[101] E. Souri, F. Khodagholi, R. Yazdanparast, Alkaline phosphatase refolding assisted by sequential use of oppositely charged detergents: a new artificial chaperone system, Int. J. Biol. Macromol. 42 (2008) 195-202.

[102] X. Wang, Z. Liu, M. Zhang, D. Lu, Z. Liu, Dextran-grafted-PNIPAAm as an artificial chaperone for protein refolding, Biochem. Eng. J. 27 (2006) 336.

[103] M.A. Esmaeili, R. Yazdanparast, Solid-phase assisted refolding of carbonic anhydrase using beta-cyclodextrin-polyurethane polymer, Protein J. 27 (2008) 334-342.

[104] S. Sugimoto, T. Nagamune, J. Honda, T. Mannen, S. Yamaguchi, Expanded-bed protein refolding using a solid-phase artificial chaperone, J. Biosci. Bioeng. 91 (2001) 403-408.

[105] S. Tsukiji, T. Nagamune, T. Mannen, S. Yamaguchi, C. Hong, Solid-phase artificial chaperone-assisted refolding using insoluble beta-cyclodextrin-acrylamide copolymer beads, Biotechnol. Lett. 26 (2004) 1787-1791.

[106] R. Khodarahmi, R. Yazdanparast, Evaluation of artificial chaperoning behavior of an insoluble cyclodextrin-rich copolymer: solid-phase assisted refolding of carbonic anhydrase, Int. J. Biol. Macromol. 40 (2007) 319-326.

[107] G. Crini, Recent developments in polysaccharide-based materials used as adsorbents in wastewater treatment, Prog. Polym. Sci. 30 (2005) 38-70.

[108] Y.T. Zhou, H.L. Nie, C. Branford-White, Z.Y. He, L.M. Zhu, Removal of Cu2+ from aqueous solution by chitosan-coated magnetic nanoparticles modified with a-ketoglutaric acid, J. Colloid Interface Sci. 330 (2009) 29-37.

[109] V.K. Gupta, Suhas, Application of low-cost adsorbents for dye removal-A review, J. Environ. Manage. 90 (2009) 2313-2342.

References

210

[110] F. Fu, Q. Wang, Removal of heavy metal ions from wastewaters: a review, J. Environ. Manage. 92 (2011) 407-418.

[111] R.W. Rousseau (Ed.), Handbook of Separation Process Technology, Wiley, New York (1987)

[112] H. Chen, Y. Zhao, A. Wang, Removal of Cu(II) from aqueous solution by adsorption onto acid-activated palygorskite, J. Hazard. Mater. 149 (2007) 346-354.

[113] G. Crini, Kinetic and equilibrium studies on the removal of cationic dyes from aqueous solution by adsorption onto a cyclodextrin polymer, Dyes and Pigments 77 (2008) 415-426.

[114] G. Crini, Studies on adsorption of dyes on beta-cyclodextrin polymer, Bioresour. Technol. 90 (2003) 193-198.

[115] B. Hameed, A. Ahmed, K. Latiff, Adsorption of basic dye (methylene blue) onto activated carbon prepared from rattan sawdust, Dyes and Pigments 75 (2007) 143.

[116] G. Crini, Non-conventional low-cost adsorbents for dye removal: a review, Bioresour. Technol. 97 (2006) 1061-1085.

[117] A.R. Hedges, Industrial applications of cyclodextrins, Chem. Rev. 98 (1998) 2035-2044.

[118] D. Zhao, L. Zhao, C.S. Zhu, X. Shen, X. Zhang, B. Sha, Comparative study of polymer containing beta-cyclodextrin and -COOH for adsorption toward aniline, 1-naphthylamine and methylene blue, J. Hazard. Mater. 171 (2009) 241-246.

[119] Q. Wang, Y. Guan, X. Ren, M. Yang, X. Liu, Application of magnetic extractant for the removal of hexavalent chromium from aqueous solution in high gradient magnetic separator, Chem. Eng. J. 183 (2012) 339-348.

[120] A. Ditsch, S. Lindenmann, P.E. Laibinis, D.I.C. Wang, T.A. Hatton, High-gradient magnetic separation of magnetic Nanoclusters, Ind. Eng. Chem. Res. 44 (2005) 6824-6836.

[121] R.D. Ambashta, M. Sillanpää, Water purification using magnetic assistance: a review, J. Hazard. Mater. 180 (2010) 38-49.

[122] S. Li, M. Wang, Z. Zhu, Q. Wang, X. Zhang, H. Song, D. Cang, Application of superconducting HGMS technology on turbid wastewater treatment from converter, Sep. Purif. Technol. 84 (2012) 56-62.

References

211

[123] K. Kondo, T. Jin, O. Miura, Removal of less biodegradable dissolved organic matters in water by superconducting magnetic separation with magnetic mesoporous carbon, Physica C: Superconductivity 470 (2010) 1808-1811.

[124] M. Ozmen, K. Can, G. Arslan, A. Tor, Y. Cengeloglu, M. Ersoz, Adsorption of Cu(II) from aqueous solution by using modified Fe3O4 magnetic nanoparticles, Desalination 254 (2010) 162-169.

[125] S.F. Lim, Y.M. Zheng, S.W. Zou, J.P. Chen, Removal of copper by calcium alginate encapsulated magnetic sorbent, Chem. Eng. J. 152 (2009) 509-513.

[126] J. Wang, S. Zheng, Y. Shao, J. Liu, Z. Xu, D. Zhu, Amino-functionalized Fe3O4@SiO2 core-shell magnetic nanomaterial as a novel adsorbent for aqueous heavy metals removal, J. Colloid Interface Sci. 349 (2010) 293-299.

[127] M. Bystrzejewski, K. Pyrzyn´ska, A. Huczko, H. Lange, Carbon-encapsulated magnetic nanoparticles as separable and mobile sorbents of heavy metal ions from aqueous solutions, Carbon 24 (1994) 19-44.

[128] S.C. Tang, P. Wang, K. Yin, I.M. Lo, Synthesis and application of magnetic hydrogel for Cr(VI) removal from contaminated water, Environ. Eng. Sci. 27 (2010) 947-954.

[129] K. Pyrzyńska, M. Bystrzejewski, Comparative study of heavy metal ions sorption onto activated carbon, carbon nanotubes, and carbon-encapsulated magnetic nanoparticles, Colloids Surf. A: Physicochem. Eng. Aspects 362 (2010) 102-109.

[130] M. Monier, D. Ayad, Y. Wei, A. Sarhan, Preparation and characterization of magnetic chelating resin based on chitosan for adsorption of Cu(II), Co(II), and Ni(II) ions, React. Funct. Polym. 70 (2010) 257-266.

[131] C.M. Chou, H.L. Lien, Dendrimer-conjugated magnetic nanoparticles for removal of zinc (II) from aqueous solutions, J. Nanopart. Res. 13 (2011) 2099-2107.

[132] M. Iram, C. Guo, Y. Guan, A. Ishfaq, H. Liu, Adsorption and magnetic removal of neutral red dye from aqueous solution using Fe3O4 hollow nanospheres, J. Hazard. Mater. 181 (2010) 1039-1050.

[133] S. Qadri, A. Ganoe, Y. Haik, Removal and recovery of acridine orange from solutions by use of magnetic nanoparticles, J. Hazard. Mater. 169 (2009) 318-323.

[134] A.A. Atia, A.M. Donia, W.A. Al-Amrani, Adsorption/desorption behavior of acid orange 10 on magnetic silica modified with amine groups, Chem. Eng. J. 150 (2009) 55-62.

References

212

[135] Y.C. Chang, D.H. Chen, Adsorption kinetics and thermodynamics of acid dyes on a carboxymethylated chitosan-conjugated magnetic nano-adsorbent, Macromol. Biosci. 5 (2005) 254-261.

[136] S. Qu, F. Huang, S. Yu, G. Chen, J. Kong, Magnetic removal of dyes from aqueous solution using multi-walled carbon nanotubes filled with Fe2O3 particles, J. Hazard. Mater. 160 (2008) 643-647.

[137] N. Yang, S. Zhu, D. Zhang, S. Xu, Synthesis and properties of magnetic Fe3O4-activated carbon nanocomposite particles for dye removal, Mater. Lett. 62 (2008) 645-647.

[138] S. Shariati, M. Faraji, Y. Yamini, A.A. Rajabi, Fe3O4 magnetic nanoparticles modified with sodium dodecyl sulfate for removal of safranin O dye from aqueous solutions, Desalination 270 2011) 160-165.

[139] X. Fu, X. Chen, J. Wang, J. Liu, Fabrication of carboxylic functionalized superparamagnetic mesoporous silica microspheres and their application for removal basic dye pollutants from water, Micropor. Mesopor. Mater. 139 (2011) 8-15.

[140] Y. Lin, H. Chen, P. Chien, C. Chiou, C. Liu, Application of bifunctional magnetic adsorbent to adsorb metal cations and anionic dyes in aqueous solution, J. Hazard. Mater. 185 (2011) 1124-1130.

[141] G. Crini, H.N. Peindy, Adsorption of C.I. Basic Blue 9 on cyclodextrin-based material containing carboxylic groups, Dyes and Pigments 70 (2006) 204-211.

[142] E.Y. Ozmen, M. Yilmaz, Use of beta-cyclodextrin and starch based polymers for sorption of Congo red from aqueous solutions, J. Hazard. Mater. 148 (2007) 303-310.

[143] A. Sharma, A. Mishra, Microwave induced beta-cyclodextrin modification of chitosan for lead sorption, Int. J. Biol. Macromol. 47 (2010) 410-419.

[144] F. Li, X. Sun, B. Li, Kinetics and mechanism of cationic dyes adsorption onto modified β-cyclodextrin microspheres, ICEE 2010.

[145] E. Yilmaz, S. Memon, M. Yilmaz, Removal of direct azo dyes and aromatic amines from aqueous solutions using two beta-cyclodextrin-based polymers, J. Hazard. Mater. 174 (2010) 592-597.

[146] L. Ducoroy, M. Bacquet, B. Martel, M. Morcellet, Removal of heavy metals from aqueous media by cation exchange nonwoven PET coated with β-cyclodextrin-polycarboxylic moieties, React. Funct. Polym. 68 (2008) 594-600.

References

213

[147] Langmuir, The adsorption of gases on plane surfaces of glass, mica and platinum, J. Am. Chem. Soc. 40 (1918) 1361-1403.

[148] H. Freundlich, Über die adsorption in lösungen, Z. Phys. Chem. 57 (1906) 385-471.

[149] Y. Ho, G. McKay, Pseudo-second order model for sorption processes, Process Biochem. 34 (1999) 451-465.

[150] Y.S. Ho, G. McKay, A comparison of chemisorption kinetic models applied to pollutant removal on various sorbents, Proc. Saf. Environ. Protect. 76 (1998) 332-340.

[151] J.Y. Tseng, C.Y. Chang, C.F. Chang, Y.H. Chen, C.C. Chang, D.R. Ji, C.Y. Chiu, P.C. Chiang, Kinetics and equilibrium of desorption removal of copper from magnetic polymer adsorbent, J. Hazard. Mater. 171 (2009) 370-377.

[152] S. Sun, A. Wang, Adsorption kinetics of Cu(II) ions using N,O-carboxymethyl-chitosan, J. Hazard. Mater. 131 (2006) 103-111.

[153] W. Weber, J. Morris, Kinetics of adsorption on carbon from solution, J. Sanit. Eng. Div. ASCE 89 (1963) 31-59.

[154] A. Kamari, W.S.W. Ngah, Isotherm, kinetic and thermodynamic studies of lead and copper uptake by H2SO4 modified chitosan, Colloids Surf. B Biointerfaces 73 (2009) 257-266.

[155] C. Chen, J. Hu, D. Shao, J. Li, X. Wang, Adsorption behavior of multiwall carbon nanotube/iron oxide magnetic composites for Ni(II) and Sr(II), J. Hazard. Mater. 164 (2009) 923-928.

[156] E. Demirbas, N. Dizge, M. Sulak, M. Kobya, Adsorption kinetics and equilibrium of copper from aqueous solutions using hazelnut shell activated carbon, Chem. Eng. J. 148 (2009) 480-487.

[157] M. Zhao, Z. Tang, P. Liu, Removal of methylene blue from aqueous solution with silica nano-sheets derived from vermiculite, J. Hazard. Mater. 158 (2008) 43-51.

[158] Z.G. Peng, K. Hidajat, M.S. Uddin, Adsorption and desorption of lysozyme on nano-sized magnetic particles and its conformational changes, Colloids Surf. B Biointerfaces 35 (2004) 169-174.

[159] W.B. Ou, Y.D. Park, H.M. Zhou, Effect of osmolytes as folding aids on creatine kinase refolding pathway, Int. J. Biochem. Cell Biol. 34 (2002) 136-147.

References

214

[160] P. Zancan, M. Sola-Penna, Trehalose and glycerol stabilize and renature yeast inorganic pyrophosphatase inactivated by very high temperatures, Arch. Biochem. Biophys. 444 (2005) 52-60.

[161] F. Meng, Y. Park, H. Zhou, Role of proline, glycerol, and heparin as protein folding aids during refolding of rabbit muscle creatine kinase, Int. J. Biochem. Cell Biol. 33 (2001) 701-709.

[162] M. Yasuda, Y. Murakami, A. Sowa, H. Ogino, H. Ishikawa, Effect of additives on refolding of a denatured protein, Biotechnol Prog 14 (1998) 601-606.

[163] J.L. Cleland, S.E. Builder, J.R. Swartz, M. Winkler, J.Y. Chang, D.I. Wang, Polyethylene glycol enhanced protein refolding, Biotechnology (NY) 10 (1992) 1013-1019.

[164] Y.X. Zhang, Y. Zhu, H.W. Xi, Y.L. Liu, H.M. Zhou, Refolding and reactivation of calf intestinal alkaline phosphatase with excess magnesium ions, Int. J. Biochem. Cell Biol. 34 (2002) 1241-1247.

[165] X.J. Tian, X.H. Song, S.L. Yan, Y.X. Zhang, H.M. Zhou, Study of refolding of calf intestinal alkaline phosphatase, J. Protein Chem. 22 (2003) 417-422.

[166] D.B. Wetlaufer, Y. Xie, Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase II, Protein Sci. 4 (1995) 1535-1543.

[167] S. Tandon, P.M. Horowitz, Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent, J. Biol. Chem. 262 (1987) 4486-4491.

[168] N. Karuppiah, A. Sharma, Cyclodextrin as protein folding aids, Biochem. Biophys. Res. Commun. 211 (1995) 60-66.

[169] G.K.E.V. Scriba, Cyclodextrins in capillary electrophoresis enantioseparations--recent developments and applications, J. Sep. Sci. 31 (2008) 1991-2011.

[170] E.Y. Ozmen, M. Sezgin, A. Yilmaz, M. Yilmaz, Synthesis of beta-cyclodextrin and starch based polymers for sorption of azo dyes from aqueous solutions, Bioresour. Technol. 99 (2008) 526-531.

[171] C. Sivakama Sundari, B. Raman, D. Balasubramanian, Artificial chaperoning of insulin, human carbonic anhydrase and hen egg lysozyme using linear dextrin chains--a sweet route to the native state of globular proteins, FEBS Lett. 443 (1999) 215-219.

References

215

[172] S. Machida, S. Ogawa, S. Xiaohua, T. Takaha, K. Fujii, K. Hayashi, Cycloamylose as an efficient artificial chaperone for protein refolding, FEBS Lett. 486 (2000) 131-135.

[173] F. Khodagholi, B. Eftekharzadeh, R. Yazdanparast, A new artificial chaperone for protein refolding: sequential use of detergent and alginate, Protein J. 27 (2008) 123-129.

[174] C. Xu, S. Sun, Monodisperse magnetic nanoparticles for biomedical applications, Polym Int 56 (2007) 821-826.

[175] T. Mannen, S. Yamaguchi, J. Honda, S. Sugimoto, T. Nagamune, Expanded-bed protein refolding using a solid-phase artificial chaperone, J. Biosci. Bioeng. 91 (2001) 403-408.

[176] R. Yazdanparast, R. Khodarahmi, Evaluation of artificial chaperoning behavior of an insoluble cyclodextrin-rich copolymer: solid-phase assisted refolding of carbonic anhydrase, Int. J. Biol. Macromol. 40 (2007) 319-326.

[177] S. Yamaguchi, C. Hong, T. Mannen, S. Tsukiji, T. Nagamune, Solid-phase artificial chaperone-assisted refolding using insoluble beta-cyclodextrin-acrylamide copolymer beads, Biotechnol. Lett. 26 (2004) 1787-1791.

[178] M.A. Esmaeili, R. Yazdanparast, Beta-cyclodextrin-bonded silica assists alkaline phosphatase and carbonic anhydrase refolding in a solid phase assisted refolding approach, Process Biochem. 45 (2010) 239-246.

[179] L.A. Belyakova, K.A. Kazdobin, V.N. Belyakov, S.V. Ryabov, A.F. Danil de Namor, Synthesis and properties of supramolecular systems based on silica, J. Colloid Interface Sci. 283 (2005) 488-494.

[180] H.N. Li, Y.X. Ci, A new rapid fluorimetric method for the determination of carbonic anhydrase, Anal. Chim. Acta. 317 (1995) 353-357.

[181] Y. Pocker, J. Stone, Biochemistry 6 (1968) 668.

[182] Z. Xu, Q. Liu, J. Finch, Silanation and stability of 3-aminopropyl triethoxy silane on nanosized superparamagnetic particles, Appl. Surf. Sci. 120 (1997) 269-278.

[183] B. Martel, A. Leckchiri, A. Pollet, M. Morcellet, Eur. Polym. J. 31 (1995) 1083.

[184] S. Tang, L. Kong, J. Ou, Y. Liu, X. Li, H. Zou, Application of cross-linked beta-cyclodextrin polymer for adsorption of aromatic amino acids, J. Mol. Recognit. 19 (2006) 39-48.

References

216

[185] B. Li, D. Jia, Y. Zhou, Q. Hu, W. Cai, In situ hybridization to chitosan magnetite nanocomposite induced by the magnetic field, J. Magn. Magn. Mater. 306 (2006) 223-227.

[186] V.S. Zaitsev, D.S. Filimonov, I.A. Presnyakov, R.J. Gambino, B. Chu, Physical and Chemical Properties of Magnetite and Magnetite-Polymer Nanoparticles and Their Colloidal Dispersions, J. Colloid Interface Sci. 212 (1999) 49-57.

[187] P.E. Hanson, S.H. Gellman, Mechanistic comparison of artificial-chaperone-assisted and unassisted refolding of urea-denatured carbonic anhydrase B, Fold. Des. 3 (1998) 457-468.

[188] R. Yazdanparast, R. Khodarahmi, E. Soori, Comparative studies of the artificial chaperone-assisted refolding of thermally denatured bovine carbonic anhydrase using different capturing ionic detergents and beta-cyclodextrin, Arch. Biochem. Biophys. 437 (2005) 178-185.

[189] R. Khodarahmi, R. Yazdanparast, Refolding of chemically denatured alpha-amylase in dilution additive mode, Biochim. Biophys. Acta 1674 (2004) 175-181.

[190] L. Stryer, J. Mol. Biol. 13 (1965) 482-495.

[191] R. Veyret, T. Delair, C. Pichot, A. Elaissari, Amino-containing magnetic nanoemulsions: elaboration and nucleic acid extraction, J. Magn. Magn. Mater. 295 (2005) 155-163.

[192] T. Helboe, S.H. Hansen, Separation of nucleosides using capillary electrochromatography, J. Chromatogr. A 836 (1999) 315-324.

[193] L. Jin, X. Ni, X. Liu, M. Wei, Selective adsorption of adenosine and guanosine by a β-Cyclodextrin/layered double hydroxide intercalation compound, Chem. Eng. Technol. 33 (2010) 82-88.

[194] J.D. Lamb, Y.K. Ye, Separation of nucleotides and nucleosides by gradient macrocycle-based ion chromatography, J. Chromatogr. 602 (1992) 189-195.

[195] H.M. Liebich, C. Di Stefano, A. Wixforth, H.R. Schmid, Quantitation of urinary nucleosides by high-performance liquid chromatography, J. Chromatogr. A 763 (1997) 193-197.

[196] T. Umemura, K.I. Tsunoda, A. Koide, T. Oshima, N. Watanabe, K. Chiba, H. Haraguchi, Amphoteric surfactant-modified stationary phase for the reversed-phase high-performance liquid chromatographic separation of nucleosides and their bases by elution with water, Anal. Chim. Acta 419 (2000) 87-92.

References

217

[197] S. Abel, M.U. Bäbler, C. Arpagaus, M. Mazzotti, J. Stadler, Two-fraction and three-fraction continuous simulated moving bed separation of nucleosides, J. Chromatogr. A 1043 (2004) 201-210.

[198] R.N. Goyal, V.K. Gupta, M. Oyama, N. Bachheti, Voltammetric determination of adenosine and guanosine using fullerene-C(60)-modified glassy carbon electrode, Talanta 71 (2007) 1110-1117.

[199] E. Fortin, J. Chane-Tune, P. Mailley, S. Szunerits, B. Marcus, J.P. Petit, M. Mermoux, E. Vieil, Nucleosides and ODN electrochemical detection onto boron doped diamond electrodes, Bioelectrochemistry 63 (2004) 303-306.

[200] H. Mouaziz, S. Braconnot, F. Ginot, A. Elaissari, Elaboration of hydrophilic aminodextran containing submicron magnetic latex particles, Colloid Polym. Sci. 287 (2009) 287-297.

[201] Y. Kang, L. Zhou, X. Li, J. Yuan, β-Cyclodextrin-modified hybrid magnetic nanoparticles for catalysis and adsorption, J. Mater. Chem. 21 (2011) 3704-3710.

[202] S.S. Banerjee, D.H. Chen, Magnetic nanoparticles grafted with cyclodextrin for hydrophobic drug delivery, Chem. Mater. 19 (2007) 6345-6349.

[203] S.S. Banerjee, D.H. Chen, Cyclodextrin-conjugated nanocarrier for magnetically guided delivery of hydrophobic drugs, J. Nanopart. Res. 11 (2008) 2071-2078.

[204] A. Badruddoza, G.S.S. Hazel, K. Hidajat, M. Uddin, Synthesis of carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbent for removal of methylene blue, Colloids Surf. A: Physicochem. Eng. Aspects 367 (2010) 85-95.

[205] A.Z.M. Badruddoza, A.S.H. Tay, P.Y. Tan, K. Hidajat, M.S. Uddin, Carboxymethyl-β-cyclodextrin conjugated magnetic nanoparticles as nano-adsorbents for removal of copper ions: Synthesis and adsorption studies, J. Hazard. Mater. 185 (2011) 1177-1186.

[206] Y. Wu, F. Zuo, Z. Zheng, X. Ding, Y. Peng, A novel approach to molecular recognition surface of magnetic nanoparticles based on host–guest effect, Nanoscale Res. Lett. 4 (2009) 738-747.

[207] H.B. Ji, Q.P. Long, H.Y. Chen, X.T. Zhou, X.F. Hu, β-Cyclodextrin inclusive interaction driven separation of organic compounds, AICHE J. 57 (2011) 2341-2352.

[208] E. Furusaki, Y. Ueno, N. Sakairi, N. Nishi, S. Tokura, Facile preparation and inclusion ability of a chitosan derivative bearing carboxymethyl-ß-cyclodextrin, Carbohydr. Polym. 29 (1996) 29-34.

References

218

[209] B.J. Ravoo, R. Darcy, A. Mazzaglia, D. Nolan, K. Gaffney, Supramolecular tapes formed by a catanionic cyclodextrin in water, Chem. Commun. (2001) 827-828.

[210] D. Caruntu, G. Caruntu, Y. Chen, C. O’Connor, G. Goloverda, V. Kolesnichenko, Synthesis of variable-sized nanocrystals of Fe3O4 with high surface reactivity, Chem. Mater. 16 (2004) 5527-5534.

[211] M.H. Kalavathy, L.R. Miranda, Comparison of copper adsorption from aqueous solution using modified and unmodified Hevea brasiliensis saw dust, Desalination 255 (2010) 165-174.

[212] J.B. Xiao, Adsorption of guanosine, cytidine, and uridine on a β-cyclodextrin derivative grafted chitosan, J. Appl. Polym. Sci. 103 (2007) 3050-3055.

[213] T. Weber, R. Chakravorti, Pore and solid diffusion models for fixed bed adsorbent, J. Am. Inst. Chem. Eng. 2 (1974) 228-238.

[214] A. Elaissari, P. Cros, C. Pichot, V. Laurent, B. Mandrand, Adsorption of oligonucleotides onto negatively and positively charged latex particles, Colloids Surf. A: Physicochem. Eng. Aspects 83 (1994) 25-31.

[215] N.H. Kim, H.Y. Lee, Y. Cho, W.S. Han, D. Kang, S.S. Lee, J.H. Jung, Thymidine-functionalized silica nanotubes for selective recognition and separation of oligoadenosines, J. Mater. Chem. 20 (2010) 2139-2144.

[216] H. Benesi, J. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703.

[217] J. Bariyanga, T. Theophanides, Synthesis, FT-IR and 1H NMR studies of alkali and alkaline earth metal complexes with guanosine, Inorg. Chim. Acta 108 (1985) 133-140.

[218] Z. Yang, K. Chai, H. Ji, Selective inclusion and separation of cinnamaldehyde and benzaldehyde by insoluble β-cyclodextrin polymer, Sep. Purif. Technol. 80 (2011) 209-216.

[219] M. Seno, M. Lin, K. Iwamoto, Chromatographic behaviour of cyclodextrin complexes of NADH and NADP, J. Chromatogr. A 508 (1990) 127-132.

[220] X. Hao, C. Liang, C. Jian-Bin, Preparation and spectroscopic studies of an inclusion complex of adenine with beta-cyclodextrin in solution and in the solid state, Analyst 127 (2002) 834-837.

[221] M. Kondo, S. Nishikawa, Inclusion kinetics of a nucleotide into a cyclodextrin cavity by means of ultrasonic relaxation, J. Phys. Chem. B 111 (2007) 13451-13454.

References

219

[222] M.E. Skold, G.D. Thyne, J.W. Drexler, J.E. McCray, Solubility enhancement of seven metal contaminants using carboxymethyl-beta-cyclodextrin (CMCD), J. Contam. Hydrol. 107 (2009) 108-113.

[223] X. Xu, H. Shen, J. Xu, M. Xie, X. Li, The colloidal stability and core-shell structure of magnetite nanoparticles coated with alginate, Appl. Surf. Sci. 253 (2006) 2158-2164.

[224] W. Zhang, L. Zhuang, H. Shen, M. Xie, Z. Hu, H. He, Controlled synthesis and properties of carboxymethyl chitosan-bound magnetic nanoparticles, J. Sci. Conf. Proc. 1 (2009) 211-214.

[225] P.I. Girginova, A.L. Daniel-da-Silva, C.B. Lopes, P. Figueira, M. Otero, V.S. Amaral, E. Pereira, T. Trindade, Silica coated magnetite particles for magnetic removal of Hg2+ from water, J. Colloid Interface Sci. 345 (2010) 234-240.

[226] L. Xiong, Y. Yang, J. Mai, W. Sun, C. Zhang, D. Wei, Q. Chen, J. Ni, Adsorption behavior of methylene blue onto titanate nanotubes, Chem. Eng. J. 156 (2010) 313-320.

[227] F.A. Pavan, E.C. Lima, S.L.P. Dias, A.C. Mazzocato, Methylene blue biosorption from aqueous solutions by yellow passion fruit waste, J. Hazard. Mater. 150 (2008) 703-712.

[228] G. Zhang, S. Shuang, C. Dong, J. Pan, Study on the interaction of methylene blue with cyclodextrin derivatives by absorption and fluorescence spectroscopy, Spectrochim. Acta A Mol. Biomol. Spectrosc. 59 (2003) 2935-2941.

[229] J. Zhi, X. Tian, W. Zhao, J. Shen, B. Tong, Y. Dong, Self-assembled film based on carboxymethyl-beta-cyclodextrin and diazoresin and its binding properties for methylene blue, J. Colloid Interface Sci. 319 (2008) 270-276.

[230] S. Karagöz, T. Tay, S. Ucar, M. Erdem, Activated carbons from waste biomass by sulfuric acid activation and their use on methylene blue adsorption, Bioresour. Technol. 99 (2008) 6214-6222.

[231] M.T. Uddin, M.A. Islam, S. Mahmud, M. Rukanuzzaman, Adsorptive removal of methylene blue by tea waste, J. Hazard. Mater. 164 (2009) 53-60.

[232] E.N.E. Qada, S.J. Allen, G.M. Walker, Adsorption of Methylene Blue onto activated carbon produced from steam activated bituminous coal A study of equilibrium adsorption isotherm, Chem. Eng. J. 124 (2006) 103-110.

[233] N. Kannan, M.M. Sundaram, Kinetics and mechanism of removal of methylene blue by adsorption on various carbons-a comparative study, Dyes and Pigments 51 (2001) 25-40.

References

220

[234] C.H. Weng, Y.F. Pan, Adsorption of a cationic dye (methylene blue) onto spent activated clay, J. Hazard. Mater. 144 (2007) 355-362.

[235] W.T. Tsai, K.J. Hsien, J.M. Yang, Silica adsorbent prepared from spent diatomaceous earth and its application to removal of dye from aqueous solution, J. Colloid Interface Sci. 275 (2004) 428-433.

[236] C. Namasivayam, S. Sumithra, Removal of direct red 12B and methylene blue from water by adsorption onto Fe(III)/Cr(III) hydroxide, an industrial solid waste, J. Environ. Manage. 74 (2005) 207-215.

[237] Y. Liu, Is the Free Energy Change of Adsorption Correctly Calculated?, J. Chem. Eng. Data 54 (2009) 1981-1985.

[238] 0. Egyed, V. Weiszfeiler, Structure determination of copper(II)-ß-cyclodextrin complex by Fourier transform infrared spectroscopy, Vib. Spectrosc. 7 (1994) 73-77.

[239] O. Ozay, S. Ekici, Y. Baran, N. Aktas, N. Sahiner, Removal of toxic metal ions with magnetic hydrogels, Water Res. 43 (2009) 4403-4411.

[240] Ç. Üzüm, T. Shahwan, A. Eroğlu, K. Hallam, T. Scott, I. Lieberwirth, Synthesis and characterization of kaolinite-supported zero-valent iron nanoparticles and their application for the removal of aqueous Cu2+ and Co2+ ions, Appl. Clay Sci. 43 (2009) 172-181.

[241] C.L. Chen, X.K. Wang, M. Nagatsu, Europium adsorption on multiwall carbon nanotube/iron oxide magnetic composite in the presence of polyacrylic acid, Environ. Sci. Technol. 43 (2009) 2362-2367.

[242] J. Hu, D. Shao, C. Chen, G. Sheng, J. Li, X. Wang, M. Nagatsu, Plasma-induced grafting of cyclodextrin onto multiwall carbon nanotube/iron oxides for adsorbent application, J. Phys. Chem. B 114 (2010) 6779-6785.

[243] L.C.A. Oliveira, D.I. Petkowicz, A. Smaniotto, S.B.C. Pergher, Magnetic zeolites: a new adsorbent for removal of metallic contaminants from water, Water Res. 38 (2004) 3699-3704.

[244] L.C.A. Oliveira, R.V.R.A. Rios, J.D. Fabris, V. Garg, K. Sapag, R.M. Lago, Activated/carbon iron oxide magnetic composites for the adsorption of contaminants in water, Carbon 40 (2002) 2177-2183.

[245] Y.T. Zhou, C. Branford-White, H.L. Nie, L.M. Zhu, Adsorption mechanism of Cu2+ from aqueous solution by chitosan-coated magnetic nanoparticles modified with a-ketoglutaric acid, Colloids Surf. B Biointerfaces 74 (2009) 244-252.

References

221

[246] E. Norkus, Metal ion complexes with native cyclodextrins: An overview, J. Inclusion Phenom. Macrocyclic Chem. 65 (2009) 237-248.

[247] X. Wang, M.L. Brusseau, Simultaneous complexation of organic compounds and heavy metals by a modified cyclodextrin, Environ. Sci. Technol. 29 (1995) 2632-2635.

[248] M.L. Brusseau, X. Wang, W.J. Wang, Simultaneous elution of heavy metals and organic compounds from soil by cyclodextrin, Environ. Sci. Technol. 31 (1997) 1087-1092.

[249] C.A. Kozlowski, T. Girek, W. Walkowiak, J. Kozlowska, The effect of β-CD polymers structure on the efficiency of copper(II) ion flotation, J. Inclusion Phenom. Macrocyclic Chem. 55 (2006) 71-77.

[250] G. Sheng, J. Li, D. Shao, J. Hu, C. Chen, Y. Chen, X. Wang, Adsorption of copper(II) on multiwalled carbon nanotubes in the absence and presence of humic or fulvic acids, J. Hazard. Mater. 178 (2010) 333-340.

[251] H. Chen, G. Dai, J. Zhao, A. Zhong, J. Wu, H. Yan, Removal of copper(II) ions by a biosorbent--Cinnamomum camphora leaves powder, J. Hazard. Mater. 177 (2010) 228-236.

[252] G. Zhao, H. Zhang, Q. Fan, X. Ren, J. Li, Y. Chen, X. Wang, Sorption of copper(II) onto super-adsorbent of bentonite-polyacrylamide composites, J. Hazard. Mater. 173 (2010) 661-668.

[253] S. Deng, R. Bai, J.P. Chen, Aminated polyacrylonitrile fibers for lead and copper removal, Langmuir 19 (2003) 5058-5064.

[254] O. Yavuz, Y. Altunkaynak, F. Güzel, Removal of copper, nickel, cobalt and manganese from aqueous solution by kaolinite, Water Res. 37 (2003) 948-952.

[255] C. Chen, X. Wang, Adsorption of Ni(II) from aqueous solution using oxidized multiwall carbon nanotubes, Ind. Eng. Chem. Res. 45 (2006) 9144-9149.

[256] S. Yang, J. Li, D. Shao, J. Hu, X. Wang, Adsorption of Ni(II) on oxidized multi-walled carbon nanotubes: effect of contact time, pH, foreign ions and PAA, J. Hazard. Mater. 166 (2009) 109-116.

[257] J. Wu, H.Q. Yu, Biosorption of 2,4-dichlorophenol from aqueous solution by Phanerochaete chrysosporium biomass: isotherms, kinetics and thermodynamics, J. Hazard. Mater. 137 (2006) 498-508.

References

222

[258] G.Z. Kyzas, M. Kostoglou, N.K. Lazaridis, Copper and chromium(VI) removal by chitosan derivatives-Equilibrium and kinetic studies, Chem. Eng. J. 152 (2009) 440-448.

[259] Y. Xia, J. Wan, Preparation and adsorption of novel cellulosic fibers modified by β-cyclodextrin, Polym. Adv. Technol. 19 (2008) 270-275.

[260] T. Aman, A.A. Kazi, M.U. Sabri, Q. Bano, Potato peels as solid waste for the removal of heavy metal copper(II) from waste water/industrial effluent, Colloids Surf. B Biointerfaces 63 (2008) 116-121.

[261] W. West, Chemical Applications of Spectroscopy, Interscience, New York, 1956.

[262] C. Liu, R. Bai, Q. San Ly, Selective removal of copper and lead ions by diethylenetriamine-functionalized adsorbent: behaviors and mechanisms, Water Res. 42 (2008) 1511-1522.

[263] S.F. Lim, Y.M. Zheng, S.W. Zou, J.P. Chen, Characterization of copper adsorption onto an alginate encapsulated magnetic sorbent by a combined FT-IR, XPS, and mathematical modeling study, Environ. Sci. Technol. 42 (2008) 2551-2556.

[264] J.C. Zheng, H.M. Feng, M.H.W. Lam, P.K.S. Lam, Y.W. Ding, H.Q. Yu, Removal of Cu(II) in aqueous media by biosorption using water hyacinth roots as a biosorbent material, J. Hazard. Mater. 171 (2009) 780-785.

[265] N. Li, R. Bai, Copper adsorption on chitosan–cellulose hydrogel beads: behaviors and mechanisms, Sep. Purif. Technol. 42 (2005) 237-247.

[266] J. Kang, H. Liu, Y.M. Zheng, J. Qu, J.P. Chen, Systematic study of synergistic and antagonistic effects on adsorption of tetracycline and copper onto a chitosan, J. Colloid Interface Sci. 344 (2010) 117-125.

[267] G. Sheng, S. Wang, J. Hu, Y. Lu, J. Li, Y. Dong, X. Wang, Adsorption of Pb(II) on diatomite as affected via aqueous solution chemistry and temperature, Colloids Surf. A: Physicochem. Eng. Aspects 339 (2009) 159-166.

[268] J. Gao, H. Gu, B. Xu, Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications, Acc. Chem. Res. 42 (2009) 1097-1107.

[269] H.S. Cho, Z. Dong, G.M. Pauletti, J. Zhang, H. Xu, H. Gu, L. Wang, R.C. Ewing, C. Huth, F. Wang, D. Shi, Fluorescent, superparamagnetic nanospheres for drug storage, targeting, and imaging: a multifunctional nanocarrier system for cancer diagnosis and treatment, ACS Nano 4 (2010) 5398-5404.

References

223

[270] T.J. Yoon, K.N. Yu, E. Kim, J.S. Kim, B.G. Kim, S.H. Yun, B.H. Sohn, M.H. Cho, J.K. Lee, S.B. Park, Specific targeting, cell sorting, and bioimaging with smart magnetic silica core-shell nanomaterials, Small 2 (2006) 209-215.

[271] J.M. Idée, M. Port, I. Raynal, M. Schaefer, B. Bonnemain, P. Prigent, P. Robert, C. Robic, C. Corot, The Superparamagnetic Iron Oxides Nanoparticles for Magnetic Resonance Imaging Applications, Wiley-VCH: Weinheim, Germany, 2007.

[272] B. Chertok, A.E. David, V.C. Yang, Brain tumor targeting of magnetic nanoparticles for potential drug delivery: effect of administration route and magnetic field topography, J. Control Release 155 (2011) 393-399.

[273] Y. Chen, H. Chen, D. Zeng, Y. Tian, F. Chen, J. Feng, J. Shi, Core/shell structured hollow mesoporous nanocapsules: a potential platform for simultaneous cell imaging and anticancer drug delivery, ACS Nano 4 (2010) 6001-6013.

[274] Z. Xu, C. Li, X. Kang, D. Yang, P. Yang, Z. Hou, J. Lin, Synthesis of a multifunctional nanocomposite with magnetic, mesoporous, and Near-IR absorption properties, J. Phys. Chem. C 114 (2010) 16343-16350.

[275] T. Yoon, K. Yu, E. Kim, J. Kim, B. Kim, S. Yun, B. Sohn, M. Cho, J. Lee, S. Park, Specific targeting, cell sorting, and bioimaging with smart magnetic silica core-shell nanomaterials, Small 2 (2006) 209-215.

[276] R. Di Corato, N. Bigall, A. Ragusa, D. Dorfs, A. Genovese, R. Marotta, L. Manna, T. Pellegrino, Multifunctional nanobeads based on quantum dots and magnetic nanoparticles: Synthesis and cancer cell targeting and sorting, ACS Nano 5 (2011) 1109-1121.

[277] Y.S. Lin, C.L. Haynes, Synthesis and characterization of biocompatible and size-tunable multifunctional porous silica nanoparticles, Chem. Mater. 21 (2009) 3979-3986.

[278] J.M. Rosenholm, A. Meinander, E. Peuhu, R. Niemi, J.E. Eriksson, C. Sahlgren, M. Lindén, Targeting of porous hybrid silica nanoparticles to cancer cells, ACS Nano 3 (2009) 197-206.

[279] L. Li, E.S.G. Choo, Z. Liu, J. Ding, J. Xue, Double-layer silica core-shell nanospheres with superparamagnetic and fluorescent functionalities, Chem. Phys. Lett. 461 (2008) 114-117.

[280] L. Sun, Y. Zang, M. Sun, H. Wang, X. Zhu, S. Xu, Q. Yang, Y. Li, Y. Shan, Synthesis of magnetic and fluorescent multifunctional hollow silica nanocomposites for live cell imaging, J. Colloid Interface Sci. 350 (2010) 90-98.

References

224

[281] S. Mohapatra, S.K. Mallick, T.K. Maiti, S.K. Ghosh, P. Pramanik, Synthesis of highly stable folic acid conjugated magnetite nanoparticles for targeting cancer cells, Nanotechnology 18 (2007) 385102-385110.

[282] S. Xuan, F. Wang, J.M.Y. Lai, K.W.Y. Sham, Y.X.J. Wang, S.F. Lee, J.C. Yu, C.H.K. Cheng, K.C.F. Leung, Synthesis of biocompatible, mesoporous Fe3O4 nano/microspheres with large surface area for magnetic resonance imaging and therapeutic applications, ACS Appl. Mater. Interfaces 3 (2011) 237-244.

[283] R. Di Corato, N.C. Bigall, A. Ragusa, D. Dorfs, A. Genovese, R. Marotta, L. Manna, T. Pellegrino, Multifunctional nanobeads based on quantum dots and magnetic nanoparticles: synthesis and cancer cell targeting and sorting, ACS Nano 5 (2011) 1109-1121.

[284] J. Kim, H. Kim, N. Lee, T. Kim, H. Kim, T. Yu, I. Song, W. Moon, T. Hyeon, Multifunctional uniform nanoparticles composed of a magnetite nanocrystal core and a mesoporous silica shell for magnetic resonance and fluorescence imaging and for drug delivery, Angew. Chem. Int. Ed 47 (2008) 8438-8441.

[285] H. Ow, D.R. Larson, M. Srivastava, B.A. Baird, W.W. Webb, U. Wiesner, Bright and stable core-shell fluorescent silica nanoparticles, Nano Lett. 5 (2005) 113-117.

[286] Y.S. Lin, S.H. Wu, Y. Hung, Y.H. Chou, C. Chang, M.L. Lin, C.P. Tsai, C.Y. Mou, Multifunctional composite nanoparticles: magnetic, luminescent, and mesoporous, Chem. Mater. 18 (2006) 5170-5172.

[287] S.A. Corr, Y.P. Rakovich, Y.K. Gun’ko, Multifunctional magnetic-fluorescent nanocomposites for biomedical applications, Nanoscale Res. Lett. 3 (2008) 87-104.

[288] H. Yang, Y. Zhuang, H. Hu, X. Du, C. Zhang, X. Shi, H. Wu, S. Yang, Silica-coated manganese oxide nanoparticles as a platform for targeted magnetic resonance and fluorescence imaging of cancer cells, Adv. Funct. Mater. 20 (2010) 1733-1742.

[289] J.E. Lee, N. Lee, H. Kim, J. Kim, S.H. Choi, J.H. Kim, T. Kim, I.C. Song, S.P. Park, W.K. Moon, T. Hyeon, Uniform mesoporous dye-doped silica nanoparticles decorated with multiple magnetite nanocrystals for simultaneous enhanced magnetic resonance imaging, fluorescence imaging, and drug delivery, J. Am. Chem. Soc. 132 (2010) 552-557.

[290] A. Baeza, E. Guisasola, E. Ruiz-Hernández, M. Vallet-Regí, Magnetically triggered multidrug release by hybrid mesoporous silica nanoparticles, Chem. Mater. 24 (2012) 517-524.

[291] F.H. Chen, L.M. Zhang, Q.T. Chen, Y. Zhang, Z.J. Zhang, Synthesis of a novel magnetic drug delivery system composed of doxorubicin-conjugated Fe3O4 nanoparticle

References

225

cores and a PEG-functionalized porous silica shell, Chem. Commun. 46 (2010) 8633-8635.

[292] K. Hayashi, K. Ono, H. Suzuki, M. Sawada, M. Moriya, W. Sakamoto, T. Yogo, High-frequency, magnetic-field-responsive drug release from magnetic nanoparticle/organic hybrid based on hyperthermic effect, ACS Appl. Mater. Interfaces 2 (2010) 1903-1911.

[293] K. Peng, C. Cui, I. Tomatsu, F. Porta, A.H. Meijer, H.P. Spaink, A. Kros, Cyclodextrin/dextran based drug carriers for a controlled release of hydrophobic drugs in zebrafish embryos, Soft Matter 6 (2010) 3778.

[294] K. Uekama, F. Hirayama, H. Arima, Recent aspect of cyclodextrin-based drug delivery system, J. Inclusion Phenom. Macrocyclic Chem. 56 (2006) 3-8.

[295] H.L. Ramírez, A. Valdivia, R. Cao, J.J. Torres-Labandeira, A. Fragoso, R. Villalonga, Cyclodextrin-grafted polysaccharides as supramolecular carrier systems for naproxen, Bioorg. Med. Chem. Lett. 16 (2006) 1499-1501.

[296] H. Cao, J. He, L. Deng, X. Gao, Fabrication of cyclodextrin-functionalized superparamagnetic Fe3O4/amino-silane core–shell nanoparticles via layer-by-layer method, Appl. Surf. Sci. 255 (2009) 7974-7980.

[297] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug Deliv. Rev. 62 (2010) 1052-1063.

[298] J. Xie, S. Lee, X. Chen, Nanoparticle-based theranostic agents, Adv. Drug Deliv. Rev. 62 (2010) 1064-1079.

[299] M.T. López-López, J.D.G. Durán, A.V. Delgado, F. González-Caballero, Stability and magnetic characterization of oleate-covered magnetite ferrofluids in different nonpolar carriers, J. Colloid Interface Sci. 291 (2005) 144-151.

[300] S. Santra, H. Yang, D. Dutta, J.T. Stanley, P.H. Holloway, W. Tan, B.M. Moudgil, R.A. Mericle, TAT conjugated, FITC doped silica nanoparticles for bioimaging applications, Chem. Commun. (2004) 2810-2811.

[301] Q.X. Guo, T. Ren, Y.P. Fang, Y.C. Liu, Binding of vitamin A by β-cyclodextrin and heptakis(2,6-O-dimethyl)-β-cyclodextrin, J. Inclusion Phenom. Mol. Recogn. Chem. 22 (1995) 251-256.

[302] Y. Liu, G.S. Chen, Y. Chen, J. Lin, Inclusion complexes of azadirachtin with native and methylated cyclodextrins: solubilization and binding ability, Bioorg. Med. Chem .13 (2005) 4037-4042.

References

226

[303] V. Crupi, R. Ficarra, M. Guardo, D. Majolino, R. Stancanelli, V. Venuti, UV-vis and FTIR-ATR spectroscopic techniques to study the inclusion complexes of genistein with beta-cyclodextrins, J. Pharm. Biomed. Anal. 44 (2007) 110-117.

[304] R. Challa, A. Ahuja, J. Ali, R. Khar, Cyclodextrins in drug delivery: an updated review, AAPS PharmSciTech 6 (2005) 329-357.

[305] A. Ascenso, R. Guedes, R. Bernardino, H. Diogo, F.A. Carvalho, N.C. Santos, A.M. Silva, H.C. Marques, Complexation and full characterization of the tretinoin and dimethyl-beta-cyclodextrin complex, AAPS PharmSciTech 12 (2011) 553-563.

[306] S.S. Banerjee, D.H. Chen, Grafting of 2-hydroxypropyl-b-cyclodextrin on Gum Arabic-modified iron oxide nanoparticles as a magnetic carrier for targeted delivery of hydrophobic anticancer drug, Int. J. Appl. Ceram. Technol. 7 (2010)

[307] B.P. Timko, T. Dvir, D.S. Kohane, Remotely triggerable drug delivery systems, Adv. Mater. 22 (2010) 4925-4943.

[308] N.A.P. Philip L. Ritger, A Simple equation for description of solute release II. Fickian and anomalous release from swellable devices, J. Control Release 5 (1987) 37-42.

[309] F.M. Koehler, M. Rossier, M. Waelle, E.K. Athanassiou, L.K. Limbach, R.N. Grass, D. Günther, W.J. Stark, Magnetic EDTA: coupling heavy metal chelators to metal nanomagnets for rapid removal of cadmium, lead and copper from contaminated water, Chem. Commun. (2009) 4862-4864.

[310] Y. Zhang, S. Xu, Y. Luo, S. Pan, H. Ding, G. Li, Synthesis of mesoporous carbon capsules encapsulated with magnetite nanoparticles and their application in wastewater treatment, J. Mater. Chem. 21 (2011) 3664.

[311] L. Sun, C. Tian, L. Wang, J. Zou, G. Mu, H. Fu, Magnetically separable porous graphitic carbon with large surface area as excellent adsorbents for metal ions and dye, J. Mater. Chem. 21 (2011) 7232.

[312] T. Ishiwata, O. Miura, K. Hosomi, K. Shimizu, D. Ito, Y. Yoda, Removal and recovery of phosphorus in wastewater by superconducting high gradient magnetic separation with ferromagnetic adsorbent, Physica C: Superconductivity 470 (2010) 1818-1821.

[313] R. Egashira, T.A. Hatton, Process for removal of hydrocarbon from water with magnetic particles, SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 12 (2005) 59-68.

References

227

[314] M. Rossier, A. Schaetz, E.K. Athanassiou, R.N. Grass, W.J. Stark, Reversible As(V) adsorption on magnetic nanoparticles and pH dependent desorption concentrates dilute solutions and realizes true moving bed reactor systems, Chem. Eng. J. 175 (2011) 244-250.

[315] G. Gubitz, M.G. Schmid, Chiral separation by chromatographic and electromigration techniques. A Review, Biopharm. Drug Dispos. 22 (2001) 291-336.

[316] S. Ghosh, A.Z.M. Badruddoza, M.S. Uddin, K. Hidajat, Adsorption of chiral aromatic amino acids onto carboxymethyl-β-cyclodextrin bonded Fe3O4/SiO2 core-shell nanoparticles, J. Colloid Interface Sci. 354 (2010) 483-492.

[317] M.J. Benotti, R.A. Trenholm, B.J. Vanderford, J.C. Holady, B.D. Stanford, S.A. Snyder, Pharmaceuticals and endocrine disrupting compounds in U.S. drinking water, Environ. Sci. Technol. 43 (2009) 597-603.

[318] Z. Yu, S. Peldszus, P.M. Huck, Adsorption characteristics of selected pharmaceuticals and an endocrine disrupting compound-Naproxen, carbamazepine and nonylphenol-on activated carbon, Water Res. 42 (2008) 2873-2882.

[319] T.A. Ternes, M. Stumpf, J. Mueller, K. Haberer, R.D. Wilken, M. Servos, Behavior and occurrence of estrogens in municipal sewage treatment plants--I. Investigations in Germany, Canada and Brazil, Sci. Total Environ. 225 (1999) 81-90.

[320] T.X. Bui, H. Choi, Adsorptive removal of selected pharmaceuticals by mesoporous silica SBA-15, J. Hazard. Mater. 168 (2009) 602-608.

[321] Y. Dong, D. Wu, X. Chen, Y. Lin, Adsorption of bisphenol A from water by surfactant-modified zeolite, J. Colloid Interface Sci. 348 (2010) 585-590.

[322] J. Zhu, J. He, X. Du, R. Lu, L. Huang, X. Ge, A facile and flexible process of β-cyclodextrin grafted on Fe3O4 magnetic nanoparticles and host–guest inclusion studies, Appl. Surf. Sci. 257 (2011) 9056-9062.

[323] H. Wang, Y. Zhou, Y. Guo, W. Liu, C. Dong, Y. Wu, S. Li, S. Shuang, β-Cyclodextrin/Fe3O4 hybrid magnetic nano-composite modified glassy carbon electrode for tryptophan sensing, Sensors and Actuators B: Chemical 163 (2012) 171-178.

[324] Y. Ji, X. Liu, M. Guan, C. Zhao, H. Huang, H. Zhang, C. Wang, Preparation of functionalized magnetic nanoparticulate sorbents for rapid extraction of biphenolic pollutants from environmental samples, J. Sep. Sci. 32 (2009) 2139-2145.

[325] K. Yang, W. Wu, Q. Jing, L. Zhu, Aqueous adsorption of aniline, phenol, and their substitutes by multi-walled carbon nanotubes, Environ. Sci. Technol. 42 (2008) 7931-7936.

References

228

[326] E. Mateo-Martí, C. Briones, E. Román, E. Briand, C.M. Pradier, J.A. Martín-Gago, Self-assembled monolayers of peptide nucleic acids on gold surfaces: a spectroscopic study, Langmuir 21 (2005) 9510-9517.

[327] N. Cottenye, F. Teixeira, A. Ponche, G. Reiter, K. Anselme, W. Meier, L. Ploux, C. Vebert-Nardin, Oligonucleotide nanostructured surfaces: effect on Escherichia coli curli expression, Macromol. Biosci. 8 (2008) 1161-1172.

[328] I. Suzuki, T. Miura, J.I. Anzai, Superiority of secondary hydroxy side modification in cyclodextrin complexation for highly hydrophilic adenine nucleotides, J. Supramol. Chem. 1 (2001) 283-288.

[329] R.P. Bagwe, C. Yang, L.R. Hilliard, W. Tan, Optimization of dye-doped silica nanoparticles prepared using a reverse microemulsion method, Langmuir 20 (2004) 8336-8342.

[330] M. Fernández, M.L. Villalonga, A. Fragoso, Caob, M. Baños, R. Villalonga, α-Chymotrypsin stabilization by chemical conjugation with O-carboxymethyl-poly-β-cyclodextrin, Process Biochem. 39 (2004) 535-539.

[331] F. Qin, B. Wen, X.Q. Shan, Y.N. Xie, T. Liu, S.Z. Zhang, S.U. Khan, Mechanisms of competitive adsorption of Pb, Cu, and Cd on peat, Environ. Pollut. 144 (2006) 669-680.

Appendix

229

Appendix A: Supporting information for Chapter 5 A-1 Magnetic properties of CMCD-APES-MNPs

The magnetic properties of the uncoated and CM-β-CD coated Fe3O4 nanoparticles

were measured by vibrating sample magnetometer (VSM) at room temperature. From

M vs. H curves (Figure A-1), the saturation magnetization value (Ms) of uncoated

MNPs was found to be 75 emu g-1 which is much less than its bulk counterpart (92 emu

g-1) [186]. For CD-APES-MNPs, the magnetization obtained at the same field was 61.5

emu g-1, lower than that of uncoated Fe3O4. This is mainly attributed to the presence of

nonmagnetic materials (APES and CM-β-CD layer) on the surface of nanoparticles,

which might have quenched the magnetic moment. The high magnetic response is

further demonstrated by applying a magnet to the outside of a vial containing the

CMCD-APES-MNPs aqueous solution (the inset of Figure A-1). This feature allows the

as-synthesized nanoparticles for highly efficient magnetic manipulation when used as

nano-adsorbents even under a relatively low external magnetic field. The magnetic

separation was accomplished efficiently within few minutes in our experiments. The

saturated magnetization value of the magnetic particles could be compared with other

magnetic adsorbents reported in literature [108,225]. In our lab-scale batch experiment

we used permanent magnet which could induce fields less than 1 T. Though this

magnetic field is suitable in small volume applications, a high gradient magnetic field

separator, however, would be required in a large scale operation. A properly designed

magnet in a HGMS separator can greatly improves the performance. Different types of

separator based on magnet type could be used as discussed in Section 2.5. However, the

separation efficiency largely depends on the magnetic field applied, magnetization of

materials, volume of liquid handled etc. For large-scale operation, the need of

Appendix

230

producing high filed generators and developing materials with high saturation

magnetization is a reality.

Figure A-1 Magnetization curves for uncoated MNPs and CMCD-APES-MNPs. A-2 XPS analysis of as-synthesized magnetic nanoparticles

XPS analysis was applied to find the chemical binding in the as-synthesized CMCD-

APES-MNPs. The C1s deconvoluted spectrum is shown in Figure A-2. The C1s

spectrum can be curve-fitted into five peak components with binding energy of about

283.1, 284.6, 286.0, 287.3 and 288.7 eV, attributable to the carbon atoms in the forms

of Si–C, C–C (aromatic), C–O (alcoholic hydroxyl and ether), O=C–N and O=C–O

species, respectively [324]. The C–O/C–O–C peak is characteristic of β-CD, confirming

that the modification of magnetic nanoparticles with β-CD derivative was

successful.The broad peak for N1s could be fitted into two peaks at 398.8 and 400.4 eV

corresponding to –NHCO and –NH2 [281]. The appearance of –NHCO peak suggests

-80

-60

-40

-20

0

20

40

60

80

-15 -10 -5 0 5 10 15

M, e

mu

/g F

e 3O

4

H, kOe

CMCD-APES-MNPs

Fe3O4 MNPs

Appendix

231

394 396 398 400 402 404 406

Cou

nts (

a.u)

Binding energy (eV)

-NH-CO

-NH2

N 1s

that CM-β-CD is grafted on the surface of magnetic nanoparticles by amide bond

formation.

Figure A-2 XPS C1s and N1s spectra of CMCD-APES-MNPs. A-3 Effects of pH The surface charge of the adsorbent and the ionization degree of adsorbate are generally

affected by solution pH values. When pH< pKa, the non-dissociated species and the

dissociated species (bases form cations by protonation at pH) are dominated for organic

acids and organic bases, respectively; whereas when pH> pKa, the dissociated species

(anions) is dominant for organic acids and the non-dissociated species is dominant for

organic bases [325]. The fraction of non-dissociated species for organic acids (fAN) and

bases (fBN) can be respectively estimated by fA

N = (1 + 10pH−pKa)−1 and fBN = (1 +

10pKa−pH)−1, and the fraction of dissociated species for organic acids (fAD) and bases

(fBD) can be respectively estimated by fA

D = (1 + 10pKa−pH)−1 and fBD = (1 + 10pH−pKa)−1,

respectively. In order to properly interpret the nucleosides adsorption behavior, the

distribution of non-dissociated and dissociated species of guanosine and adenosine as a

function of pH is shown in Figure A-3.

279 281 283 285 287 289 291

Cou

nts (

a.u)

Binding enegry (eV)

C 1s

Si-C

C-C/C-H

C-O/C-O-

O=C-NO=C-O

Appendix

232

(A)

(B)

Figure A-3 (A) Distribution of guanosine (G) and adenosine (A) species as a function of

pH; (B) Ionization mechanism of A and G.

A-4 Spectral evidence of nucleoside adsorption onto CMCD-APES-MNPs

High resolution XPS spectra provide information on the chemical environment of each

of the detected atoms on the surface. In our study, the N 1s high-resolution spectra were

studied to examine the possible existence of chemical interaction in the adsorption of

nucleosides onto magnetic nanoparticles. Three chemical environments were

distinguishable in the N 1s high resolution spectrum after nucleoside adsorption (Figure

A-4). Deconvolution of the N 1s peak of CMCD-APES-MNPs before nucleoside

Appendix

233

adsorption produces two peaks of binding energies 398.8 and 400.4 eV corresponding

to –NHCO and –NH2 [281]. As guanine and adenine residues both contain 5 nitrogen

atoms but under different forms (3 –N=, 1 –NH– & 1 –NH2 for adenine residues and 2

–N=, 2 –NH– & 1 –NH2 for guanine residues), it was expected to find all the

corresponding N 1s deconvoluted peaks for CMCD-APES-MNPs after adsorption with

A or G. The N 1s spectrum for CMCD-APES-MNPs after A or G adsorption was fit

with three peaks at BEs of 398.2 eV, 399.5 eV and 400.5 eV corresponding to

conjugated nitrogen (–N=) in the nucleic bases, non-conjugated nitrogen (–NH– and N

with three single bonds) and the (–NH2), respectively, [326,327]. The presence of –N=

peaks on A or G-loaded CMCD-APES-MNPs confirms the adsorption of nucleosides

onto magnetic surfaces.

Figure A-4 N 1s high resolution peaks and peak fitting: (a) CMCD-APES-MNPs before

adsorption; (b) after guanosine adsorption; (c) after adenosine adsorption. A-5 Determination of binding constants between nucleoside and CDs

The inclusion capability of β-CD towards nucleosides was investigated by circular

dichroism spectra, respectively. Circular dichroism (CD) spectra were obtained on a

Jacob J810 spectropolarimeter at 25oC. The concentrations of adenosine (A) and

guanosine (G) were controlled at 0.075 mM and concentration of β-CD was varied from

2 mM to 18 mM. The molar ellipticities of β-CD:A and β-CD:G solutions at

Appendix

234

wavelength from 220 nm to 320 nm were measured separately by circular dichroism at

25oC with a 5-mm path length sample cell (data not shown here). Molar ellipticity

differences, ∆θ, were recorded at the wavelength at which the differences between pure

and complexed nucleosides were maximized (approx. 260 nm) as a function of β-CD

concentration. These spectral variations induced by β-CD and its derivatives would be

caused by the insertion of the nucleosides into optically active β-CD cavity and by the

conformational changes of the purine derivatives. The binding constants were obtained

from double reciprocal plots of 1/∆θ vs 1/[β-CD] according to the following equation:

∆ ∆ AB ‐ ∆ AB

(A-1)

where K is the binding constant and ∆θAB is the coefficient for right and left circularly

polarized light between the free and complexed nucleosides. From the slope and

intercept in Figure A-5 the binding constants of β-CD for A and G are calculated to be

17.9 and 30.3 M-1 respectively. The 1:1 stoichiometry of complexes of β-CD with A or

G is also suggested by UV-circular dichroism spectra. The formation constant of A is

very close to the literature value (16 M-1) reported by Suzuki et al. using the same

method [328]. The other study by Xiang and Anderson using solubility method also

yielded a similar value of 13 M-1 for adenosine complex [56].

Appendix

235

Figure A-5 Double reciprocal plots for A and G complexes to β-CD measured from

circular dichroism spectral data.

A-6 Preparation of inclusion complexes and DSC study

The required amount of A or G and aqueous solution of CM-β-CD (2.0 mM) were

mixed (1:1 molar ratio) and stirred at 25oC for 2 days with a thermostatic water-bath

shaker. After this period the solid residue was separated by centrifugation at 12,000 rpm

for 15 min and the upper liquid layer was filtered over 0.45 µm membrane. The

resulting solution was frozen at -18 oC for 24 h and then dried 48 h by lyophilization for

the solid inclusion complex to be collected.

Figure A-6 illustrates the DSC thermograms of β-CD, adenosine, guanosine and their

physical mixtures and inclusion complexes. The DSC curve of β-CD showed a broad

endothermic peak in the range of 130-170oC, which could be attributed to the release of

water molecule from the cavity (desolvation). The thermograms of adenosine and

guanosine were typical of highly crystalline compounds, characterized by a sharp

endothermic peak at 237.2 and 249.9oC corresponding to their melting point,

Appendix

236

respectively. In their physical mixtures, the DSC pattern revealed the presence of peaks

of both pure compounds except that the melting endotherm of adenosine and guanosine

slightly shifted to 234.2 and 254oC, respectively with a decrease in the peak intensities.

However, the absence of the characteristic peaks of adenosine and guanosine in the

adenosine:β-CD and guanosine:β-CD complexes is a strong evidence of the inclusion of

nucleosides inside the cyclodextrin cavities.

Figure A-6 DSC curves of (a) adenosine and (b) guanosine and their physical mixtures and inclusion complexes with CM-β-CD.

25 75 125 175 225 275

Hea

t flo

w (m

W)

Temperature (oC)

Inclusion complex

(237.2oC)

CM-β-CD

Adenosine

Physical mixture

(a)

25 75 125 175 225 275

Hea

t fl

ow (m

W)

Temperature (oC)

Inclusion complex

Physical mixture

CM-β-CD

Guanosine

249.9oC

(b)

Appendix

237

Appendix B: Supporting information for Chapter 8

Figure B-1 XRD patterns of a) Fe3O4 nanoparticles and b) Fe3O4@SiO2(FITC)

nanoparticles. The symbol of * indicates the broad peak of amorphous silica

Figure B-2 FTIR spectra of oleic acid capped-Fe3O4 (OA-MNPs) and fluorescent dye-

doped magnetic silica nanoparticles [Fe3O4@SiO2(FITC) NPs].

Appendix

238

Figure B-3 Field-dependent magnetization curve of Fe3O4@SiO2(FITC)]-FA/CMCD NPs at 300K. Effects of water-surfactant molar ratio on sizes of core-shell silica magnetic nanoparticles Bagwe et al. found that the microemulsion parameters such as reactant concentrations

(ammonium hydroxide), nature of surfactant molecules, and molar ratios of water to

surfactant and cosurfactant to surfactant influenced the fluorescence spectra, particle

size, and size distribution of Ru(bpy) dye-doped silica nanoparticles [329]. In this

study, in order to optimize the properties of the as-synthesized nanoprobes, a series of

nanoprobes were prepared with different thickness of the silica shell just by changing

the ratio of water to surfactant (triton X-100) in the synthetic process. When increasing

the ratio from 6.2 to 15, the average size of the as-synthesized MNPs decreases (Figure

B-4, TEM images). A thick silica shell allows high payloads of dye molecules and

therefore improving the fluorescent property of the nanoparticles, which is confirmed

by fluorescence emission spectra (Figure B-4). In case of smaller particles, the outer

silica shells in which the dye molecules are incorporated come nearer to the magnetic

Appendix

239

core resulting in fluorescence quenching. Also the outer layer contains low payloads of

dye molecules because of thin silica layer.

Figure B-4 TEM images Fe3O4@SiO2(FITC) nanoparticles (a-c) and their fluorescence emission spectra (recorded at 480 nm excitation).

Appendix

240

Figure B-5 (a) Hydrodynamic size distribution of Fe3O4@SiO2(FITC)]-FA/CMCD

NPs (b) Hydrodynamic diameter of Fe3O4@SiO2(FITC)]-FA/CMCD NPs measured by dynamic light scattering technique in deionized water.

Appendix

241

Appendix C

This section reports the synthesis of a novel nanoadsorbent- carboxymethyl-β-

cyclodextrin polymer grafted magnetic nanoparticles (CDpoly-MNPs). This polymer

having numerous carboxyl groups (Mp= 13,000, 40% COOH groups) has the ability to

adsorb heavy metal ions through complexation reaction with surface groups, mainly

carboxyl groups in the polymer matrix (Figure C-1). Competitive adsorption

experiments were conducted to elucidate the adsorption selectivity of CDpoly-MNPs on

lead ions.

C-1 Experimental

Synthesis of CM-β-CD polymer

CM-β-CD polymer was prepared following the procedure of literature [330], with the

detailed description as follows: β-cyclodextrin (5 g) was dissolved in 50 ml of 10%

(w/v) NaOH and 10 ml of epichlorohydrin was added. The system was vigorously

stirred for 8 h before another 5 ml of epichlorohydrin was added with stirring and the

mixture kept overnight at room temperature. The solution was concentrated to about 15

ml and precipitated by addition of cold ethanol (500 ml). The gummy precipitate was

crushed several times with ethanol in a mortar until a fine precipitate was obtained. The

precipitate was then washed again with ethanol and acetone and dried under high

vacuum overnight. The yield of β-CD/epichlorohydrin co-polymer was 80%. Two

grams of the above polymer was further dissolved in 50 ml 5% (w/v) NaOH and 2 g of

monochloroacetic acid was added. The system was vigorously stirred for 24 h,

neutralized with 2 M HCl, concentrated to about 15 ml and cooled to 4 oC. The

precipitated NaCl was filtered off and the supernatant was precipitated by addition of

Appendix

242

cold ethanol (500 ml). The gummy precipitate was crushed several times with ethanol

in a mortar until a fine precipitate was obtained. The precipitate was then washed two

more times with ethanol and acetone and dried under high vacuum overnight. The yield

of CM-β-CD polymer was 60%. The molecular weight of this polymer (Mp = 13000)

was determined by gel permeation chromatography on Fractogel EMD BioSEC (S)

(1.6×100 cm) calibrated with dextran standards as previously described [330].

Potentiometric titration of the –OCH2COOH groups with alkali gave a degree of

substitution of 40% (mol/mol D-glucose) for this polymer.

Synthesis of CM-β-CD polymer coated magnetic nanoparticles (CDpoly-MNPs)

CDpoly-MNPs were fabricated by one step co-precipitation method. Briefly, 0.86 g of

FeCl2.4H2O, 2.36 g FeCl3.6H2O and 1.5 g CM-β-CD polymer were dissolved in 40 ml

of de-aerated Milli-Q water with vigorous stirring at a speed of 1,200 rpm. 5 ml of

NH4OH (25%) was added after the solution was heated to 90 ºC. The reaction was

continued for 1 h at 90 ºC under constant stirring and nitrogen environment. The

resulting nanoparticles were then washed with Milli-Q water 5 to 6 times to remove any

unreacted chemicals and dried in a vacuum oven.

The step-by-step reaction procedures to synthesize CM-β-CD polymer modified

magnetic nanoparticles are shown in Figure C-1.

Appendix

243

Figure C-1 (A) Schematic presentation of CM-β-CD polymer grafting on Fe3O4 nanoparticles and (B) possible mechanism for adsorption of metal ions by CDpoly-MNPs.

Competitive Adsorption of heavy metal ions

Metal ions (Pb2+, Cd2+ and Ni2+) adsorption experiments were carried out using batch

equilibrium technique in aqueous solutions at pH 5.5 and at 25 oC. In general, an

average of 120 mg of wet magnetic nanoadsorbents (20% dry particle content) was

added to 10 ml of single, binary and ternary metals solution with each metal

concentration of 10 mg/ml and shaken in a thermostatic water-bath shaker operated at

230 rpm. After equilibrium was reached (~2 h), magnetic nanoadsorbents were

removed using a permanent Nd-Fe-B magnet and the supernatant was collected. The

Appendix

244

concentrations of Pb2+, Cd2+ and Ni2+ ions were measured using Inductive Couple

Plasma Mass Spectrometry (Agilent ICP-MS 7700 series).

C-2 Results and discussion

Characterization of magnetic nanoparticles

The grafting of CM-β-CD polymer on magnetic nanoparticles was confirmed by FTIR

and XPS analysis. Figure C-2 shows the FTIR spectra of CM-β-CD polymer, bare and

polymer coated Fe3O4 nanoparticles in the 4000-400 cm-1 wavenumber range. The

spectrum of CM-β-CD polymer shows the characteristic peaks at 1028, 1155 and 1710

cm−1. The peaks at 1028 and 1155 cm−1 correspond to the antisymmetric glycosidic

νa(C-O-C) vibrations and coupled ν (C-C/C-O) stretch vibration. The peak at 1710 cm-1

corresponds to carbonyl group (= CO) stretching which confirms the incorporation of

the carboxymethyl group (-COOCH3) into CM-β-CD polymer. The characteristic

adsorption band of magnetic nanoparticles is 586 cm-1 which is due to Fe–O bonds in

the tetrahedral sites. All the significant peaks of CM-β-CD polymer in the range of

900–1200 cm−1 are present in the spectrum of CDpoly-MNPs with a small shift.

Moreover, as shown in Fig. 1c, two main characteristic peaks appeared at 1628 and

1400 cm−1 due to bands of COOM (M represents metal ions) groups, which indicates

that the COOH groups of CM-β-CD polymer reacted with the surface OH groups of

Fe3O4 particles resulting in the formation of the iron carboxylate [205].

Appendix

245

Figure C-2 FTIR spectra of (a) CM-β-CD polymer, (b) uncoated MNPs and (b) CDpoly-MNPs.

Figure C-3 XPS C 1s spectrum of CDpoly-MNPs.

280 282 284 286 288 290 292 294

Cou

nts

(a.u

.)

Binding energy (eV)

C 1s

C-C/C-H

C-O/C-O-C

C=O

COO-

Appendix

246

The C 1s deconvoluted spectrum is shown in Figure C-3. The C 1s spectrum can be

curve-fitted into four peak components with binding energy of about 284.6, 286.1,

287.9 and 288.7 eV, attributable to the carbon atoms in the forms of C–C (aromatic),

C–O (alcoholic hydroxyl and ether), C=O (carbonyl) and COO- (carboxyl and ester)

species, respectively [264]. The C–O/C–O–C and C=O peaks are the characteristic

peaks of CM-β-CD polymer. Moreover, the presence of COO- peak at 288.7 eV

indicates that the COOH functional groups on CM-β-CD polymer reacted with surface

OH groups to form metal carboxylate (COOM). Thus, the modification of magnetic

particle surface with CM-β-CD polymer was confirmed.

Competitive adsorption of metal ions

To examine the competitive effects the metals exert on each other in multi-metal

solutions, the removal efficiencies of CDpoly-MNPs for each metal in single, binary

and ternary solutions were compared and are shown in Figure C-4. In can be seen that

the percentage removal of Pb2+, Cd2+ and Ni2+ ions in single-metal system (non-

competitive) were 99.5%, 55.9% and 24.3%, respectively. In binary metal solution

(Pb2+ - Cd2+ and Pb2+ - Ni2+), Pb2+ metal adsorption was slightly reduced (to 95.6% and

96.4% respectively) by the presence of Cd2+ or Ni2+. However, the percentage removal

of Cd2+ and Ni2+ were reduced significantly to 12.7% and 9.4% respectively in the same

binary mixtures. This indicates that Pb2+ ions were preferentially adsorbed on the

surface of CDpoly-MNPS as compared to Cd2+ or Ni2+ ions. In the binary Cd2+ - Ni2+

mixture, the removal of Cd2+ and Ni2+ were also decreased to 30.1% and 14.4%

respectively, showing the competitiveness of the two metals. In the ternary system, the

percentage removal of Pb2+ was slightly reduced (94.9%), whereas the percentage

Appendix

247

removal of Cd2+ and Ni2+ were lower than that of single or binary metal mixtures

(10.3% and 7.3% respectively).

Figure C-4 Percentage removal of Ni2+, Cd2+ and Pb2+ from single, binary and ternary mixtures (Each metal concentration: 100 mg/l, adsorbents: 120 mg, temperature: 25 °C, pH: 5.5 and contact time: 2 h).

The decrease in sorption capacity of same adsorbent in multi-metal solution than that of

single metal ion may be ascribed to the less availability of binding sites. The results

from the binary and ternary metal mixtures show that the presence of Cd2+ and/or Ni2+

has little influence on the adsorption of Pb2+ onto the CDpoly-MNPs, whereas the

adsorption of Cd2+ and Ni2+ are significantly reduced when in a competitive metal ion

environment. Hence, the order of removal efficiency for the three metal ions was Pb2+

>>Cd2+ > Ni2+, implying the stronger affinity of the adsorbent for Pb2+ than Cd2+ and

Ni2+. This tendency of higher adsorption of Pb2+ on different adsorbents containing –

COOH and –OH functional groups in multi-metal solutions was reported by other

studies [146,331].

Appendix

248

Recyclability study

The reusability was checked by following the adsorption–desorption process for four

cycles for Pb2+ ions and the adsorption efficiency in each cycle was analyzed and

presented in Figure C-5. In each cycle, 120 mg of CDpoly-MNPs and 10 ml of an initial

lead concentration of 300 ppm was used for the adsorption process, and 10 ml of 0.01

M nitric acid was used as desorption eluent. 0.01 M HNO3 could desorb around 96.0%

of Pb2+ ions from metal-loaded nanoparticles. The CDpoly-MNPs adsorbent kept its

adsorption capability after repeated adsorption–regeneration cycles with negligible

changes, indicating that there are almost no irreversible sites on the surface of CDpoly-

MNPs. Our recyclability studies suggest that these nanoadsorbents can be repeatedly

used as efficient adsorbents in wastewater treatment.

Figure C-5 Four consecutive adsorption–desorption cycles of CDpoly-MNPs adsorbent for Pb2+ (initial concentration: 300 mg/l, pH: 5.5, desorption agent: 10 ml of 0.01 mol/l HNO3).

0

20

40

60

1 2 3 4

q e(m

g/g)

No. of Cycles

Lead Adsorption Lead Desorption

Appendix

249

C-3 Conclusions

In this work, carboxymethyl-β-cyclodextrin polymer grafted magnetic nanoparticles

(CDpoly-MNPs) were synthesized successfully and used as easily separable, recyclable

and highly selective nanoadsorbent for the removal of Pb2+ ions from contaminated

water.

List of Publications

250

List of publications

Journal Articles

A.Z.M. Badruddoza, K. Hidajat, M.S. Uddin, Synthesis and characterization of beta-

cyclodextrin-conjugated magnetic nanoparticles and their uses as solid-phase artificial

chaperones in refolding of carbonic anhydrase bovine, J. Colloid Interface Sci. 346(2) (2010)

337-346.

Sudipa Ghosh, A.Z.M. Badruddoza, M.S. Uddin, K. Hidajat, Adsorption of chiral aromatic

amino acids onto carboxymethyl-β-cyclodextrin bonded Fe3O4/SiO2 core-shell nanoparticles, J.

Colloid Interface Sci. 354(2) (2011) 483-492.

A.Z.M. Badruddoza, Goh Si Si Hazel, K. Hidajat, M.S. Uddin, Synthesis of carboxymethyl-β-

cyclodextrin conjugated magnetic nano-adsorbent for removal of methylene blue, Colloids Surf.

A: Physicochem Eng. Aspects 367 (2010) 85-95.

A.Z.M. Badruddoza, A.S.H. Tay, P.Y. Tan, K. Hidajat, M.S. Uddin, Carboxymethyl-β-

cyclodextrin conjugated magnetic nanoparticles as nano-adsorbents for removal of copper ions:

Synthesis and adsorption studies, J. Hazard. Mater. 185(2-3) (2011) 1177-1186.

A.Z.M. Badruddoza, L. Junwen, K. Hidajat, M.S. Uddin, Selective recognition and separation

of nucleosides using carboxymethyl-β-cyclodextrin functionalized hybrid magnetic

nanoparticles, Colloids and Surfaces B: Biointerfaces, 92 (2012) 223-231.

A.Z.M. Badruddoza, Sudipa Ghosh, K. Hidajat, M.S. Uddin, Multifunctional core-shell SiO2

nanoparticles with magnetic, fluorescent, specific cell targeting and inclusion properties for

biomedical applications, Submitted.

Abu Zayed M. Badruddoza, Zayed Bin Zakir Shawon, Tay Wei Jin Daniel, Kus Hidajat,

Mohammad Shahab Uddin, Single and multi-component adsorption of lead, cadmium and

nickel onto cyclodextrin polymer modified magnetic nanoparticles, Manuscript in Preparation.

List of Publications

251

Conference Proceedings

Abu Zayed M. Badruddoza, Zayed Bin Zakir Shawon, Tay Wei Jin Daniel, Kus Hidajat and

Mohammad Shahab Uddin, Carboxymethyl-β-cyclodextrin polymer modified magnetic

nanoadsorbents for removal of endocrine disrupter and toxic metal ions, Proceedings of the

International Conference on Chemical Engineering 2011, ICChE2011, 29-30 December,

Dhaka, Bangladesh.

Zayed Bin Zakir Shawon, Abu Zayed M. Badruddoza, Soh Wei Min Louis, Kus Hidajat,

Mohammad Shahab Uddin, Accelerated procedure for synthesizing Janus magnetic

nanoparticles, Proceedings of the International Conference on Chemical Engineering 2011,

ICChE2011, 29-30 December, Dhaka, Bangladesh.

Conference Presentations Liang Hong, Abu Zayed Md. Badruddoza , Mohammad S Uddin and Kus Hidajat, Synthesis

and characterization of magnetic nano-particles functionalized with β-cyclodextrin for

biomolecule refolding, Particle, Florida, USA, May 10-13, 2008.

K Hidajat, A Z M Badruddoza, M S Uddin, L Hong, N T K Thuyen, Magnetic nano-sized

particles functionalized with beta-cyclodextrin and its use in lysozyme refolding process,

CHISA, Prague, Czech Republic, August 24-28, 2008.

A Z M Badruddoza, K Hidajat and M S Uddin, Solid-phase artificial chaperone assisted

refolding of carbonic anhydrase using β-cyclodextrin conjugated magnetic nanoparticles, VIII

European Symposium of the Protein Society, Zurich, Switzerland, June 14-18, 2009.

A Z M Badruddoza, K Hidajat and M S Uddin, Beta-cyclodextrin bonded magnetic

nanoparticles as solid-phase artificial chaperone for protein refolding, ICBN 2010, Biopolis,

Singapore, August 1-4, 2010.

beta-cyclodextrin conjugated magnetic nanoparticles for … · 2018. 1. 10. · beta-cyclodextrin...

Documents