Project Proposal for:

Modification of Carbone Nanotube (CNTs) with metal

nanoparticles for electrochemical immunoassay of

alpha-fetoprotein

By

Awad Nasser Albalwi

Departement of Chemistry

King Saud Univerisity

Contents

Subject Page N0#

Review for modification methods of CNTsIntroductionEndohedral Functionalization Non-Covalent FunctionalizationDefect FunctionalizationCovalent Sidewall FunctionalizationThe chemical modificationCarboxylation of the terminal carbons and defect sites of CNTsHalogenation of Carbon NanotubesRadical additionsCycloadditions Carbene AdditionAddition of NitrenesNucleophilic Cyclopropanation:Physical functionalizationFunctionalization of SWNTs with oligomers and polymersApplication: biosensor for of detecting glucose:

Project Proposal for:GoldMag -Modified Carbone Nanotube (CNTs) sensing for electrochemical

immunoassay of alpha-fetoprotein1- Abstract2- Introduction

An immunosensor multi-walled carbon nanotubes ( MWCNTs) Alpha-fetoprotein (AFP): Some Latest review 3- Objective:

4-Methodology Modification Carbon Nanotube with Carboxylic Acid group : Carbon Nanotubes modified with Magnetic Nanoparticles Synthesis and bioconjugation of GoldMag-functionalized CNTs Principle the magneto-controlled electrochemical immunoassay 5- Caractrization of bio- GMCNTs

6- References:

2

General Review for modification Methods of CNTS

Introduction:

Carbon nanotubes (CNTs) (fig.1) were discovered by Iijima in 1991, and then have attracted the fancy

of many scientists worldwide. Their small size and unusual physical properties make them become a

unique material with a whole range of promising applications [1].

Large aspect ratios of CNTs with high chemical stability, thermal conductivity, and high

mechanical strength are advantageous for applications to the field emitter [2].

Carbon nanotubes were found to have great potential applications in various fields Such as

biosensors and nanobiotechnology and many others applications, due to the electrical

conductivity and stability toward chemical reaction.[2] Moreover , carbon nanotube (CNT)

consider one of the best sensing materials for electrochemical and biochemical

applications [3,4]. Moreover, the unique chemical, physical, electronic (metallic or semiconducting) and high thermal

properties of carbon nanotubes (CNTs) made them interesting materials for widespread application in the fields such as

electrochemical sensors, biosensors, supports for heterogeneous metal catalysts in organic synthesis, fuel cells,

semiconductors, batteries, random access memory cells, field effect transistor, field emission display, atomic force microscopy

probes, microelectrodes, specific adsorbents to remove organic pollutants from water and waste water and as a potential drug

carriers in cancer therapy. [5]

A major barrier for the preparation of CNTs-based biosensors is the insolubility of CNTs in

most solvents [4]. Moreover, the poor solubility of carbon nanotubes in organic solvents restricts them to be used as

drug delivery agents into living systems in drug therapy. Hence many modification approaches like physical, chemical or

combined have been exploited for their homogeneous dispersion in common solvents to improve their solubility [6]. The

reports appeared till now in the literature reveals that the modification is required to control the dispersion and such

modification introduces specific functionalities as molecular wedges onto the surface of the carbon nanotubes. Thus the

functional groups present on the surface control the lateral interactions between the bundles and separate indivisual tubes.

Hence homogeneous dispersion can be achieved by breaking the close lateral contact between them which enhances the

affinity towards solvents and other related matrices [6 ]. Due to these specific properties, many research groups explored the

development of novel methods for the modification of nanotubes and investigated the mechanistic aspects of these new class

of novel materials called chemically modified carbon nanotubes (CMCNTs).[5]

There are different strategies addressing the surface of CNTs which can be divided in two

general classes (Figure 2). a) The supramolecular approach including the self assembled

construction using non-covalent interactions b) the chemical addition of reactive molecules,

resulting in the formation of a new covalent bond and. [7 ]

The supramolecular approach comprises

1) the endohedral filling

2) the non-covalent functionalization

The chemical approach includes

3) the defect functionalization and

4) the covalent sidewall functionalization.

4

Fig.1

Endohedral Functionalization

One possibility of CNT functionalization is the filling of the inner cavity with molecules,

called endohedral functionalization, which especially is interesting for the development of

nanowires, storage applications of e.g. fuel and nanocontainers. Additionally,

metallofullerenes were encapsulated in CNTs as well as noble metals (e.g. Au, Ag, Pt, Pd)

yielding metallic nanowires. In opened MWCNTs with an inner diameter of 2-10 nm also

bio molecules such as carotene or proteins (lactamase) were channeled in, showing a

41

Fig.2

catalytic activity inside the CNTs. Even the polymerization of conductive polymers inside a

carbon nanotube was achieved.[7]

Non-Covalent Functionalization

One major drawback of CNTs is the poor solubility in organic solvents or water, due to their

strong interfacial π- π -interactions. These properties can also be utilized for a further kind of

functionalization (non-covalent) which coincidently results in an efficient debundling,.

Hereby, a broad variety of e.g. amphiphilic- or -surfactants as well as different kinds of

polymers are added to a CNT dispersion yielding significantly individualization after ultra

sonication or stirring. An important benefit of this kind of functionalization is the

reversibility and the structural integrity of the nanotube as no covalent bonds between the

addend molecules and the nanotube surface are formed. Another invaluable advantage is the

fact that besides the design of the corresponding addend molecules, no special chemical

equipment and knowledge is necessary, making this method very convenient and scalable.

First basic findings in this field exhibited that indeed π - π -interactions are fundamental for

the attaching of addends to the CNT surface, including experiments with small aromatic

molecules such as cyclohexane, cyclohexene. Further on, classical detergents such as

SDS(Sodium dodecylsulfate) or SDBS(Sodium dodecylbenzenesulfonate) have been

extensively becoming standard surfactants in carbon nanotube chemistry and processing,

due to their unlimited availability and low prices in contrast to other surfactant molecules.

As for the classical surfactants, another substance class successfully attached on the CNT

surface, yielding water soluble CNT materials are ionic liquids. Especially the detergent

6

molecules SDS and SDBS can not only interact via π-π -interactions with the CNT surface,

but their unpolar tail can additionally wrap around the nanotube. This binding motif can be

used wrapping a huge variety of synthetic- and bio-polymers as for example DNA around

nanotubes, tailoring the degree of debundling and the solubility in different solvents. The

DNA functionalized samples could be separated into nanotube batches with different

diameters, as well as by ion exchange chromatography and density ultra centrifugation .[7]

Defect Functionalization

One convenient method is the oxidation of the impurities possessing a higher reactivity than

the 1D sp2 lattice of the nanotubes. The fact that nanotubes have two fullerene like end-caps

and in reality are not perfect sp2 cylinders, additionally results in a partial oxidation of the

CNT material. Next to their end-caps nanotubes display various disorders from topological-,

rehybridization- and incomplete bonding defects right up to intramolecular junctions and

Stone-Wales defects. These defects occur preferentially on small diameter CNTs and in

general are locations with an increased reactivity which are oxidized during the purification

process. The oxidation mainly yields an opening of the sp2 carbon lattice and the generation

of carboxylic acid or other oxygen containing functionalities at the defect sites.

There are different oxidation approaches including nitric and sulfuric acid, mixtures of

hydrogen peroxide and sulfuric acid, gaseous oxygen and ozone as well as potassium

permanganate. Additionally ultra sonication steps, further shorten the oxidized nanotubes

yielding an even higher degree of introduced oxygen containing functionalities. Materials

which was treated in such a fashion, afterwards exhibit increased solubility in polar organic

solvents. [7]

Covalent Sidewall Functionalization

One of the outstanding properties of CNTs is their high aspect ratio. In the course of defect

functionalization only the ends and the defects of CNTs can be functionalized, whereby only

a small surface area of the CNT can be addressed. As a consequence, the degree of

functionalization is relatively low and the introduction of defect sites deteriorates the

electronic and mechanical properties of the nanotube. By direct covalent sidewall

functionalization, the complete CNT surface can be addressed, resulting in a very high

degree of functionalization and the preservation of the structural integrity of the CNT

framework. The addition only yields a rehybridization of sp2 into sp3 carbon atoms which

has a minor influence on the mechanic stability of the CNT. the reactivity of the 1D carbon

allotrope (SWCNT) is by far lower in comparison to the 0D representative, the fullerenes.

Although some reaction sequences were successfully transferred from fullerene to CNT

chemistry, “hot addened ” needed. Another opportunity is the activation of CNTs in

combination with subsequently mild reactions, as in the case of fluorinated ones (Figure 3).[7]

8

The chemical modification:

Fig.3

The modification protocol was generally achieved by attaching specific molecule or entity

which imparts chemical specificity to the substrate material [8]. These chemical modifications can be

easily achieved in many ways However, in this work the modification routes are mainly classified into two types namely

surface modification and bulk modification. The surface modification includes electrochemical induced method, polymer

grafting and metal nanoparticle deposition. The later includes chemical reduction of diazonium salts using hypo phosphorous

acid as a reducing agent, thermally activated covalent modification, microwave assisted modification and ball milling

modification.[2]

Here in Fig. 4 is a summarize of the covalent surface chemistry of CNTs,

Fig. 4 Surface functionalization of carbon nanotubes.[2]

Carboxylation of the terminal carbons and defect sites of CNTs:

10

One of the most common functionalization techniques is the “oxidative purification” of

nanotubes by liquid-phase or gas-phase oxidation, introducing carboxylic groups and some

other oxygen-bearing functionalities such as hydroxy, carbonyl, ester and nitro groups into

the tubes[9].

Acid treatment typically involves the use of a strong oxidizing agent . SWNTs have been

refluxed with HNO3, H2SO4, HCl or mixtures of these acids . Many groups have varied

many parameters for this treatment such as duration, concentration, and repeated cycles. It is

known that the use of these processes can cause defects and/or shortening of SWNTs.

Oxidation usually results in the formation of carboxylate groups on the surface of the tube.[2].

the Haddon group using the acid moieties for attaching long alkyl chains to SWNTs via

amide linkages or carboxylate-ammonium salt ionic interactions [10] .

Halogenation of Carbon Nanotubes

Being the strongest element, fluorine can fluorinate the sidewalls of CNTs between room

temperature and 600°C (Fig. 1). Fluorinated CNTs have been extensively characterized

by (TEM), (STM), (EELS), and (XPS), . The sidewall carbons on which fluorine atoms

attached adopt sp3 hybridization and possess tetrahedral configuration. This destroys the

Fig.5 section of an oxidized SWCNT , reflecting terminal and sidewall oxidation [9] [7]

electronic band structure of metallic or semiconducting CNTs and generates an insulating

material. Although there is controversy regarding the favorable pattern of F addition onto

the sidewall of CNTs being either 1,2-addition or 1,4-addition, . The highest degree of

functionalization was estimated to be C2F by elemental analysis. Fluorinated CNTs were

reported to have a moderate solubility ( 1 mg/mL) in

alcoholic solvents.The fluorination reaction is very useful because further substitution of F

can introduce useful functional groups. The fluorine atom can be replaced with alkyl groups

using Grignard or organolithium reagents.The alkylated CNTs are well dispersed in

organic solvents and can be completely dealkylated upon heating at 500 °C in inert

atmosphere, thus recovering pristine CNTs. Several diamines and diols were also reported to

substitute fluorine atoms on fluorinated CNTs. Infrared (IR) spectroscopy was used to

confirm the disappearance of the C–F bond stretching at 1225 cm−1 as the indication of F-

substitution on CNTs. These reacted CNTs, such as amino alkylated CNTs, can be further

modified on the free amino groups to introduce more sophisticated functionalities which

may bind biomolecules for biological applications. Chlorination or bromination reactions to

CNTs were also accomplished through electrochemical means but the products contain

significant amounts of carboxyl or hydroxyl groups. [10]

By treatment Fluorinated CNTs with hydrazine or LiBH4/LiAlH4, the majority of the

covalently bound fluorine could be removed (fig 7, b) ], restoring most of the conductivity

and spectroscopic properties of the pristine material.[9 ].

12

Radical additions:

Molecular dynamics simulations showed that there is a great probability of reaction of

radicals on the sidewalls of CNTs. Experimentally the covalent sidewall

functionalization via radical addition was achieved with diazonium salts.

Electrochemical reduction of substituted aryl diazonium salts in organic media generates an

aryl radical in situ which covalently attaches to the surface of CNTs. The formation of aryl

radicals was triggered by electron transfer between CNT and the aryl diazonium salts in a

self-catalyzed reaction. A similar reaction was later reported, utilizing water-soluble

diazonium salts, which have been shown to react selectively with metallic CNTs. These

diazonium salt functionalized CNTs are well-dispersed in DMF or aqueous solutions. In situ

electrochemical modification of individual CNTs was demonstrated by attachment of

substituted phenyl groups. Two types of coupling reactions were proposed, namely the

reductive coupling of aryl diazonium salts and the oxidative coupling of aromatic amines. In

fig.6

fig.7

both cases a radical species is produced on the surface of the nanotube, which attacks the

carbon lattice to form a covalent bond. In the former case, the reaction resulted in a C–C

bond formation at the graphitic surface, whereas in the latter, amines were directly attached

to CNTs. Reductive alkylation of SWNTs with lithium metal in liquid ammonia followed by

the addition of either alkyl iodides/sulfides or aryl iodides/sulfides is also proposed to be a

radical rocess. The reductive intercalation of lithium ion onto the nanotube surface

in ammonia or in polar aprotic solvents has been observed by TEM and AFM. The

negatively charged tubes were found to exchange electrons with long chain

aryl/alkyliodides, resulting in transient aryl/alkyl radicals. The latter were covalently added

to the graphitic surface of the nanotubes to afford modified products. A similar reaction was

reported for the functionalization of CNTs via one-

electron reduction of benzophenone by potassium. A radical anion is generated from the

reaction of a potassium atom with benzophenone molecule that results in transferring one

electron from the potassium to the benzophenone. The radical anion adds readily to

sidewalls to yield diphenylcarbinol-functionalized SWNTs. Thermal and photochemical

methods have also been applied to the successful covalent functionalization of CNTs with

radicals. Alkyl or aryl peroxides were decomposed thermally and the resulting radicals

added to the graphitic network. The reaction of CNTs with succinic or glutaric acid acyl

peroxides resulted in the addition of carboxyalkyl radicals onto the sidewalls. These acid-

functionalized CNTs can be converted to materials with new functions. Addition of

perfluoroalkyl radicals to CNTs was obtained by photo induced reactions. [10]

Tour et al 2001, reacted a series of aryl diazonium salts with purified

14

SWCNTs in an electrochemical reaction, using a bucky paper as working electrode, to

prepare a variety of functionalized SWCNTs . The corresponding reactive aryl radicals are

generated from the diazonium salts via one-electron reduction (Scheme 8, a) & latter the

authors described in similar reactions the in situ generation of the diazonium ions from the

corresponding anilines (Scheme 8, b).[9]

Marcoux et al 2004, described the covalent exohedral derivatizations of HiPco nanotubes,

through electrochemical reduction of aryldiazonium salts, After the evolution of the spectra

(area of the D-band), as a function of the number of grafted groups, led to the conclusion

that the electrochemical reduction of aryldiazonium salts into radicals gives rise to the

growth of aryl chains on the sidewalls of the nanotubes (scheme 9) [8].

Scheme18

Cycloadditions

Carbene Addition

In the course of a study on organic functionalization of CNTs, Haddon et al. discovered in

1998 that dichlorocarbene was covalently bound to soluble SWCNTs (Scheme 10) [11]. The

carbene was first generated from chloroform with potassium hydroxide [12 656], and later from

phenyl(bromodichloromethyl)mercury [11]. However, the degree of functionalization was

low: a chlorine atom amount of only 1.6 wt % was determined by XPS [.Because of impure

starting material and a large amount of amorphous carbon, the site of reaction could not be

ascertained [9].

16

scheme 9Illustrations (b) and (c) schematize two samples giving rise to D-bands that have the sameintensity (same number of covalent bonds on the sidewalls of the nanotubes)but show different amounts of bromine in the XPS spectra. This difference can be explained through the growth of aryl chains on SWNTs.[8]

Addition of Nitrenes

Sidewall functionalization of SWCNTs was achieved via the addition of reactive

alkyloxycarbonyl nitrenes obtained from alkoxycarbonyl azides. The driving force for this

reaction is the thermally induced N2-extrusion (Scheme 11). Such nitrenes attack nanotube

sidewalls in a [2+1] cycloaddition forming an aziridine ring at the tubes’ sidewalls [9].

Jiang et al 2011 reported a new approach to produce amino-CNTs by cycloaddition of

nitrenes as in the Scheme [13]

Fig.10 Functionalization of carbon nanotubes by cycloaddition reactions.

7

Scheme.12 produce amino-CNTs by cycloaddition of nitrenes

Scheme.11

Nucleophilic Cyclopropanation:

Fullerenes are known to react easily with bromomalonates to form cyclopropanated

methanofullerenes .A similar reaction was performed by Coleman et al. [14] using purified

SWCNTs and diethyl bromomalonate as addend (Scheme 13). The authors have developed a

chemical tagging technique which allows the functional groups to be visualized by

AFM.Immobilized SWCNT derivatives were transesterified with 2-(methylthio)ethanol, and

by exploiting the gold–sulfur binding interaction the cyclopropane group was “tagged” using

preformed 5 nm gold colloids [14].

Scheme 13 . Nucleophilic Cyclopropanation

18

Physical functionalization

Functionalization of CNTs using covalent method can provide useful functional groups onto

the CNT surface. However, these methods have two major drawbacks: firstly, during the

functionalization reaction, especially along with damaging ultrasonication process, a large

number of defects are inevitably created on the CNT sidewalls, and in some extreme cases,

CNTs are fragmented into smaller pieces. These damaging effects result in severe

degradation in mechanical properties of CNTs as well as disruption of p electron system in

nanotubes. The disruption of p electrons is detrimental to transport properties of CNTs

because defect sites scatter electrons and phonons that are responsible for the electrical and

thermal conductions of CNTs, respectively. Secondly, concentrated acids or strong oxidants

are often used for CNT functionalization, which are environmentally unfriendly. Therefore,

many efforts have been put forward to developing methods that are convenient to use, of

low cost and less damage to CNT structure. Non-covalent functionalization is an alternative

method for tuning the interfacial properties of nanotubes. The suspension of CNTs in the

presence of polymers, such as poly(phenylene vinylene) [15] or polystyrene [16], lead to the

wrapping of polymer around the CNTs to form supermolecular complexes of CNTs. This is

a typical example of non-covalent functionalization of CNTs (Fig. 13A). The polymer

wrapping process is achieved through the van der Waals interactions and π-π stacking

between CNTs and polymer chains containing aromatic rings. [17]

Functionalization of SWNTs with oligomers and polymers

SWNTs are considered to be the ideal reinforcing fibers due to their exceptional mechanical,

electronic and thermal properties, low density and high aspect ratio. However, the

incorporation of SWNTs into the polymer matrix is often problematic due to the chemical

inertness of SWNTs. The covalent functionalization of SWNTs is a valuable route towards

the development of high-performance composites. It provides homogeneously dispersed

SWNTs incorporated in the polymer and a strong interfacial bonding between the polymer

and SWNTs. Bekyarova et al 2004reported appling chemically functionalized SWNTs to

prepare a number of SWNT-polymer composite materials. In one approach, poly (m-

aminobenzene sulphonic acid), PABS, a conducting water soluble polymer, was covalently

attached to chemically functionalized SWNTs (Scheme 1).[18]

In another study Sen et al 2004 & co-workers have prepared SWNT-reinforced

polyurethane (PU) and polystyrene (PS) membranes.[ 19]

20

SChem14 CNTs functionalization using non-covalent method a)polymer wrapping ,b)surfactant adsorption c) ndohedral method[17]

Scheme15

Scheme16 , Functionalization of SWNTs with PU & PS [ 19 ]

Carbon Nanotubes Decorated with Magnetic Nanoparticles

Hybrid systems based on iron oxides/carbon nanotubes have many potential applications in

electric device, magnetic data storage, and heterogeneous catalysis. The removal of azo dyes

is an important issue. In fact, most dyes used in the manufacturing industries contain

aromatic rings that are generally toxic or potentially carcinogenic/mutagenic agents . With

the aim to remove azo dyes (i.e., methyl orange) dissolved in aqueous solution, magnetic

CNTs have been also prepared by a straightforward Fenton’s reagent method (Figure 17)

[43]. This method consists in the slow addition of H2O2 to a solution of FeSO4, in which

CNTs have been suspended. The resulting solution is the so-called Fenton’s reagent.

Fig.17

The oxidant solution not only allows the conversion of Fe(II) to Fe(III) but also the

generation of reactive functional groups on the CNT’s surface. Further precipitation of

Fe(OH)3 followed by heat treatment under a nitrogen/hydrogen flow, produced Fe2O3

nanoparticles that uniformly dispersed on the surface of CNTs with high loading (>50%).

The advantage of this method consists in the preparation of magnetic CNTs without the use

of strong acids or exploiting reactions for the formation of covalent bonds. Moreover, this

system is able to remove the azo dye methyl orange from aqueous solution by adsorption, to

be separated by an external magnetic field and easily regenerated by UV photocatalysis.

Decoration of CNTs by spinel ferrites nanoparticles with the chemical formula MFe2O4 (M

= Mn, Co, Ni, Mg, or Zn) has been reported to improve optical, magnetic and

electrochemical properties of pristine CNTs. In a recent work, a special electrode has been

designed with the aim to determine analytically the concentration of the antibiotic cefixime

with voltammetric techniques .

Pristine CNTs (diameter of 10–30 nm, length of 5–15 μm) have been treated with HNO3 to

introduce reactive functional groups then they have been dropped onto the surface of a

glassy carbon disk electrode. To obtain magnetic metal functionalized CNTs the authors

developed an in situ chemical citrate gel method. This procedure consists in the treatment of

functionalized CNTs into 1M citric acid followed by addition of a (1:2) solution of Ni/Fe

nitrate. The pH was increased to 9 with ammonia and stirred at 30 °C for 48 h to complete

the reaction. The substance was finally calcinated at 620 °C for 2 h in argon atmosphere to

obtain a powder of NiFe2O4-MWCNTs. This powder has been supported on a carbon disk

electrode and employed in the analytical determination of cefixime (in tablet, blood

22

plasma, and urine samples) by exploiting the presence of its NH2 groups that can be oxidized

by anodic reactions .

A recent paper dealing with the decoration of CNTs with magnetic iron oxide nanoparticles

and exploiting two different reactions (ligand exchange and chemo-selective ligation or

“click chemistry”) has been reported (Figures 7 and 8) ]. The authors suggested that these

systems can be employed in applications for cell labeling, MRI cell tracking and magnetic

manipulations. First, iron oxide superparamagnetic nanoparticles were synthesized by

thermal decomposition of iron stearate in octyl ether and oleic acid. Then, the ligand

exchange reaction took place by adding oxidized CNTs (bearing COOH groups) and stirring

the suspension in THF for 24 h (Figure 18).

Fig.18

The click ligation has been performed in mild conditions exploiting the Cu(I)-catalyzed

azide–alkyne Huisgen 1,3-dipolar cycloaddition reaction in an efficient and highly selective

way.

CNTs have been functionalized with alkyne moieties, whereas iron oxide nanoparticles have

been coated with a dendrimer bearing a terminal azide group. Both derivatized CNTs and

nanoparticles have been reacted with sodium ascorbate and Cu(II) sulphate in a THF/H2O

(3:1) solution (Figure 19).

The degree of functionalization is different for the two reactions owing to the different

degree of active groups (COOH and alkyne groups) on the surface of the CNTs. Oxidized

CNTs have a greater percentage of substitution but alkyne derivatives favor a more

homogeneous derivatization. Moreover, aggregation is reduced when the click chemistry

approach is followed. These compounds enter easily into cells, are moderately toxic and

display good magnetic properties. These properties can be exploited to monitor these

systems by MRI techniques and to manipulate them with magnetic devices.

24

The control of CNTs by external magnetic fields and the monitoring with non-invasive

techniques opens new perspectives for targeted therapy or tissue engineering.

Application:

Amino-carbon nanotubes (amino-CNTs) can conjugate with the DNA by electrostatic

interactions and shuttle the DNA to the cell cytoplasm or even the nucleus.[20] The

functionalized-amino-CNTs are very soluble in water and can be conjugated with nucleic

acids and transfer them into the mammalian cells to produce biological effects (Pantarotto et

al. 2004[20]; Singh et al. 2005[21]).

Huang et al 2013 demonstrated simultaneous attainment of high sensitivity and selectivity in

thin-film field effect transistors (TFTs) based on outer-wall selectively functionalized

double-walled carbon nanotubes (DWCNTs). With carboxylic acid functionalized DWCNT

TFTs, , the author and co-workers obtained excellent gate modulation (on/off ratio as high

as 4000) with relatively high ON currents at a CNT areal density as low as 35 ng/cm2. The

devices displayed an NH3 sensitivity of 60 nM (or 1 ppb), which is comparable to small

molecule aqueous solution detection using state-of-the-art SWCNT TFT sensors while

concomitantly achieving 6000 times higher chemical selectivity toward a variety of amine-

containing analyte molecules over that of other small molecules. These results highlight the

potential of using covalently functionalized double-walled carbon nanotubes for

simultaneous ultrahigh selective and sensitive detection of chemicals and illustrate some of

the structural advantages of this double-wall materials strategy to nanoelectronics. [22]

biosensor for of detecting glucose:

It is reported that a novel amperometric glassy carbon biosensing electrode for glucose

which is based on the immobilization of a highly sensitive glucose oxidase (GOx) by

affinity interaction on carbon nanotubes (CNTs) functionalized with iminodiacetic acid and

metal chelates. The new technique for immobilization is exploiting the affinity of Co(II)

ions to the histidine and cysteine moieties on the surface of GOx. The direct

electrochemistry of immobilized GOx revealed that the functionalized CNTs greatly

improve the direct electron transfer between GOx and the surface of the electrode to give a

pair of well-defined and almost reversible redox peaks and undergoes fast heterogeneous

electron transfer with a rate constant (ks) of 0.59 s−1. The GOx immobilized in this way

fully retained its activity for the oxidation of glucose. The resulting biosensor is capable of

detecting glucose at levels as low as 0.01 mM, and has excellent operational stability (with

no decrease in the activity of enzyme over a 10 days period). The method of immobilizing

GOx is easy and also provides a model technique for potential use with other redox enzymes

26

and proteins (Scheme1).(Tu et al 2012)[23

28

Project Proposal for:

Modification of Carbone Nanotube (CNTs) with metal

nanoparticles for electrochemical immunoassay of alpha-

fetoprotein

Abstract

A new flow-through electrochemical immunosensor is suggesting to be designed for

sensitive detection of alphafetoprotein (AFP) in human serum by using nanogold-

functionalized magnetic Carbon Nanotube (CNTs) as immunosensing probes. Initially,

CNTs will be modified with Carboxylic acid group then amino functionalized magnetic

beads will be covalently immobilized on the surface of CNTs (MCNTs), then nanogold

particles will be adsorbed on the amino groups of the MCNTs to construct GoldMag

functionalized Carbone nanotube (GMCNTs), and then horseradish peroxidase-anti-AFP

conjugates (HRP-anti-AFP) will be assembled onto the surface of nanogold particles

(bio-GMCNTs). With the aid of an external magnet, the formed bio-GMCNTs will be

attached onto the base electrode in the flow system. Sample containing AFP antigens can

be tested by capability of the producing transparent immunoaffinity reaction with the

immobilized HRP-anti-AFP on the bio-GMCNTs.

Introduction

An immunosensor is a kind of biosensor that provides concentration-dependent signals by

using antibodies (Ab) or antigens (Ag) as the specific sensing element [24, 25]. Recently,

electrochemical immunosensors have incited the interest of scholars because of their

sensitivity, highly selectivity, convenience and inexpensiveness, and they have been

successfully applied in environmental analysis [26], the food industry [27,28 ], and clinical

chemistry [29,30].

Sensitive determination of disease-related proteins based on the immunoassay has gained

increasing attention in early disease diagnostic and highly reliable predictions (Choi et al.,

2011)[31]. Among these immunoassays, the homogeneous immunoassays usually involve in

the immobilization of the biomolecules on the nano-/microbeads, and take place in the

solution, thus allowing the integration of multiple liquid handling processes (Song et al.,

2011)[32]. Especially combining with microfluidic device, the homogeneous immunoassay

can be used for the detection of complex samples, such as urine or blood, without the large

sample consumption and sample pretreatment, resulting in a relatively inexpensive and easy

performance (Kang and Li, 2009; Lin et al., 2010)[33,34]. Microfluidic lab-on-a-chip

technology has the advantages of portability, portability, inte- gration, and automation

(Wong and Ho, 2009)[35].

30

multi-walled carbon nanotubes ( MWCNTs)

As is well known, semiconductor multi-walled carbon nanotubes ( MWCNTs), have unique

electrical and mechanical properties, high surface area, and are proven to promote electron

transfer between electrochemically active compounds and electrodes [36,38]. Cao et al. have

developed an electrochemical immunosensor using poly(L-arginine)/multi-walled carbon

nanotubes composite film with functionalized gold nanoparticles for the sensitive detection

of casein [39 ].

The review papers by Masotti et al 2013 emphasized the role of magnetic carbon

nanotubes (Mag-CNTs) as novel and promising drug delivery vectors for applications in

biomedical and biotechnological applications. [40]

fluidMAG-nanoparticles

fluidMAG-nanoparticles (such as fluidMAG-Amine )are ferrofluids consisting of an

aqueous dispersion of magnetic iron oxides with diameters of 50 nm, 100 nm and 200 nm.

The particles are covered with hydrophilic polymers which protect them against aggregation

by foreign ions. Terminal functional groups such as ion-exchange groups or reactive groups

for covalent immobilization can be used for binding to biomolecules. Ferrofluids can further

be used for MRI-diagnostics and magnetic drug targeting applications. fluidMAG-Amine is

used in Covalent coupling of biomolecules application.[64]

Alpha-fetoprotein (AFP):

Alpha-fetoprotein (AFP) is a major plasma protein produced by the yolk sac and the liver.

The AFP expression is often associated with diseases of hepatocellular, testicular

nonseminomatous origin, and occasionally of other entodermal origin and has been widely

accepted as a tumor marker for monitoring the therapeutic effectiveness of hepatocellular

cancer and nonseminomatous testicular cancer (Tomasi, 1977).[41]

Alpha-fetoprotein (AFP) is a glycoprotein that is produced in early fetal life by the liver and

by a variety of tumors including hepatocellular carcinoma, hepatoblastoma, and

nonseminomatous germ cell tumors of the ovary and testis (eg, yolk sac and embryonal

carcinoma). Most studies report elevated AFP concentrations in approximately 70% of

patients with hepatocelllular carcinoma. Elevated AFP concentrations are found in 50% to

70% of patients with nonseminomatous testicular tumors[42]

AFP is elevated during pregnancy. Persistence of AFP in the mother following birth is a rare

hereditary condition.[43] Neonates have markedly elevated AFP levels (>100,000 ng/mL) that

rapidly fall to below 100 ng/mL by 150 days and gradually return to normal over their first

year.[43] Concentrations of AFP above the reference range also have been found in serum of

32

patients with benign liver disease (eg, viral hepatitis, cirrhosis), gastrointestinal tract tumors

and, along with carcinoembryonic antigen, in ataxia telangiectasia.

The biological half-life of AFP is approximately 5 days. <6.0 ng/mL Reference values are

for nonpregnant subjects only; fetal production of AFP elevates values in pregnant women.

Range for newborns is not available, but concentrations over 100,000 ng/mL have been

reported in normal newborns, and the values rapidly decline in the first 6 months of life.

Serum markers are not specific for malignancy, and values may vary by method. Alpha-

fetoprotein (AFP) levels may be elevated in association with a variety of malignancies or

benign diseases. Failure of the AFP value to return to normal by approximately 1 month

after surgery suggests the presence of residual tumor. Elevation of AFP after remission

suggests tumor recurrence; however, tumors originally producing AFP may recur without an

increase in AFP.[ 45,42,46]

Alpha-fetoprotein (AFP) is an oncofetal protein found in high concentration in fetal and

maternal blood and in patient with certain neoplastic and non-neoplastic disorders[47]. AFP

was first identified in 1956, in 2 separete laboratories during electrophoretic experiments on

plasma proteins of neonates, as a protein which migrated between albumin and alpha-

globulin[48] . clinical interist in AFP developed when it was discovered that transplantable

hepatocellular carcinoma of the mouse synthesized and secreted AFP into the blood[49]. High

serum levels of AFP were subsequently detected in patients with hepatoma,germ cell

tumors[47,50,51] . in addition to mammals , birds and even several species of sharks have been

found to synthesize a fetal specific plasma alpha-globulin analogous to mammalian AFP[51].

Ding etal 2009, reported preparing a novel and effective electrochemical immunosensor for

the rapid determination of alpha-fetoprotein (AFP) based on carbon paste electrode (CPE)

consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate

(BPPF(6)) and graphite. The surface of the CPE was modified with gold nanoparticles for

the immobilization of the alpha-fetoprotein antibody (anti-AFP). By sandwiching the

antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary

antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled

anti-AFP), the immunoassay was established. The concentration of AFP was determined

based on differential pulse voltammetry (DPV) signal, which was generated in the reaction

between O-aminophenol (OAP) and H(2)O(2) catalyzed by HRP labeled on the sandwich

immunosensor. The immunosensor exhibited high sensitivity and good stability, and would

be valuable for clinical assay of AFP. [52]

Alpha-fetoprotein (AFP) detection by using a localized surface plasmon coupled

fluorescence (LSPCF) fiber-optic biosensor is setup and experimentally demonstrated by

Chang et al 2008. It is based on gold nanoparticle (GNP) and coupled with localized surface

plasmon wave on the surface of GNP. In this experiment, the fluorophores are labeled on

anti-AFP which are bound to protein A conjugated GNP. Experimentally, the ability of real

time measurement in the range of AFP concentration from 0.1ng/ml to 100ng/ml was

detected. To compare with conventional methods such as enzyme-linked immunosorbent

assay (ELISA) or radioimmunoassay (RIA), the LSPCF fiber-optic biosensor performs

higher or comparable detection sensitivity, respectively. [53]

Hsu et al 2011 reported a new highly sensitive biosensor approach which has the ability to

determine 5~100 ng/ml alpha-fetoprotein (AFP) quantitatively using gold nanoparticle

arrays slightly embedded in glass substrates which can be quickly prepared by one-step

microwave-plasma dewetting process [54].

34

Huang et al 2011reported investigation of high sensitive and quantitative detection of alpha-

fetoprotein (AFP) by biosensor based on imaging ellipsometry (BIE) through biological

amplification.. AFP firstly reacted with the rat monoclonal antibody (rat-mAb) initially

immobilized on glutaraldehyde modified silicon surface, then rabbit anti-human AFP

polyclonal antibodies (Rabbit-pAb) and goat anti-rabbit IgG (goat-IgG) were sequentially

applied to amplify signal. Results revealed that signal was enhanced approximately six fold.

. The cross-reaction rate was less than 5.2% evaluated by biomarker (carcinoembryonic

antigen, carbohydrate 19-9 and carbohydrate antigen 242) and two common proteins (human

serum albumin, fibrinogen) and their mixture. [55]

Xia et al 2012 reported droplet methods that have been successfully applied in DNA

hybridization analysis and protein-protein interaction. Existing assay methods implemented

in droplet platforms are severely limited by expensive and high-maintenance equipment. As

a convenient detection method, colorimetry provides a new path for microscale assay since it

can enhance assay efficiency and simplify the detection procedure. a microscale

immunoassay for α-fetoprotein (AFP) was developed for the first time by the incorporation

of colorimetry and droplet platform. Ru(bpy)2(mcbpy-O-Su-ster)(PF6)2 complex (Ru) was

coupled with the monoclonal antibody (Ab) of AFP to form a stable red Ru–Ab complex

both as a quencher for green CdTe quantum dots (QDs) and as a capture probe for AFP. In

the absence of AFP, the mixed droplet showed a red color. With the increase of AFP

concentration, the color change of the droplet was from red to green as a result of the

competition of AFP with QDs for Ru–Ab. The biosensor exhibited not only good sensitivity

and specificity for AFP with a detection limit of 0.06 ng ml−1, but also satisfactory

performance in diluted human sera with a detection limit of 0.4 ng ml−1. [56]

Objective:

In this research proposal , we propose to design a new Gold- Mag-modified CNTs

(SWCNTs, DWCNTs and MWCNT) as biosensor for electrochemical detection of Alpha-

fetoprotein (AFP) in a flow system (human-blood).

The aim of this work is to explore a new electrochemical immunoassay method using

(Gold- Mag-modified CNTs) for the determination of cancer marker in human serum .

Hopefully, the methodology can be applied for the determination of real samples with high

sensitivity and feasibility in clinical screening of cancer markers and diagnostics.

Methodology

Modification Carbon Nanotube with Carboxylic Acid group :

The multi-wall CNTs will be pretreated according to the procedure with minor

modifications According to the method used by (Tang et al 2009, Yu et al 1998, [57,58]. The

multi-wall carbon nanotubes (95%) with outer diameters of ca 10–20 nm and lengths of 5–

10 mm will be oxidized by refluxing in concentrated HNO3 and H2SO4 (volume ratio of 1:3)

for 12 h. The resultant mixture then will be diluted with pure water to about three times of

the original volume and stirred for 24 h. The mixture will be subjected to high-speed

centrifugal sedimentation.

36

Fig. modification CNTs with –COOH group

Carbon Nanotubes modified with Magnetic Nanoparticles

After modifying CNTs with Carboxylic group, Magnetic CNTS can be synthesized

according to the literatures with some modification (Koo et al., 2011; He and Gao, 2010 and

Zhang etal 2011). Amount of CNTs can be initially dispersed into distilled water, and then

sonicated for certin time at room temperature . Following that, amount ( mg) of NHS (N

-ydroxysulfosuccinimide) and amount (mg) of EDC (N-(3- dimethylaminopropyl)-N

-ethylcarbodiimidhydrochlorid) will be added into the mixture, and incubated for certain

time at 4 ℃ to activate the –COOH groups on the CNTS. Afterwards, amount ( mg) of

amino functionalized magnetic nanoparticles (fluidMAG-Amine) will be added into the

mixture, and further stirred for 12 h at 4 ℃. The unconjugated magnetic nanoparticles were

removed by filtration for 10 times with the distilled water. The obtained CNTs then will be

dried in a vacuum oven at 60 ◦C.[59,60,61 ]

Synthesis and bioconjugation of GoldMag -functionalized CNTs

According to the literatures by ( Zhang etal 2011) with miner modification ,

Bioconjugation of GoldMag -functionalized CNTs, can be Synthesized as following,

certain amount (mg) of Magnetic CNTS will be dispersed into amount ( mL) of distilled

water, and the mixture will be then sonicated for 30 min at RT. Following that, amount

(mL) of gold colloids (C[Au] ≈ 24 mM) will be added into the mixture, and incubated for 6

h at 4 ◦C with slight stirring to make nanogold particles assemble on the Magnetic CNTS

surface. Afterwards, the GoldMag -functionalized Magnetic CNTS will be separated and

purified by using an external magnetic field. The obtained GoldMag -functionalized

Magnetic CNTS ( it can be called as GMCNTs) will be then dispersed into amount of

distilled water, and used for the conjugation of HRP-anti-AFP. Amount of HRP-anti-AFP

will be injected into the GMCNTs suspension, and incubated for 6 h at 4 ◦C with slight

stirring. Afterwards, HRP-anti-AFP-labeled GMCNTs (designed as bio- GMCNTs) will be

collected through an external magnet. The obtained pellet will be re-suspended into 2.5 wt%

BSA(bovine serum albumin ) for 60 min at RT to eliminate non-specific binding effect and

block the remaining active groups. Finally, the bio- GMCNTs will be e suspended into

certine amount of pH 6.0 PBS(phosphate-buffer saline (PBS)) containing sodium azide,

and then can be stored at 4 ◦C .[61] Noticeably , this method was applied for grapheme

oxide , and it can be apply for CNTs as a result of the similarity in properties between the

two.

38

Scheme 1. Schematic representation of the process of the bio-GMGPs

Principle the magneto-controlled electrochemical immunoassay

In this research We intend to follow the similar explanation of the principle and

charactrisitic of the magneto-electrochemical immunoassay experiment which was reported

by Zhang et al 2011 and others.

Since AFP and anti-AFP are biomacromolecules with weak conductivity, the formed

immunocomplex can act as an inert layer and partly shield the active center of the HRP

enzyme, which weaken the catalytic efficiency of the HRP toward the reduction of H2O2 in

the solution. As a result, the current response is decreased. The decrease in the current

depends on the concentration of AFP in the sample. The assay principle can be summarized

as follows:

ImmunoreactionHRP-anti-AFP +AFP→ HRP-anti-AFP-AFP (1)Electrochemical measurementH2O2 +HRP(red)→ H2O +HRP(ox) (2)HRP(ox) +2[Fe(CN)6]3−→ HRP(red) +2[Fe(CN)6]4− (3)[Fe(CN)6]4−→ [Fe(CN)]3− + e− (4)

Thus, the access of the active center of HRP catalyzing the oxidation reaction of

[Fe(CN)6]4−/3− by H2O2 was partly inhibited by AFP, which connected on the surface of the

immunosensor by immunoreaction. In addition, the assay principle was also described in

detail in these literatures (Zhao et al., 2007; Liang and Mu, 2006)[62,63]

Caractrization of bio- GMCNTs

To verify the successful synthesis of the bio- GMCNTs, we intend to use some techniques

such as (1)transmission electron microscopy (TEM) to characterize the synthesized

GMCNTs. Because of Nanometer-sized particles could be formed on the surface of CNTS.

(2) the UV–vis absorption spectroscopy of variously functionalized CNTs ( such as (a)

CNTs, (b) MCNTS, (c) GMCNTs and (d) bio-GMCNTS Especially, when HRP-anti-AFP

molecules are assembled on the GMCNTs. On the basis of the results of these methods , we

might make a conclusion that the bio-GMCN Ts could be synthesized by using this new

developed method.

40

Electrochemical characteristics of of bio- GMCNTs

According to the method which explained by Zhang et al 2011 and others, we will use

AFP as an example to investigate the feasibility of the electrochemical immunoassay. In

the experiment, we will investigated the cyclic voltammograms of the bio- GMCNTs-

modified electrode in pH 6.0 PBS-[Fe(CN)6]4−/3− solution at the absence and presence of

H2O2.[61]

Electrochemical characteristics of the magneto-controlled immunosensor

Fig. 2. (A) Cyclic voltammograms of (a) BSA/Au, (b) MGP/BSA/Au, (c) GMGP/BSA/Au and (d) bio-

GMGP/BSA/Au in 0.1 M pH 6.0 PBS-[Fe(CN)6]4−/3− solution; (B) cyclic voltammograms of the bio-

GMGP/BSA/Au at various scan rates (20, 30, 40, 50, 60, 70, 80, 90, and 100 mV s−1 from inner to

outer) in 0.1 M pH 6.0 PBS-[Fe(CN)6]4−/3− solution (Inset: dependence of peak current on square

root of potential sweep rate); and (C and D) cyclic voltammograms of the bio-GMGP/BSA/Au at the

absence (solid dots) and presence (hollow dots) of 1.5 mM H2O2 in 0.1 M pH 6.0 PBS-

[Fe(CN)6]4−/3− solution (Note: before (C) and after (D) incubation with 10 ng mL−1 AFP).

42

Zhang et al 2011 explained the Electrochemical properties of the magneto-controlled

immunosensor using GoldMag -functionalized graphene saying that.

Fig. 2A displays cyclic voltammograms of various nanostructures-modified gold electrodes

in 0.1 M PBS (pH 6.0) containing 5 mM [Fe(CN)6]4−/3− at 50 mV s−1.

During the measurement, 100 µL of MGPs, GMGPs and bio-GMGPs were injected

in the detection cell, respectively (C[GP] ≈ 1.5 mg mL−1). A couple of well-defined redox

peak was observed at the BSA-modified gold electrode (curve ‘a’ in Fig. 2A).

The difference between the cathodic- and anodic-peak potential (_Ep) was 70 mV,

indicating the ferricyanide was a good electron mediator. When the MGPs were

immobilized on the BSA-modified gold electrode, the currents were decreased (curve ‘b’ in

Fig. 2A), suggesting that the amino functionalized magnetic nanoparticles hindered the

electron transfer. Moreover, the peak currents were decreased again when nanogold particles

were assembled onto the MGPs (curve ‘c’ in Fig. 2A). The reason may be the fact that gold

nanoparticles are a highly negatively charged species as a result of the adsorption of citrate

in the fabrication process, and they repulse the negatively charged ferricyanide (Tang et al.,

2011c). The decrease in the current was obviously attained when HRP-anti-AFP was

conjugated to the GMGPs (curve ‘d’ in Fig. 2A). These results adequately revealed that

HRP-anti-AFP could be immobilized on the GMGPs. Typical cyclic voltammograms of the

bio-GMGP-modified gold electrode at different scan rates of 20–100 mV s−1 were studied

(Fig. 2B). As shown the inset of Fig. 2B, the peak currents were linearly proportional to the

square root of the relevant scan rate. These phenomena can be explained by the Randles

Sevcik equation (Bard and Faulkner, 1980), applicable to the case of a semi-infinite volume

of a diffusing reactant in contact with the electrode.

References:

[1] Ai Zhong XU, Ming Shu YANG, "Flow Field Induced Steady Alignment of Oxidized Multi-walled Carbon Nanotubes", Chinese Chemical Letters, Vol. 16, No. 6, 2005, pp 849-852.[2] Lee, S., Oda, T., Shin, P. & Lee, B. 2009, "Chemical modification of carbon nanotube for improvement of field emission property", Microelectronic Engineering, vol. 86, no. 10, pp. 2110-2113.

[3]Wan, Q., Yang, P., Cai, H., Song, H. & Yang, N. 2013, "Voltammetry of nanomolar leveled environmental hazards on the polymer/CNT coated electrodes", Journal of Electroanalytical Chemistry, vol. 689, no. 0, pp. 252-256.

[4]Zhou, H., Wang, T. & Duan, Y.Y. 2013, "A simple method for amino-functionalization of carbon nanotubes and electrodeposition to modify neural microelectrodes", Journal of Electroanalytical Chemistry, vol. 688, no. 0, pp. 69-75.

[5]Malingappa Pandurangappa and Gunigollahalli Kempegowda Raghu (2011)." Chemically Modified Carbon Nanotubes: Derivatization and Their Applications", Carbon Nanotubes Applications on Electron Devices.

[6] Hwa-Jeong Lee, Sang-Wook Han, Young-Do Kwon, Loon-Seng Tan, Jong-Beom Baeka," Functionalization of multi-walled carbon nanotubes with various 4-substituted benzoic acids in mild polyphosphoric acid/phosphorous pentoxide" C A R B ON 4 6 ( 2 0 0 8 ) 1 8 5 0 –1 8 5 9

[7] Gebhardt,B 2012 "ype Selective Functionalization of Single-Walled Carbon Nanotubes" Friedrich-Alexander-Universität Erlangen-Nürnberg

[8] Pierre R. Marcoux, Philippe Hapiot, Patrick Batail and Jean Pinsonc "Electrochemical functionalization of nanotube films: growth of aryl chains on single-walled carbon nanotubes" New. J. Chem. , 2004, 28, 302–307.

[9] Hirsch, A · Vostrowsky,O 2005, "Functionalization of Carbon Nanotubes", Top Curr Chem (2005) 245: 193–237

[10] Wu.H.C, Chang.X, Liu.L, Zhao.F and Zhao.Y2010 "Chemistry of carbon nanotubes in biomedical applications", J. Mater. Chem. , 2010, 20, 1036-1052

[11]. Chen J, Hamon MA, Hu H, Chen Y, Rao AM, Eklund PC, Haddon RC (1998)" Solution properties of single-walled carbon nanotubes " Science 282:95

44

[12]Chen Y, Haddon RC, Fang S, Rao AM, Eklund PC, Lee WH, Dickey EC, Grulke EC,Pendergrass JC, Chavan A,Haley BE, Smalley RE (1998) " Chemical attachment of organic functional groups to single-walled carbon nanotube material" J Mater Res 13:2423

[13]Jiang, Y., Jin, C., Yang, F., Yu, X., Wang, G., Cheng, S., Di, Y., Li, J., Fu, D. & Ni, Q. 2011, "A new approach to produce amino-carbon nanotubes as plasmid transfection vector by [2 + 1] cycloaddition of nitrenes",Journal of Nanoparticle Research, vol. 13, no. 1, pp. 33-38.

[14] Hirsch A and Vostrowsky.O, "C-60 hexakisadducts with an octahedral addition pattern - A new structure motif in organic chemistry", EUR J ORG C, (5), 2001, pp. 829-848

[15] McCarthy B, Coleman JN, Czerw R, Dalton AB, Carroll DL, Blau WJ. Microscopy studies of nanotube-conjugated polymer interactions. Synth Met 2001;121:1225–6.

[16] Hill DE, Lin Y, Rao AM, Allard LF, Sun YP. Functionalization of carbon nanotubes with polystyrene. Macromolecules 2002;35:9466–71.

[17]Ma, P., Siddiqui, N.A., Marom, G. & Kim, J. 2010, "Dispersion and functionalization of carbon nanotubes for polymer-based nanocomposites: A review", Composites Part A: Applied Science and Manufacturing, vol. 41, no. 10, pp. 1345-1367.

[18] Elena Bekyarova, Bin Zhao, Rahul Sen, Mikhail E. Itkis, Robert C. addon 2004"APPLICATIONS OF FUNCTIONALIZED SINGLEWALLED CARBON NANOTUBES" Prepr. Pap.-Am. Chem. Soc., Div. Fuel Chem. 2004, 49(2), 936

[19]Sen,R; Zhao,B;Perea,D;Itkis,M;Hu,H;Love,J; Bekyarova,E;Haddon,R.C, preparation of Single-Walled Crbon Nanotube Reinforced polystyrene and polyurethane Nanofibers and Membranes by Electrospining. Nano letters 2004,4,(3) ,459-464.

[20]Pantarotto D, Singh R, McCarthy D, Erhardt M, Briand J-P, Prato M, Kostarelos K, Bianco A (2004) Functionalized carbon nanotubes for plasmid DNA gene delivery. Angew Chem Int Ed Engl 43:5242–5246

[21] Singh R, Pantarotto D, McCarthy D, Chaloin O, Hoebeke J, Partidos CD, Briand J-P, Prato M, Bianco A, Kostarelos K (2005) Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors. J Am Chem Soc 127:4388–4396

[22]Jia Huang, Allen L. Ng, Yanmei Piao, Chien-Fu Chen, Alexander A. Green, Chuan-Fu Sun, Mark C. Hersam, Cheng S. Lee, and YuHuang Wang 2013 "Covalently Functionalized Double-Walled Carbon Nanotubes Combine High Sensitivity and Selectivity in the

Electrical Detection of Small Molecules"Journal of the American Chemical Society 2013 135 (6), 2306-2312

[23]Tu, X., Zhao, Y., Luo, S., Luo, X. & Feng, L. 2012, "Direct electrochemical sensing of glucose using glucose oxidase immobilized on functionalized carbon nanotubes via a novel metal chelate-based affinity method",Microchimica Acta, vol. 177, no. 1, pp. 159-166.

[24]Jiang, X.S.; Li, D.Y.; Xu, X.; Ying, Y.B.; Li, Y.B.; Ye, Z.Z.; Wang, J.P. Immunosensors for detection of pesticide residues. Biosens. Bioelectron. 2008, 23, 1577–1587.

[25]. Li, X.L.; Yuan, R.; Chai, Y.Q.; Zhang, L.Y.; Zhuo, Y.; Zhang, Y. Amperometric immunosensor based on toluidine blue/nano-Au through electrostatic interaction for determination of carcinoembryonic antigen. J. Biotechnol. 2006, 123, 356–366.

[26] Campanella, L.; Eremin, S.; Lelo, D.; Martini, E.; Tomassetti, M. Reliable new immunosensor for atrazine pesticide analysis. Sens. Actuators B Chem. 2011, 156, 50–62.

[27] Mello, L.D.; Kubota, L.T. Review of the use of biosensors as analytical tools in the food and drink industries. Food Chem. 2002, 77, 237–256.

[28] Li, Y.; Cheng, P.; Gong, J.H.; Fang, L.C.; Deng, J.; Liang, W.B.; Zheng, J.S. Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens. Anal. Biochem.2012, 421, 227–233.

[29] Pupim, F.A.A.; Colli, W.; Inácio da Costa, P.; Yamanaka, H. Immunosensor for the diagnosis of Chagas’ disease. Biosens. Bioelectron. 2005, 21, 175–181.

[30]. Luppa, P.B.; Sokoll, L.J.; Chan, D.W. Immunosensors-principles and applications to clinical chemistry. Clin. Chim. Acta 2001, 314, 1–26.

[31]Choi, S., Goryll, M., Sin, L., Wong, P., Chae, J., 2011." Microfluidic-based biosensors toward point-of-care detection of nucleic acids and proteins " Microfluid. Nanofluid. 10, 231–247.

[32] Song,Y H. Zhang, C. Chon, X Pan and D. Li, "Nanoparticle Detection by Microfluidic Resistive Pulse Sensor with a submicron sensing gate and dual detecting channels-two stage differential amplifier", Sensors and Actuators, B, 155 (2011) 930-936.

[33] Kang, Y., Li, D., 2009." Electrokinetic motion of particles in microchannels”" Microfluid. Nanofluid. 6, 431–460.,

[34] Lin, F., Gao, Y., Li, D., Sherman, P., 2010 “Development of microfluidic-based heterogeneous immunoassays" . Front. Biosci. S2, 73–84.

46

[35] Wong, T.-S. and Ho, C.-M., “Dependence of Macroscopic Wetting on Nanoscopic Surface Textures”, Langmuir, vol. 25, pp. 12851 – 12854, 2009.

[36] Trocino, S.; Donato, A.; Latino, M.; Donato, N.; Leonardi, S.G.; Neri, G. Pt-TiO2/MWCNTs hybrid composites for monitoring low hydrogen concentrations in air. Sensors 2012, 12, 12361–12373.

[37] Zhao, G.Y.; Zhan, X.J.; Dou, W.C. A disposable immunosensor for Shigella flexneri based on multiwalled carbon nanotube/sodium alginate composite electrode. Anal. Biochem. 2011, 408, 53–58.

[38] Sui, K.Y.; Li, Y.J.; Liu, R.Z.; Zhang, Y.; Zhao, X.; Liang, H.C.; Xia, Y.Z. Biocomposite fiber of calcium alginate/multi-walled carbon nanotubes with enhanced adsorption properties for ionic dyes. Carbohydr. Polym. 2012, 90, 399–406.

[39] Cao, Q.; Zhao, H.; Yang, Y.M.; He, Y.J.; Ding, N.; Wang, J.; Wu, Z.J.; Xiang, K.X.; Wang, G.W. Electrochemical immunosensor for casein based on gold nanoparticles and poly(l-Arginine)/ multi-walled carbon nanotubes composite film functionalized interface. Biosens. Bioelectron.2011, 26, 3469–3474.20. Sun, A.L.; Chen, G

[40] Andrea Masotti 1,* and Andrea Caporali 2 2013 " Preparation of Magnetic Carbon Nanotubes (Mag-CNTs) for Biomedical and Biotechnological Applications" Int. J. Mol. Sci. 2013, 14, 24619-24642; doi:10.3390/ijms141224619

[41] Tomasi TB Jr: Structure and function of alpha –fetoprotein. Ann Rev Med 28:453-465,1977.

[42]Yachnin.S the clinical significant of human alpha-fetoprotein Ann Clin Lab Sci8:84-90,1978.

[43]Bergstrand CG, Czar B: Demonstration of anew protein fraction in serum from the human fetus. Scand J Clin Lab Invest 8:174-4,1956

[44] Abelev GI, perova SD,Khramkova NI, Postnikova ZA, Irlin IS: Production of embryonal alpha-globulin by transplantable mouse hepatoma. Transpl Bull 1:174-180, 1963.

[45]Alpert E: Human alpha-fetoprotein (AFP) ; Developmental biology and clinical significance . prog Liv Dis 5:337-349,1976

[46] Lester EP, Miller JB, Yachnin S, Human alpha-fetoprotein as a modulator of human lymphocyte transformation :Correlation of biological potency with electrophoretic variants . Proc Natl Acad Sci USA 73:4645-4648,1976

[47]Yachnin.S the clinical significant of human alpha-fetoprotein Ann Clin Lab Sci8:84-90,1978.

[48] Bergstrand CG, Czar B: Demonstration of anew protein fraction in serum from the human fetus. Scand J Clin Lab Invest 8:174-4,1956

[49] Abelev GI, perova SD,Khramkova NI, Postnikova ZA, Irlin IS: Production of embryonal alpha-globulin by transplantable mouse hepatoma. Transpl Bull 1:174-180, 1963.

[50] Tomasi TB Jr: Structure and function of alpha –fetoprotein. Ann Rev Med 28:453-465,1977.

[51]Alpert E: Human alpha-fetoprotein (AFP) ; Developmental biology and clinical significance . prog Liv Dis 5:337-349,1976

[52]Ding C, Zhao F, Ren R, Lin JM. An electrochemical biosensor for alpha-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles. Talanta. 2009 May 15;78(3):1148-54.

[53] ChangY.F ; ChenR.C ; Li.Y.C ; YuC.J ;HsiehB.Y, et al. 2008"Alpha-fetoprotein detection by using a localized surface plasmon coupled fluorescence fiber-optic biosensor", Proc. SPIE 6826, Optics in Health Care and Biomedical Optics III, 68261B (January 08, 2008); doi:10.1117/12.754617;

[54] Hsu C.Y, Huang N.W and KLin.K.J,2011 "High sensitivity and selectivity of human antibody attachment at the interstices between substrate-bound gold nanoparticles"Chem. Commun., 47 (2011) 872-874.

[55] Huang.C, Chen.Y, Wang.C, Zhu.W, Ma.H,Gang Jin 2011"Detection of alpha-fetoprotein through biological signal amplification by biosensor based on imaging ellipsometry" ,Thin Solid Films (Impact Factor: 1.6). 01/2011; 519(9):2763-2767. DOI:10.1016/j.tsf.2010.11.064

[56] Xia Xiang, Lu Chen, Cuiling Zhang, Ming Luo, Xinghu Ji and Zhike He 2012" A fluorescence-based colorimetric droplet platform for biosensor application to the detection of α-fetoprotein " Analyst, 2012,137, 5586-5591

[57] Tang.C.R,Tian.G, WangY.J, Su.Z.H, LiC.X, Lin.B.G, Huang.H.W, Yu.X.Y, Li.X.F, Long.Y.F and Zeng.Y.L, 2009 " selective pesponse of dopamine in the presence of ascorbic acid and uric acid at gold nanoparticles and MWCNTS grafted with tetraacitic acid modified electrode" Bull. Chem. Soc. Ethiop. 2009, 23(3), 317-326.

[58] Yu RQ, Chen LW, Liu QP, Lin JY, Tan KL, Xu GQ, et al. Platinum deposition on carbon nanotubes via chemical modification. Chem Mater 1998;10(3):718–22.27.

48

[59] Koo.H.Y, Lee.H.J, Go.H.A, Lee.Y.B, Bae.T.S , Kim.J.K and Choi .W.S 2011 " Graphene-Based Multifunctional Iron Oxide Nanosheets with Tunable Properties" Chem. Eur. J. 2011, 17, 1214 – 1219

[60] He, H., Gao, C., 2010. "3210Supraparamagnetic, Conductive, and Processable Multifunctional Graphene Nanosheets Coated with High-Density Fe3O4 Nanoparticles " ACS Appl. Mater. Interface 2, 3201–3210

[61]Zhang, B., Tang, D., Liu, B., Chen, H., Cui, Y. & Chen, G. 2011, "GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein", Biosensors and Bioelectronics, vol. 28, no. 1, pp. 174-180.

[62] Liang, K., Mu, W., 2006.". flow-injection immune-bioassay for interleukin-6 in human based on gold nanoparticales modified screen-printed graphite electrod" Anal. Chim. Acta 580, 128–135.

[63] Zhao, G., Xing, F., Deng, S., 2007"A disposable amperometric enzyme immunosensor for rapid detection of Vibrio parahaemolyticus in food based on agarose/Nano-Au membrane and screen-printed electrode ." . Electrochem. Commun. 12, 1263–1268.[64] http://www.chemicell.com/products/ferrofluid/ferrofluids.html