microfluidic development of bubble-templated microstructured … · 2012-11-01 · this thesis...

Microfluidic Development of Bubble-templated Microstructured

Materials

By

Jai Il Park

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Chemistry

University of Toronto

© Copyright by Jai Il Park, 2010

II

Microfluidic Development of Bubble-templated

Microstructured Materials

Jai Il Park

Doctor of Philosophy

Department of Chemistry

University of Toronto

2010

Abstract

This thesis presented a microfluidic preparation of bubbles-templated micro-size materials.

In particular, this thesis focused on the microfluidic formation and dissolution of CO2

bubbles. First, this thesis described pH-regulated behaviours of CO2 bubbles in the

microfluidic channel. This method opened a new way to generate small (<10 µm in

diameter) with a narrow size distribution (CV<5%). Second, the microfluidic dissolution of

CO2 bubbles possessed the important feature: the local change of pH on the bubble surface.

This allowed us to encapsulate the bubbles with various colloidal particles. The bubbles

coated with particles showed a high stability against coalescences and Ostwald ripening.

The dimensions and shapes of bubbles with a shell of colloidal particle were manipulated

III

by the hydrodynamic and chemical means, respectively. Third, we proposed a microfluidic

method for the generation of small and stable bubbles coated with a lysozyme-alginate shell.

The local pH decrease at the periphery of CO2 bubbles led to the electrostatic attraction

between lysozyme on the bubble surface and alginate in the continuous phase. This

produced the bubbles with a shell of biopolymers, which gave a long-term stability (up to a

month, at least) against the dissolution and coalescence. Fourth, we presented a single-step

method to functionalize bubbles with a variety of nanoparticles. The bubbles showed the

corresponding properties of nanoparticles on their surface. Further, we explored the

potential applications of these bubbles as contrast agents in ultrasound and magnetic

resonance imaging.

Key words: microfluidics, microreactors, microbubble, polymer particles, carbon dioxide,

colloidal assembly, Pickering emulsions, nanoparticles, ultrasound imaging, magnetic

resonance imaging.

IV

Acknowledgements

First of all, I cannot express fully my gratitude to my Ph.D. supervisor, Prof. Eugenia

Kumacheva. With her great enthusiasm, she continuously teaches, advises and supports me

throughout my graduate life. This thesis would not have been completed without her

encouragement. I am also thankful to Prof. Mitchell A. Winnik and Prof. Michael K.

Georges for being my supervisory committee members and for providing me with valuable

advice.

I would also like to thank all current and former members in Prof. Kumacheva’s group

and my friends in the chemistry department at University of Toronto. It was a truly

experience of a lifetime working with such nice, kind and intelligent people: Dr. Zhihong

Nie, Dr. Alla Petukhova, Dr. Wei Li, Dr. Kun Liu, Dr. Jesse Greener, Dr. Ryan Simms, Dr.

Hung Pham, Dr. Nana Zhao, Dr. Hong Zhang, Dr. Daniele Fava, Dr. Mallika Das, Dr.

Hyunwoo Kim, Dr. Jungho Kim, Dr. Yonghwan Cho, Dr. Sungyeon Choi, Dr. Kyungtaek

Kim, Dr. Sunmok So, Dr. Minseok Seo, Dr. Jeffrey Haley, Dr. Jinshe Song, Mr. Ilya

Gourevich, Mr. Ivan Gorelikov, Ms. Lindsey Fiddes, Ms. Anna Lee, Mr. Ethan Tumarkin,

Mr. Stanislav Dubinski, Mr. Andrew Paton, Ms. Siyon Chung, Ms. Neta Raz, and Ms. Lsan

Tzadu.

I also acknowledge Prof. Axel Günther and Prof. Arron Wheeler for being one of my

committee members and giving valuable suggestions for my completion of Ph.D. I also

wish to acknowledge my collaborators Prof. Howard A. Stone, Prof. Bernard P. Binks, Prof.

Stefan A. F. Bon, Prof. Greg Staniz, Dr. Naomi Matsuura, Mr. Ross Williams, and Ms.

Wendy Oakden who provided valuable input for my different research projects.

My very special thanks belong to my wife, Min Hee, Han, and my 2 year-old son, Shin

Young, Park for their being with me and giving me limitless love. I also give my full

appreciation to my parents, parents-in-law, sister, brothers-in-law, uncles, aunts, cousins

and grandmothers for their constant love and support.

I am thankful to University of Toronto for the financial support.

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Preface

This thesis was written in part based on a series of papers (see the list below), which

have been published in (or submitted to) peer-reviewed scientific journals. As identified by

primary authorship, all manuscripts were written by Jai Il Park with critical comments and

revision by Prof. Eugenia Kumacheva and corresponding collaborators. Jai Il Park

contributed to the papers by designing and carrying out key experiments, data analysis and

interpretation, and manuscript writing. The contributions of other authors are provided in

detail below.

Chapter 3. Microfluidic Generation and Dissolution of CO2 bubbles

The results in this chapter are from manuscripts published in J.I. Park, Z.H. Nie, A.

Kumachev, E. Kumacheva. A Microfluidic Route to Small CO2 Microbubbles with

Narrow Size Distribution. Soft Matter 2010, 6, 630.

Contribution: Z. H. Nie conducted preliminary work on the generation and dissolution of

CO2 bubbles. He also contributed to data analysis and provided critical comments. A.

Kumachev helped with making devices and conducting experiments.

Chapter 4. Assembly of Colloidal Particles at Gas-Water and Water-Oil Interfaces

The results in this chapter are from manuscripts published in J.I. Park, Z.H. Nie, A.

Kumachev, A. I. Abdelrahman, H. A. Stone, B. P. Binks, E. Kumacheva. A Microfluidic

Approach to Chemically Driven Assembly of Colloidal Particles at Gas-Liquid

Interfaces. Angewandte Chemie International Edition 2009, 48, 5300.,1 and Z.H. Nie, J.I.

Park (co-first author), W. Li, S. Bon, E. Kumacheva. An "Inside-Out" Microfluidic

Approach to Monodisperse Emulsions Stabilized by Solid Particles. Journal of the

American Chemical Society 2008, 130, 16508.2

Contribution: 1Z. H. Nie designed and conducted several key experiments. A. I.

Abdelrahman provided carboxylated polystyrene particles. A. Kumachev contributed to

make devices and conduct preliminary experiments. Prof. H. A. Stone designed a

theoretical model for the deposition of particles on the surface of bubbles. Prof. B. P. Bink

gave a critical input to manuscript writing. 2J.I. Park conducted several key experiments

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and finalized the manuscript. W. Li helped to obtain the confocal microscope images. Prof.

S. Bon initiated the project and provided guidance.

Chapter 5. Bubbles Encapsulated with a Shell of Biopolymers

The results in this chapter are from manuscripts published in J.I. Park, E. Tumarkin, E.

Kumacheva. Small, Stable, and Monodispersed Bubbles Encapsulated with

Biopolymers. Macromolecular Rapid Communications 2010, 31, 222.

Contribution: E. Tumarkin conducted several key experiments and helped to polish the

manuscript.

Chapter 6. A Single-step Microfluidic Route to Producing Multifunctional

Microbubbles The results in this chapter are from manuscripts submitted to J.I. Park, D.

Jagadeesan, R. Williams, W. Oakden, S. Chung, G. Stanisz, E. Kumacheva. A Single-step

Microfluidic Route to Producing Multifunctional Microbubbles. Journal of the

American Chemical Society 2010.

Contribution: D. Jagadeesan and S. Chung conducted experiments and gave valuable

suggestions to the manuscript. R. Williams performed ultrasound imaging experiments,

data analysis and provided critical inputs to the manuscript. W. Oakden conducted

magnetic resonance imaging experiments and analyzed the data. G. Stanisz gave valuable

direction to magnetic resonance imaging experiments.

VII

Publications during PhD Study

Peer-reviewed Papers:

1. E. Tumarkin, Z. Nie, J.I. Park. Greener, E. Kumacheva, Microfluidic Mimicking

Solubility Pump. 2010, in preparation. 2. J.I. Park, D. Jagadeesan, R. Williams, W. Oakden, S. Chung, G. Stanisz and E.

Kumacheva. A Single-step Microfluidic Route to Producing Multifunctional

Microbubbles. Journal of the American Chemical Society 2010, submitted.

3. J.I. Park, A. Saffari, S. Kumar, A. Günther, E. Kumacheva. Microfluidic Synthesis

of Polymer and Inorganic Particulate Materials. Annual Review of Materials

Research 2010, 40, 415, invited review article.

4. J.I. Park, E. Tumarkin, E. Kumacheva. Small, Stable, and Monodispersed

Bubbles Encapsulated with Biopolymers. Macromolecular Rapid

Communications 2010, 31, 222.

5. J.I. Park, Z.H. Nie, A. Kumachev, E. Kumacheva. A Microfluidic Route to Small

CO2 Microbubbles with Narrow Size Distribution. Soft Matter 2010, 6, 630.

6. J.I. Park, Z.H. Nie, A. Kumachev, A. I. Abdelrahman, H. A. Stone, B. P. Binks, E.

Kumacheva. A Microfluidic Approach to Chemically Driven Assembly of

Colloidal Particles at Gas-Liquid Interfaces. Angewandte Chemie International

Edition 2009, 48, 5300, cover page.

7. S. Dubinsky, J.I. Park, I. Gourevich, H.K.C. Chan, M. Deetz, E. Kumacheva,

Towards Controlling the Surface Morphology of Macroporous Copolymer

Particles. Macromolecules 2009, 42, 1990.

8. Z.H. Nie, J.I. Park (co-first author), W. Li, S. Bon, E. Kumacheva. An "Inside-

Out" Microfluidic Approach to Monodisperse Emulsions Stabilized by Solid

Particles. Journal of the American Chemical Society 2008, 130, 16508.

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Oral Presentations in Conferences:

1. J.I. Park, E. Tumarkin, E. Kumacheva. Small, Stable, and Monodisperse Bubbles

Encapsulated with Biopolymers, Canadian Chemistry Conference, Toronto,

Canada, 2010.

2. J.I. Park, Z.H. Nie, Kumachev. A. I. Abdelrahman, B. P. Binks, H. A. Stone, E.

Kumacheva, A Microfluidic Approach to Chemically Driven Assembly of

Colloidal Particles at Gas-Liquid Interfaces, MicroTas, Jeju, Korea, 2009.

3. J.I. Park, Z.H. Nie, E. Kumacheva, Using Microfluidics to Study Dissolution of

Carbon Dioxide, Canadian Chemistry Conference, Hamilton, Canada, 2009

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Table of Content Chapter 1 .......................................................................................................................... 1

Introduction to Microfluidic Synthesis of Particulate Materials .................................... 1

1.1 Overview of Microfluidics .................................................................................... 1

1.2 Different Modes of Microfluidic Synthesis of Polymer Particles ........................... 2

1.3 Microfluidic Synthesis of Polymer Particles ......................................................... 5

1.3.1 Microfluidic Synthesis of Solid Polymer Microspheres .............................. 6

1.3.2 Microfluidic Synthesis of Spherical Polymer Microgels ............................. 8

1.3.3 Microfluidic Synthesis of Polymer Particles with Different Morphologies 10

1.3.4 Microfluidic Synthesis of Non-spherical Polymer Particles ...................... 14

1.3.5. Microfluidic Synthesis of Bubble-templated Polymer Particles ............... 17

1.3.6 Productivity of Continuous Microfluidic Reactors .................................... 18

1.4 Outlook .............................................................................................................. 20

References ................................................................................................................ 21

Chapter 2 ........................................................................................................................ 30

Materials and Methods .................................................................................................. 30

2.1 Materials ............................................................................................................ 30

2.2 Methods.............................................................................................................. 31

2.2.1 Mask Design ............................................................................................ 31

2.2.2 Microfabrication of Negative Masters ...................................................... 32

2.2.3 Fabrication of Microfluidic Devices ......................................................... 32

2.2.4 Microfluidic Experiments ......................................................................... 33

2.2.5 Photopolymerization Experiments ............................................................ 34

2.3. Characterization ................................................................................................. 34

2.3.1 Optical and Fluorescence Microscopy Imaging ........................................ 34

2.3.2 Size Distribution of Bubbles, Droplets and Particles ................................. 35

2.3.3 Scanning and Transmission Electron Microscopy imaging ....................... 35

2.3.4 Laser Confocal Fluorescence Microscopy imaging ................................... 35

2.3.5 Contact angle measurement ...................................................................... 36

2.3.6 Circular Dichroism Spectroscopy ............................................................. 36

X

2.3.7 Fluorescence Spectroscopy ...................................................................... 37

References ................................................................................................................ 37

Chapter 3 ........................................................................................................................ 40

Microfluidic Generation and Dissolution of CO2 bubbles ............................................ 40

3.1 Introduction ........................................................................................................ 40

3.2 Results and Discussions ...................................................................................... 42

3.2.1 Generation of CO2 bubbles ....................................................................... 42

3.2.2 Dissolution of CO2 Bubbles ..................................................................... 49

3.2.3 Generation of Small Bubbles .................................................................... 55

3.3 Conclusion ......................................................................................................... 56

References ................................................................................................................ 57

Chapter 4 ........................................................................................................................ 60

Assembly of Colloidal Particles at Gas-Water and Water-Oil Interfaces .................... 60

4.1 Introduction ........................................................................................................ 60

4.2 Results and Discussion ....................................................................................... 63

4.2.1 Synthesis of Colloidal Particle Armoured Bubbles ................................... 63

4.2.1.1 Experimental Design ..................................................................... 63

4.2.1.2 Generation of Armoured Bubbles .................................................. 64

4.2.1.3 Effect of Flow Rate on the Dimensions of Armoured Bubbles ....... 68

4.2.1.4 Effect of pH and Particle Concentration on the Dimension and

Morphologies of Armoured Bubbles.......................................................... 69

4.2.1.5 Generality of the Assembly of Colloidal Particles at Gas-Water

Interface .................................................................................................... 73

4.2.2 Synthesis of Colloidal Particle-Coated Droplets ....................................... 74

4.2.2.1 Experimental Design ..................................................................... 75

4.2.2.2 Microfluidic Control of Particle Coverage at Water-Oil Interfaces . 76

4.3. Conclusion......................................................................................................... 81

References ................................................................................................................ 82

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Chapter 5 ........................................................................................................................ 85

Bubbles Encapsulated with a Shell of Biopolymers ...................................................... 85

5.1 Introduction ........................................................................................................ 85

5.2 Results and Discussions ...................................................................................... 88

5.2.1 Preparation of Bubbles Encapsulated with a Mixture of Biopolymers ....... 88

5.2.2. Long-term Stability of Bubbles ............................................................... 91

5.2.3 Control of Bubble Dimensions ................................................................. 93

5.2.4 Characteristic Properties of the Bubbles ................................................... 97

5.3 Conclusion ....................................................................................................... 100

References .............................................................................................................. 101

Chapter 6 ...................................................................................................................... 104

A Single-step Microfluidic Route to Producing Multifunctional Microbubbles ........ 104

6.1 Introduction ...................................................................................................... 104

6.2 Results and Discussions .................................................................................... 106

6.2.1 Experimental Design .............................................................................. 106

6.2.2 Long-term Stability of Biopolymer Encapsulated-Bubbles Functionalized

with NPs ......................................................................................................... 109

6.2.3 Control of the Dimension of Biopolymer Encapsulated-Bubbles

Functionalized with NPs ................................................................................. 110

6.2.4 Control Experiments ............................................................................... 111

6.2.5 Characterization of Biopolymer Encapsulated-Bubbles Functionalized with

various NPs ..................................................................................................... 111

6.2.6 Control over the Amount of NPs on the Bubble Surface ......................... 113

6.2.7 Properties of NP-functionalized Bubbles ................................................ 113

6.2.8 Application of NP-functionalized Bubbles in US Imaging ...................... 114

6.2.9 Application of NP-functionalized Bubbles in MRI ................................. 116

6.3 Conclusions ...................................................................................................... 117

References .............................................................................................................. 118

Chapter 7 ...................................................................................................................... 121

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Conclusion and Outlook .............................................................................................. 121

Appendix ...................................................................................................................... 125

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List of Figures

Chapter 1 Introduction to Microfluidic Synthesis of Particulate Materials

Figure 1.1 Synthesis of particulate materials (A) in a batch reactor and (B) in a flow-

based microfluidic (MF) reactor. Adapted with permission from reference 9.

Copyright 2010, Annual Reviews…………………………………………..…2

Figure 1.2 Schematic illustrations of (A) single-phase and (B) multiphase formats for the

MF synthesis of polymer particles synthesized (C) in droplets (i), and (D) at the

interface between the two phases (ii). Adapted with permission from reference

9. Copyright 2010, Annual Reviews…………………………………………..3

Figure 1.3 Rigid polymer particles synthesized in continuous multiphase MF reactors.

(A) SEM images of poly(tripropyleneglycol diacrylate) particles. (B) Size

distribution of the particles shown in panel (A). d denotes particle diameter. (C)

Optical fluorescence microscopy images of poly(tripropylene glycol diacrylate)

microspheres loaded with CdSe quantum dots. (D) Optical polarization

microscopy images of poly(tripropyleneglycol diacrylate) microspheres with 5–

20 wt. % of liquid crystals made of 4-cyano-4’-pentylbiphenyl. Panels (A–D)

are reproduced with permission from reference 20. Copyright 2005, Wiley-

VCH. (E) SEM image of porous poly(glycidyl methacrylate–co-ethylene glycol

dimethacrylate). Panel (E) is adapted with permission from reference 67.

Copyright 2009, American Chemical Society. (F) Confocal microscopy image

of poly(tripropyleneglycol diacrylate)/poly(urethane) particles with an

interpenetrating network (IPN) structure. Panel (F) is adapted with permission

from reference 68. Copyright 2008, American Chemical Society……………7

Figure 1.4 Microgel particles synthesized in continuous multiphase MF reactors. (A)

Optical microscopy images of the formation of droplets of poly(N-

isopropylacrylamide) microgel particles (left) and the resulting microgel

particles labeled with fluorescein isothiocyanate (right). The inset shows a

schematic of the microfluidic reactor (OF, outer fluid; MF, middle fluid).

Reproduced with permission from reference 55. Copyright 2007, Wiley-VCH.

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(B) Optical microscopy images of ionically cross-linked alginate (left), k-

carrageenan (middle), and carboxymethylcellulose (right) microgels. Scale bar:

100 μm. Adapted with permission from reference 56. Copyright 2006,

American Chemical Society……………………………………………………9

Figure 1.5 MF synthesis of polymer particles with different morphologies. (A) Schematic

of the microfluidic synthesis of Janus microgels. The inset shows an optical

fluorescence image of poly(acrylamide) particles encapsulating silica particles

labeled with two different dyes in distinct compartments. Adapted with

permission from reference 72. Copyright 2006, American Chemical Society.

(B) Optical microscopy image of bicolored poly(isobornyl acrylate) particles

containing carbon black and titanium oxide in two distinct hemispheres. The

inset shows electrically actuated particles displaying their white-colored side in

the display panel. Adapted with permission from reference 79. Copyright 2006,

Wiley-VCH. (C) SEM images of microcapsules prepared by microfluidic

interfacial polymerization. The inset shows a high magnification image of the

interior of the capsular wall. Adapted with permission from reference 54.

Copyright 2005, American Chemical Society. (D) Optical microscopy image of

the colloidal crystalline array confined in polymeric shells. The inset shows the

particles with three colloidal crystal cores. Scale bars: 200 μm. Adapted with

permission from reference 99. Copyright 2008, American Chemical Society..11

Figure 1.6 MF synthesis of particles with non-spherical shapes. (A,B) SEM images of

poly(tripropyleneglycol diacrylate) particles with (A) disk and (B) rod shapes.

Insets show the relationship between the particle shape and the channel

dimensions. Adapted with permission from references 20 and 103. Copyright

2005, Wiley-VCH. Copyright 2005, American Chemical Society. (C) SEM

image of poly(tripropyleneglycol diacrylate) particles with bowl-like shapes.

The inset shows the precursor droplet with a small fraction of nonpolymerizable

liquid (indicated by the arrow). Scale bar: 40 μm. Adapted with permission

from reference 85. Copyright 2005, American Chemical Society. (D–F) SEM

images of poly(ethylene glycol diacrylate) particles with various shapes

synthesized in one-phase MF synthesis using continuous-flow lithography.

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Insets show the corresponding photomasks. Scale bars: 10 μm. Adapted with

permission from reference 15. Copyright 2006, Macmillan…………….…..15

Chapter 3 Microfluidic Generation and Dissolution of CO2 bubbles

Figure 3.1 Schematic of the MF device and the dissolution of CO2 bubbles. Inset shows a

zoomed in schematic of the orifice and the generation of bubbles……….…42

Figure 3.2 Schematic of pH-dependent dissolution of CO2 bubble leading to bubble

shrinkage……………………………………………………………………...43

Figure 3.3 Representative optical microscopy images taken immediately after the

generation of CO2 bubbles (A,B) and N2 bubbles (C,D) and in the downstream

channel 107 mm (A’-D’) away from the orifice. The initial pH value of the

continuous phase was 1.5 (A,A’,C,C’) and 13.2 (B,B’,D,D’). The bubbles were

generated at PCO2=27.6 kPa and QL=6 mL/h. Scale bar is 200 μm………….44

Figure 3.4 Variation in initial (●, ), and final (○,) volumes of CO2 (●,○) and N2

(,) bubbles plotted as a function of the initial pH of the continuous aqueous

phase……………………………………………………………………….….46

Figure 3.5 pH-dependence of the relative frequency of bubble generation with respect to

the frequency of the formation of bubbles at pH=13.2 for CO2(■) and N2()

bubbles. The bubbles were generated at P= 27.6 kPa, QL=6 mL/h……….…47

Figure 3.6 (A-C) Optical microscopy images of CO2 bubbles taken at varying distances

away from the orifice in the device at (A) pH=1.5, (B) pH=9, and (C) pH=13.2.

Scale bar is 50 μm. (D) Variation in the volume of CO2 bubbles examined at

different pH values, plotted vs the distance, d, away from the orifice……....48

Figure 3.7 (A) Comparison of theoretical (■) and experimental () amounts of dissolved

CO2 (mol/L), (B) Relative change in bubble volume plotted as a function of pH

of the continuous phase. PCO2=27.6 kPa, QL=6 mL/h……………………....52

Figure 3.8 Effect of the flow rate of the continuous phase, QL, on (A) the initial and (B)

final volume of CO2 bubbles generated at different pH values of the continuous

phase. pH=1.5 (,), pH=5(,), pH=7 (,), pH=9 (,), pH=11

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(,), and pH=13.2 (,). PCO2=27.6 kPa. The lines are given for eye

guidance……………………………………………………………………....53

Figure 3.9 Variation in the final diameter of CO2 bubbles, plotted as a function of pH and

QL (each represents the QL range from 5 to 7 mL/h, respectively)………….54

Figure 3.10 (A) Generation of 30 μm-diameter CO2 bubbles. (B) Bubbles with a

stabilized diameter of 8 μm in the downstream channel. The bubbles were

generated in the microfluidic device with the length, width, and height of the

orifice of 60, 22 and 40 μm, respectively, at PCO2=55.2 kPa, QL=12 mL/h, and

pH=13.2……………………………………………………………………….55

Chapter 4 Assembly of Colloidal Particles at Gas-Water and Water-Oil Interfaces

Figure 4.1 (A) A schematic of a MF T-junction bubble generator. The widths of the main

and the side channels are 220 and 40 μm, respectively. The height of the

channels is 130 μm. (B) A schematic of the formation of colloidal particle shell

during the dissolution of CO2 bubbles……………………………………….62

Figure 4.2 Progression of the plugs to spherical armoured bubbles. Optical microscopy

images of bubbles at a distance of 0 (A), 30 (B), 80 (C) and 150 (D) mm from

the T-junction. Scale bars are 200 μm. Bubbles generated at pH=14, PCO2=34.5

kPa, QL=10.5 mL/h, initial particle concentration, Cp,=1.5 wt. %, and

28.8oC…………………………………………………………………………63

Figure 4.3 (A) Optical microscopy image of armoured bubbles generated as in Figure 4.2

and collected at the outlet of the MF device. Scale bar is 200 μm (B) Optical

microscopy image of close-packed crystalline shell of armoured bubble. Scale

bar is 25 μm (C) SEM image (side view) of the shell of the microtomed

armored bubble infiltrated with poly(ethylene glycol) diacrylate. Scale bar is 10

μm. The inset shows an LCFM image of the bubble coated with HY-labeled

PS-co-PAA particles. Scale bar is 25 μm……………………………………..64

Figure 4.4 Optical microscopy images of bubbles and plugs flowing through the

microchannels. (A) Dissolution of CO2 plugs in particle-free at pH=14. (B)

Plugs of gaseous N2 formed in a dispersion of anionic PS-co-PAA particles. (C)

XVII

Bubbles of CO2 generated in a dispersion of PS-co-PAA particles containing 2

wt. % of the non-ionic surfactant (Triton X-100). (D) Plugs of N2 formed in a

dispersion of 700 nm diameter cationic PMMA-co-P4VP particles. (E) Bubbles

of CO2 dispersed in an aqueous dispersion of 700 nm diameter PMMA-co-

P4VP particles. In all experiments, the bubbles were generated at 28.8oC,

pH=14, PCO2/N2=34.5kPa, QL=10.5mL/h, and Cp=1.5wt. %. Optical microscope

images of collected bubbles (F),(G) from (D),(E), respectively. Scale bars: 200

μm………………………………………………………………………….…..65

Figure 4.5 Variation in contact angle (θ) of an aqueous solution of NaOH in air on the

PS-co-PAA film, measured at different pH values..........................................66

Figure 4.6 (A) Effect of the flow rate, QL, of the continuous phase on the fractional

volume change (ΔV/V0) of CO2 plugs and the final diameter Df of armoured

bubbles in the microchannel. The produced armoured bubbles at QL= 8.5 (B)

and 10.5 mL/h (C). Scale bars are 50 μm. The insets show the initial plugs of

CO2 bubbles. Scale bars are 200 μm. Bubbles were generated at pH=14,

PCO2=34.5 kPa and Cp=1.5 wt. %....................................................................67

Figure 4.7 (A) Effect of the initial pH of the continuous phase on the fractional volume

reduction (ΔV/V0) of CO2 plugs and the final diameter, Df, of armoured bubbles

in the microchannel. The produced armoured bubbles at initial pH=14 (B) 8 (C)

and 5 (D). Scale bars are 100 μm. The insets show the bubbles before the exit of

the microchannel. Scale bars are 200 μm. Bubbles were generated at PCO2=34.5

kPa, QL=9.0 mL/h and Cp=1.5 wt. %..............................................................68

Figure 4.8 (A) Effect of the initial concentration of particles, Cp, of the continuous phase

on the fractional volume reduction, ΔV/V0, of CO2 plugs and the final diameter,

Df, of armoured bubbles in the microchannel. The produced armoured bubbles

at initial Cp =0.1 (B) 1.5 (C) and 5 wt. % (D). Scale bars are 100 μm. The insets

show the bubbles before the exit of the microchannel. Scale bars are 200 μm.

Bubbles were generated at PCO2=34.5 kPa, QL=10.0 mL/h and pH=14…….70

Figure 4.9 Generation of bubbles with various types of colloidal armour. (A) Optical

fluorescence microscopy image of bubbles coated with a shell of 2.8 μm

diameter HY-labelled PS-co-PAA particles (Cp=0.5 wt. %) and 3.5 μm

XVIII

diameter PS-co-PAA particles (Cp=1.0 wt. %), PCO2=41.4 kPa, QL=12 mL/h,

pH=14. Scale bar is 50 μm. (B) Optical microscopy image of bubbles coated

with 3 μm diameter carboxylated silica particles. PCO2=82.7 kPa, QL =24 mL/h,

pH=14, and Cp=1.5 wt. %. Scale bar is 100 μm. The inset shows a fluorescence

microscopy image of the surface of an armoured bubble coated with 3 μm

diameter carboxylated silica particles and 2.8 μm diameter HY-labelled PS-co-

PAA polymer particles in the continuous phase in the weight ratio 3:1,

respectively. Scale bar is 25 μm. (C) LCFM image of bubbles encapsulated

with 20 nm diameter carboxylated silica nanoparticles loaded with CdSe/ZnS

QDs, λex=480 nm. PCO2=55.2 kPa, QL=23 mL/h, pH= 10, and Cp =0.12 wt. %.

Scale bar is 100 μm. (D) Fluorescence optical microscopy image of armoured

bubbles engulfed with the FITC–BSA shell, λex=495 nm. The bubbles were

generated at PCO2=44.8 kPa, QL=13 mL/, pH=7, and a protein concentration of

0.02 wt. %. Scale bar is 100 μm………………………………………………73

Figure 4.10 (A) Generation of droplets from the water-ethanol mixture (85/15, v/v)

containing 4 wt. % of poly (DVB-co-MAA) particles. The flow rates of the

droplet and continuous phases are 0.5 and 3.5 mL/h, respectively. Scale bar is

200 μm. (B) Collected particle-coated droplets at the exit of the microchannel.

Scale bar is 150 μm……………………………………………………………75

Figure 4.11 A diagram of the surface coverage of water-ethanol droplets with 3.5 μm-

diameter poly(DVB-co-MAA) particles. The dashed line shows the theoretical

conditions for the complete coverage of the droplets with a monolayer of 2D

hexagonally packed particles. Filled circles show the experimental data

points…………………………………………………………….……………76

Figure 4.12 Plug-shaped particle-covered droplets. Scale bar is 100 µm. Inset shows the

corresponding elliptical droplets flowing in the microchannel. Scale bar is 200

μm. Cp=14 wt. %. The flow rate of the droplet and continuous phases are 0.5

and 5.5 mL/h, respectively…………………………………………………..77

Figure 4.13 (A-C) Optical and (D-F) LCFM images of the water-ethanol droplets

armoured with a shell of poly(DVB-co-MAA) particles at the not-complete

(A,D) and complete (B,E) surface coverage, and at the excess of particles in

XIX

the droplet interior (C,F). In (C) an excess of particles appears as the large dark

region on the background of the droplet coated with a monolayer of particles. In

(D-F) poly(DVB-co-MAA) microbeads were labelled with anthryl methacrylate.

LCFM images show the plane located in the centre of the droplets. Cp =8 wt. %.

λex=380 nm. Scale bars in (A-E) and in (F) are 50 and 100 μm, respectively.

Scale bars in insets are 5 μm…………………………………………………78

Figure 4.14 SEM (A) and LCFM (B) images of poly(TPGDA) particles armoured with

poly(DVB-MAA) particles. Cp=14 wt. %. Scale bars are 40 μm in (A) and 60

μm in (B)……………………………………………………………………...79

Chapter 5 Bubbles Encapsulated with a Shell of Biopolymers

Figure 5.1 Schematic of co-adsorption of lysozyme and alginate on the surface of CO2

bubble during its dissolution…………………………………………………88

Figure 5.2 A) Schematic of a MF flow-focusing bubble generator, B) Representative

optical microscopy images of the generation (top) and dissolution of CO2

bubbles in a MF channel (bottom). The image is taken 250 mm away from the

orifice of the MF device. The width and height of the orifice are 50 and 120 μm,

respectively. PCO2=48.3 kPa, QL=6 mL/h. Clys=Calg=0.2 wt. %, pH=12. Scale

bar is 200 μm………………………………………………………………….89

Figure 5.3 A-C) Optical microscopy images of the bubbles at the exit of the microchannel

(inset shows the image of initial bubble at the orifice, scale bar: 100 μm) (A),

after 0.3(B), 2 (C), 24 (D) and 720 h (E) storage. The bubbles were formed at

PCO2=48.3 kPa and QL=6 mL/h. Clys=Calg=0.2 wt. %. Scale bars: 50 μm. D)

Change in the diameter of bubbles plotted as a function of time; the dashed line

separates on-chip (left) and off-chip (right) bubble storage…………………91

Figure 5.4 N2 bubbles after 24 h storage. Scale bar: 200 μm. N2 bubbles generated at

PN2=48.3 kPa, QL=6 mL/h, pH=12, Clys=Calg=0.2 wt. %...............................92

Figure 5.5 A) Effect of QL of the continuous aqueous phase on the initial (Di) and final

dimension (Df) of bubbles. PCO2=48.3 kPa, Clys=Calg=0.2 wt. %, B-D) Optical

microscopy images of the bubbles after 24 h (insets show the image of initial

XX

bubble at the orifice, scale bars: 100 μm) at QL=4.5 (B), 5.5 (C) and 6.5 mL/h

(D). Scale bars: 15 μm……………………………………………………….93

Figure 5.6 A) Effect of Calg on the initial and final dimensions of bubbles, PCO2=48.3 kPa,

QL=6 mL/h. Clys=0.2 wt. %, B-D) Optical microscopy images of the bubbles

after 24h (insets show the image of initial bubble at the orifice, scale bars: 100

μm) at Calg=0 (B, scale bar: 30 μm), 0.15 (C) and 0.2 wt. % (D). Scale bars: 15

μm……………………………………………………………………………94

Figure 5.7 A) Effect of Clys on the initial and final dimensions of bubbles, PCO2=48.3 kPa,

QL=6 mL/h. Calg=0.2 wt. %, B-D) Optical microscopy images of the bubbles

after 24h (insets show the image of initial bubble at the orifice, scale bars: 100

μm) at Clys=0 (B), 0.05 (C) and 0.15 wt. % (D). Scale bars: 15 μm…………95

Figure 5.8 The preparation of 7 μm-diameter bubbles produced at PCO2=72.4 kPa,

QL=10.5 mL/h. Clys=0.05 wt. % and Calg=2 wt. %. Optical microscopy image of

the bubbles after 24 (A). (inset: the initial CO2 bubble at the orifice. scale bar:

80 μm) and 720 h (B). Scale bars: 15 μm…………………………………….95

Figure 5.9 A) LCFM image of the bubble encapsulated with a lysozyme-alginate shell

and stored for 10 days (PCO2=48.3 kPa, QL=6 mL/h, Clys=Calg=0.2 wt. %). The

focal plane is located at 8 μm below the surface of the bubble. B) SEM images

of the bubble produced under the same conditions as in A). In A) and B) the

scale bar is 7 μm. C) A high magnification SEM image of the surface of the

bubble shown in (B). The scale bar is 2 μm. D) SEM image of the fractured

bubble surface. The bubble was stored for 28 days. The scale bar is 4 μm….97

Figure 5.10 Comparison of CD spectra of the native lysozyme solution (Clys=0.003

wt. %) (━) and the bubbles encapsulated with a lysozyme-alginate shells

(,). The bubbles were stored for 5 days () and 28 days ().

Conditions of bubble formations: PCO2=48.3 kPa, QL=5 mL/h, Clys=Calg=0.2

wt. %...............................................................................................................98

Figure 5.11 A) Polarization optical microscopy image of the bubbles coated with a

lysozyme-alginate shell. The bubbles were produced under PCO2=48.3 kPa,

QL=6 mL/h, Clys=Calg=0.2 wt. % and stored for 28 days. The scale bar is 50 μm.

B) Fluorescence intensity profile of 50 μM ThT solution (), freshly prepared

XXI

lysozyme (Clys=0.003 wt. %) dissolved in 50 μM ThT solution (), freshly

prepared alginate (Calg=0.003 wt. %) dissolved in 50 μM ThT solution () and

the dispersion of biopolymer-encapsulated bubbles in 50 μM ThT solution ().

The bubbles were stored for 28 days………………………………………...99

Chapter 6 A Single-step Microfluidic Route to Producing Multifunctional

Microbubbles

Figure 6.1 Schematics of the microfluidic (MF) generation of multifunctional bubbles.

(A) Schematic of a MF reactor. The height of the MF device is 120 µm. The

width of the orifice and the length of the downstream microchannel are 50 µm

and 260 mm, respectively. The top and the bottom insets show optical

microcopy images of the bubbles at the beginning and the end of the process,

respectively. Bubbles were generated at PCO2=48.3 kPa and QL=9.5 mL/h.

Scale bars in insets are 200 µm. (B) Schematic of the formation of NP

functionalized bubbles stabilized with a mixed lysozyme-alginate layer….106

Figure 6.2 Optical microscopy images of the bubbles functionalized with Fe3O4 NPs

after different storage times: (A) 3 sec, (B) 1 h and (C) 2000 h. The scale

bars in (A), (B) and (C) are 50, 15 and 15 µm, respectively. Inset in (A)

shows the bubble imaged immediately after its generation in the orifice of

the MF device. Scale bar is 100 µm. Bubbles were generated at PCO2=48.3

kPa and QL=9.5 mL/h………………………………………………………108

Figure 6.3 (A) Variations in the initial,Di, and final dimensions, Df, of microbubbles

are plotted as a function of the flow rate of the continuous phase, QL. (B-D)

Representative optical microscope images of the initial (insets) and final

dimension of bubbles coated with the biopolymers and Fe3O4 NPs at

different QL s (A) 6.5, (B) 7.5 and (C) 8.5 mL/h. Clysozyme=0.05 wt. %.

Calginate=0.1 wt. %, and CFe3O4 dispersion = 1 wt. %. PCO2=48.3 kPa. Scales

bars are 15 µm (100 µm, insets)…………………………………………109

Figure 6.4 Optical microscope images of (A) aggregated bubbles generated with

lysozyme and Fe3O4 NPs. Scale bar is 15 µm and (B) coalesced N2 bubble

generated with lysozyme, alginate and Fe3O4 NPs. Scale bar is 200 µm…110

XXII

Figure 6.5 Scanning transmission electron microscopy (STEM) images of the bubbles

coated with the lysozyme-alginate shell and (A) Fe3O4 NPs, (B) Au NPs, and

(C) SiO2-encapsulated CdSe/ZnS NPs, Scale bars are 6 µm. Insets in (A-C)

show corresponding high magnification images of the surface of the bubbles.

Scale bars in insets are 150 nm. (D-F) Energy dispersive X-ray (EDX)

spectrometry line scanning profiles for the system shown as a red line in (A),

(B) and (C). Bubbles were generated at PCO2 = 48.3 kPa and QL = 8.5

mL/h………………………………………………………………………111

Figure 6.6 (A-C) STEM images of the surface of bubbles coated with Fe3O4 NPs at

surface density of (A) 1.5x105, (B) 6.6x105 and (C) 1.5x106 NPs/µm2. Scale

bars are 300 nm. Insets show the corresponding bubbles. Scale bars are 3 µm.

Bubbles were generated at PCO2=48.3 kPa and and QL=8 mL/h……….112

Figure 6.7 Properties of NP-coated bubbles. (A) Magnetic actuation of bubbles

functionalized with Fe3O4 NPs. Scale bar is 50 µm. Bubbles were generated at

PCO2=48.3 kPa and and QL=8.5 mL/h. (B) Extinction spectra of Au NPs (red

spectrum, top) and of the bubbles coated with these Au NPs (blue spectrum,

bottom). Bubbles were generated at PCO2=48.3 kPa and and QL =8.5 mL/h.

(C) Confocal fluorescence microscopy image of bubbles carrying SiO2-

encapsulated CdSe/ZnS NPs. Scale bar is 30 µm. λex=364 nm. Inset shows an

image of the individual bubble. Scale bar is 5 µm. Bubbles were generated at

PCO2=48.3 kPa and and QL=9.5 mL/h…………………………………….113

Figure 6.8 In-vitro US imaging of the dispersion of dispersion of biopolymer-coated

bubbles at 90 % receive gain. The dispersion is placed in the Opticell chamber.

(A) NP-free bubbles, (B-D) bubbles coated with Fe3O4 NPs (B), Au NPs (C)

and SiO2-encapsulated CdSe/ZnS NPs (D). (E) US signal enhancement over

background for the systems shown in A-D. The concentration of bubbles in all

systems was 104 bubbles/mL……………………………………………….115

Figure 6.9 (A) In-vitro MRI images (top-view) of the dispersions of biopolymer-

encapsulated bubbles coated with different amounts of Fe3O4 NPs. The images

were obtained at 6.9 ms echo time. (B) Variation in T2* relaxation rate plotted

XXIII

as a function of surface density of Fe3O4 NPs on the surface of bubbles. The

concentration of bubbles in all systems was 104 bubbles/mL……………116

XXIV

Abbreviation

Alginate Alginic acid sodium salt BSA Bovine albumin serum CV Coefficient of variation DMPA 2,2-dimethoxy-2-phenyl-acetophenone DVB Divinylbenzene FITC Fluorescein isothiocyanate 4VP 4-vinylpyridine HMPP 2-hydroxy-2- methylpropiophenone HY Hostasol Yellow LCFM Laser confocal fluorescence microscopy Lysozyme Hen egg white lysozyme MF Microfluidic MAA Methacrylic acid MMA Methyl methacrylate MRI Magnetic resonance imaging NP Nanoparticle PAA Poly(acrylic acid) PDMS Poly(dimethylsiloxane) PEGDA Poly(ethylene glycol) diacrylate PS Poly(styrene) SEM Scanning electron microscopy STEM Scanning Transmission electron microscopy ThT Thioflavin-T TPGDA Tripropyleneglycol diacrylate TX-100 Triton X-100 US Ultrasound

1

Chapter 1

Introduction to Microfluidic Synthesis of Particulate Materials

1.1 Overview of Microfluidics

Microfluidic devices manipulate small (from 10–9 to 10–18 L) volumes of fluids, using

channels with dimensions from tens to hundreds of micrometers.1 Research in the field of

microfluidics covers physics, chemistry, engineering, materials science, and biology.2-6 In

particular, the last decade has witnessed rapid advancement in the area of materials

chemistry. Continuous microfluidic (MF) synthesis of colloidal materials offered a number

of advantages in comparison with conventional batch synthesis, such as a significant

reduction of reagent consumption, fast and controlled heat and mass transfer, and the

capability of performing complex parallel or consecutive reactions in a highly controlled

manner.7 Moreover, the capability to effectively screen chemical formulations and the

ability to produce products of chemical reactions with enhanced properties, e.g., a narrow

molecular mass distribution of polymer molecules or highly monodisperse colloidal

particles, made MF synthesis very beneficial in the development of new materials.8,9 The

dawbacks in MF preparation of materials include low productivity, the use of fast chemical

reactions and the difficulty in interfacing conventional equipment with MF devices. The

purpose of this chapter is to review recent progress in the MF synthesis of polymeric

particulate materials. This chapter focuses on the synthesis of particles conducted in the

continuous mode, that is, in the MF reactor, in contrast to numerous comprehensive papers

describing MF synthesis combined with the batch synthesis.7, 10-14

2

1.2 Different Modes of Microfluidic Synthesis of Polymer Particles

Figure 1.1 schematically shows a conventional batch reactor and a MF reactor.

Compared to their batch counterparts (Figure 1.1A), MF reactors (Figure 1.1B) offer

superior control over broad reaction conditions such as mixing, residence times and

reaction temperatures. These properties result from rapid heat and mass transfer, short

mixing times and well-defined temperature profiles, which are the consequences of small

reaction volumes in a flow-based reaction format. The flow-based concept can also be

incorporated with in-flow analysis. With respect to the production of particulate materials,

MF reactors provide a continuous-flow alternative to conventional batch synthesis for

preparing micrometer and nanometer-sized particles in a single or in multi-phase solution.

Figure 1.1 Synthesis of particulate materials (A) in a batch reactor and (B) in a flow-based, microfluidic (MF) reactor. Adapted with permission from reference 9. Copyright 2010, Annual Reviews.

3

MF synthesis of particulate materials can be realized in a single- or a multi-phase flow

as shown in Figure 1.2. Single-phase MF synthesis (Figure 1.2A) occurs either in a single

liquid or in a mixture of miscible liquids. The reagents required for the reaction can be

supplied to the reactor simultaneously, or added in the course of reaction.8, 15-17 In the first

case, the reaction is generally preceded by a mixing step, which rapidly and uniformly

distributes reactant molecules within the microchannel cross-section. Since the reactant

flow is considered to be of laminar nature, mixing across the channel occurs either by

diffusion, or convection. The synthetic process can be triggered in a particular location of

the reactor, e.g., by applying a particular temperature profile along the microchannel length,

by radiative heating, or by diffusion of reactants to the area of the reaction. The particles

are generated in the continuous phase and are carried towards the outlet of the MF reactor.

Figure 1.2 Schematic illustrations of (A) single-phase and (B) multiphase formats for the MF synthesis of polymer particles synthesized (C) in droplets (i), and (D) at the interface between the two phases (ii). Adapted with permission from reference 9. Copyright 2010, Annual Reviews.

4

Multi-phase MF synthesis of polymeric particles starts by forming droplets of a gas or

a liquid which serve to induce convective mixing, to confine the synthesis to miniature

compartments or even to define particle size and shape. An analogy to chemical synthesis

can be drawn by applying individual reaction compartments to their path through the

microreactor. The multiphase flows of interest here are sometimes referred to as segmented

flows and either consists of continuously alternating liquid droplets and liquid segments, or

of liquid segments and gas bubbles. Regular and stable multiphase flows can be obtained

by varying reactant flow rates, viscosities and microchannel sizes/configurations.18,19

Generally, polymeric particles can be synthesized in one of two locations schematically

shown in Figure 1.2B: i) inside droplets (Figure 1.2C),20-22 or ii) at the interface between

the phases (Figure 1.2D).23,24

Compared to a single-phase approach, droplet-based synthesis is characterized by rapid

heat and mass transfer, and by efficient mixing. The effect of axial dispersion is either

eliminated or minimized.18, 25 Generally, droplets are generated in one of four types of MF

droplet generators, namely, terrace-like devices,26, 27 T-junctions or Y-junctions,28, 29 flow-

focusing droplet or bubble generators,30-32 and capillary-based co-flow devices.33, 34

The size of droplets can be controlled by tuning the dimensions of microchannels,

macroscopic properties of liquids and the flow rates of liquids. Regardless of the geometry

of MF reactors, the control of wetting of microchannel with liquids is critical in the

generation of droplets: the continuous fluid should wet the channel walls preferentially over

the dispersed phase.35, 36

5

Both single- and multi-phase MF syntheses of polymer particles provide the following

advantages: i) the ability to control particle dimensions (and hence the corresponding size-

dependent properties of the particles), ii) the ability to generate particles with a variety of

shapes that cannot be implemented in conventional (non-microfluidic) synthesis, iii) the

capability to control particle morphologies by the hydrodynamic means, iv) the ability to

control particle morphologies by the uniform supply of energy to precursor droplets and v)

a simple production of composite particles.

1.3 Microfluidic Synthesis of Polymer Particles

Polymer particles with narrow size distributions find a broad range of applications in

chromatography as ion exchange resins, toners, spacers, and calibration standards.37-39

Advanced applications of monodisperse polymer particles include optical data storage,40,41

biometrics,42,43 stabilization of Pickering systems,44 production of dielectric resonators,45,46

encapsulation of drugs and biological molecules,47 medical diagnostics48 and photonics.49

Control of particle compositions, dimensions, and internal structure is highly desirable and

sometimes it is vital in realizing a particular application.

Conventional methods of particle synthesis, such as emulsion, dispersion, precipitation,

suspension and miniemulsion polymerizations either lack control of particle size,

morphology, and shapes, or require multi-step processes, or are material-specific.50-52

Sometimes, lack of control of particle size necessitates post-synthetic fractionation of

polymer beads, which is both time- and material-consuming.

6

MF synthesis of polymer particles enables control of particle size, shape, composition

and internal structure. Polymer particles have been synthesized in both a single-phase15,16

and a multi-phase system.24, 53 Single-phase MF generation of polymer particles utilized

UV-illumination onto the continuous stream of a polymerizable liquid through a patterned

mask.15 Another example of the single-phase MF production of polymeric particles

includes the nanoprecipitation of block copolymers, e.g., poly(lactic-co-glycolic acid)-b-

poly(ethylene glycol), which was induced by the controlled mixing between two miscible

fluids.16 Multi-phase MF generation of polymer particles relies on the (i) emulsification of

liquid monomers or polymers and (ii) the solidification or gelation of the precursor droplets

by the chemical or physical means.

Polymerization mechanisms for the production of particles include

polycondensation24,54 and radical polymerization.21,55 In addition to chemical methods,

ionic crosslinking,56,57 thermosetting,58 or solvent extraction and evaporation from the

droplets transform precursor droplets into polymer particles.59,60

The following sections describe the recent accomplishments in the MF production of i)

rigid polymer particles with narrow size distributions, ii) gel particles (microgels), iii)

particles with controlled internal structures, iv) particles with non-spherical shapes, and v)

particle with gaseous core. In addition, the productivity of MF synthesis of polymeric

particles is discussed in section 1.3.6.

1.3.1 Microfluidic Synthesis of Solid Polymer Microspheres

Historically, droplet-based MF emulsification of monomers and subsequent post-

polymerization of monomer droplets in a batch process was proposed in 2002.26 In 2005,

7

continuous on-chip production of monodisperse solid polymer particles with various

compositions was realized by emulsifying monomers mixed with a photoinitiator and

various organic or inorganic additives and solidifying the precursor droplets by photo-

initiated polymerization (Figure 1.3A).20,21 The diameter of particles generated by the MF

synthesis was in the range from tens to hundreds of micrometers. In contrast to

conventional methods, the narrow size distribution of the precursor droplets was preserved

by polymerizing “one particle at a time”, that is, when droplets moving through the

downstream microchannel were separated with a well-defined gap of the continuous phase.

Particle polydispersity (coefficient of variation, CV, defined by a standard deviation

divided by the diameter of the droplets or particles) was typically below 5-6% (Figure

1.3B).

Figure 1.3 Rigid polymer particles synthesized in continuous multiphase MF reactors. (A) SEM images of poly(tripropyleneglycol diacrylate) particles. (B) Size distribution of the particles shown in panel (A). d denotes particle diameter. (C) Optical fluorescence microscopy images of poly(tripropylene glycol diacrylate) microspheres loaded with CdSe quantum dots. (D) Optical polarization microscopy images of poly(tripropyleneglycol diacrylate) microspheres with 5–20 wt. % of liquid crystals made of 4-cyano-4’-pentylbiphenyl. Panels (A–D) are reproduced with permission from Ref. 20. Copyright 2005, Wiley-VCH. (E) SEM image of porous poly(glycidyl methacrylate–co-ethylene glycol dimethacrylate). Panel (E) is adapted with permission from Ref. 67. Copyright 2009, American Chemical Society. (F) Confocal microscopy image of poly(tripropyleneglycol diacrylate)/poly(urethane) particles with an interpenetrating network (IPN) structure. Panel (F) is adapted with permission from Ref. 68. Copyright 2008, American Chemical Society.

8

MF synthesis allowed efficient production of rigid polymer microspheres with various

compositions by emulsifying mixtures of several monomers or mixtures of monomers with

inorganic nanoparticles, small molecules, or solvents and by conducting subsequent

continuous solidification of the precursor droplets.20,61-63 In this method, the requirements

to the liquid-to-be-emulsified included: (i) the absence of precipitation in the inlet tubing

and (ii) the appropriate values of viscosity and interfacial tension with the continuous phase.

Examples of composite rigid polymer particles produced by the MF synthesis included

copolymer particles,61,62 and polymer particles with inorganic nanoparticles20,63,64 and liquid

crystals.20,65 Porous polymer particles were prepared by mixing a monomer with a porogen

solvent, polymerizing the monomer and removing the solvent.20,66,67 Several examples of

composite rigid polymer particles are presented in Figure 1.3 (C-E).

Polymer particles with an interpenetrating polymer network (IPN) structure (Figure

1.3F) were synthesized by performing multi-step reactions in the precursor droplets: heat

generated in the exothermic photo-initiated free radical polymerization of the diacrylate

monomer triggered the polycondensation of the poly(urethane) oligomer with

diethanolamine.68

1.3.2 Microfluidic Synthesis of Spherical Polymer Microgels

Polymer microgel particles produced in continuous MF reactors have attracted a lot of

interest owing to the ability to encapsulate in their interior biological molecules and

cells.56,57,69,70 In chemically induced gelation, the MF emulsification of aqueous solutions of

e.g., acrylamides or poly(ethylene glycol) diacrylate was followed by the rapid on-chip

photoinitiated polymerization or crosslinking reactions, which yielded microgel

9

particles.55,71-73 Figure 1.4A shows the microgels of poly(N-isopropylacrylamide)

synthesized by redox polymerization.55 The proposed approach was based on generating

core-shell droplets composed of the aqueous core containing the redox initiator and the

aqueous shell containing a mixture of monomers with the reaction accelerator. Crossliking

of the microgels was ensured by complete mixing between the two aqueous phases.

Photoinitiated MF synthesis also allowed for the continuous production of fluorescent and

magnetic microgels.72,73

Figure 1.4 Microgel particles synthesized in continuous multiphase MF reactors. (A) Optical microscopy images of the formation of droplets of poly(N-isopropylacrylamide) microgel particles (left) and the resulting microgel particles labeled with fluorescein isothiocyanate (right). The inset shows a schematic of the microfluidic reactor (OF, outer fluid; MF, middle fluid). Reproduced with permission from reference 55. Copyright 2007, Wiley-VCH. (B) Optical microscopy images of ionically cross-linked alginate (left), k-carrageenan (middle), and carboxymethylcellulose (right) microgels. Scale bar: 100 μm. Adapted with permission from reference 56. Copyright 2006, American Chemical Society.

Physically-induced gelation was used to form microgels from biopolymers.74 The

methods that were used to gel precursor droplets included thermosetting (of e.g., agarose)20

or ionic crosslinking (of e.g., alginate, κ-carrageenan, or carboxymethyl cellulose).56,69,75

Since ionic gelation led to the rapid build-up in the viscosity of the droplet phase,

10

emulsification of the gelling solution was problematic.74 One of the ways to circumvent this

problem has utilized the diffusion of the cross-linking agent, e.g., Ca+2 or Fe+3 ions, from

the continuous phase into the precursor droplets. Examples of particles generated by

diffusion-controlled ionic crosslinking are shown in Figure 1.4B.56,75 Alternatively,

ionically-mediated gelation was achieved by generating precursor droplets

compartmentalizing a crosslinking agent in an inactive state and a polymer, and extracting

from the continuous phase a reagent that triggered gelation of the polymer in the droplets .

For example, droplets containing sodium alginate and CaCO3 underwent gelation due to

diffusion of acetic acid from the continuous phase into the droplets, thereby reducing pH

and liberating Ca+2 ions which crosslinked the alginate molecules.69,75 In another method,

coalescence of precursor droplets of polymer solution and droplets containing a

crosslinking agent also yielded alginate microgels.57

1.3.3 Microfluidic Synthesis of Polymer Particles with Different

Morphologies

Microfluidic synthesis can be used to generate particles with a variety of internal

structures that can be controlled by the hydrodynamic means. One of the examples is the

synthesis of Janus particles, that is, particles with two compartments having distinct

compositions. These particles are produced by generating precursor droplets from two

parallel co-flowing streams of two immiscible monomers and performing subsequent

polymerization.76,77 The volume fraction of each compartment of the particles can be

precisely controlled by varying the flow rate ratio of the constituent monomers.76-78

11

Furthermore, since at low Reynolds number (Re<1) convective transport across the

interface between the two adjacent streams is suppressed, Janus droplets can be formed

from the miscible solutions of the same monomer, which contain distinct additives, e.g.,

colloid particles labeled with different dyes.72,79 Rapid photopolymerization of the

monomer results in the formation of Janus particles.72,76,77 Figure 1.5A illustrates the

formation of Janus microgel particles from the aqueous solutions of acrylamide containing

silica particles labeled with rhodamine isothiocyanate (red color) and fluorescein

isothiocyanate (green color).72

Figure 1.5 MF synthesis of polymer particles with different morphologies. (A) Schematic of the microfluidic synthesis of Janus microgels. The inset shows an optical fluorescence image of poly(acrylamide) particles encapsulating silica particles labeled with two different dyes in distinct compartments. Adapted with permission from reference 72. Copyright 2006, American Chemical Society. (B) Optical microscopy image of bicolored poly(isobornyl acrylate) particles containing carbon black and titanium oxide in two distinct hemispheres. The inset shows electrically actuated particles displaying their white-colored side in the display panel. Adapted with permission from reference 79. Copyright 2006, Wiley-VCH. (C) SEM images of microcapsules prepared by microfluidic interfacial polymerization. The inset shows a high magnification image of the interior of the capsular wall. Adapted with permission from reference 54. Copyright 2005, American Chemical Society. (D) Optical microscopy image of the colloidal crystalline array confined in polymeric shells. The inset shows the particles with three colloidal crystal cores. Scale bars: 200 μm. Adapted with permission from reference 99. Copyright 2008, American Chemical Society.

12

The surface of each compartment of Janus particles can be selectively functionalized

with e.g., biological molecules.76 Alternatively, the constituent phases of the Janus particles

generated by MF synthesis can be loaded with two distinct species.79 For example,

poly(isobornyl acrylate) Janus particles were synthesized with two halves loaded with

carbon black and titanium oxide. In an alternating electric field, the particles were rotated

thereby giving the ability to change the color (black or white) of the display panel. These

experimental results showed potential application of such particles as a constituent of

electronic paper (Figure 1.5B).80

MF synthesis and assembly also proved to be useful in the generation of polymer

capsules. Polymer capsules find applications in cosmetics, nutrition, agricultural,

pharmaceutical and food industries and in biomedical engineering.81,82 Polymer particles

with a core-shell structure (capsules) have been produced by a number of MF methods that

are described below.

One of the most general approaches to polymer capsules is the formation of double

emulsions, e.g., water-in-oil-in-water or oil-in-water-in-oil droplets and subsequent

solidification of the shell of these droplets to yield core-shell particles.33,83,84 Double

emulsions have been produced in MF devices with different geometries such as a two

consecutive T-junctions or flow-focusing devices,83,84 a single flow-focusing device,85 a

combination of flow-focusing and T-junction devices,86 and a capillary-based device.33,55

Polymerization of the shell of these droplets led to the formation of capsules with rigid or

soft polymer shell.33,55,85 Alternatively, core-shell droplets containing a solution of diblock

copolymer or colloidal particles in the shell transformed into capsules when the solvent was

evaporated from the shell.87,88 Manipulation of the hydrodynamic conditions during the

formation of double emulsions, namely, the variation of the flow rates of the liquids

13

allowed control of the size of particles, shell thicknesses and the number of cores per

capsule.85,87,89

Another strategy used for the generation of capsules relies on reactions occurring at the

interface between the dispersed and the continuous phases.24,54,90 For example, interfacial

polymerization between aqueous 1,6-diaminohexane (a droplet phase) and adipoyl chloride

dissolved in a mixture of solvents (a continuous phase) yielded monodisperse capsules with

a nylon-6,6 membrane.24 Interfacial polymerization was also used to fabricate polyamide

capsules with a fibrous shell structure by emulsifying a mixture of acid chlorides in the

aqueous solution of polyethyleneimine and carrying out an interfacial condensation reaction

(Figure 1.5C).54

Diffusion-controlled reactions have also generated polymer capsules.56,91 For example,

diffusion of Ca2+ ions from the non-polar continuous phase into the aqueous droplets of

sodium alginate resulted in the limited crosslinking of the aliginate molecules and the

formation of microgel capsules.56 The thickness of the gelled shell depended on the time of

residence of the microgels in the microchannels and the concentration of the crosslinking

agent in the continuous phase. Diffusion of photo-activated initiators from the continuous

oil phase into the aqueous droplets of poly(N-isopropylacrylamide) also resulted in the

formation of gel capsules with a thin shell membrane.91

Finally, capsules have been generated by assembling particles or depositing polymeric

molecules at the interface between the dispersed phase and the continuous phase.92-96 For

example, the close-packed assembly of polymer particles at the surface of droplets resulted

in the formation of Pickering emulsions that were resistant to coalescence and Ostwald

Ripening.92,94 Sequential adsorption of poly(methacrylic acid) and poly(N-

14

vinylpyrrolidone) to the surface of liquid crystal of 4’-pentyl-4-cyanobiphenyl as droplets

produced capsules with the shell comprising alternating polymer layers.96

Another group of particles with interesting, non-conventional morphologies included

polymer microspheres with a supracolloidal structure.97-99 These particles were formed by

generating core-shell droplets compartmentalizing colloidal beads in the aqueous core and

conducting continuous polymerization of the shell composed of a photo-curable monomer.

The resulting composite microspheres are shown in Figure 1.5D. Due to the confinement-

induced crystallization of the microbeads in the particle core, the composite capsules

exhibited distinct diffraction patterns in the visible range that were independent of the

orientation of the spherical surface.

1.3.4 Microfluidic Synthesis of Non-spherical Polymer Particles

Many interesting applications of polymer particles are governed by their shapes.100,101

Single- and multi-phase MF synthesis allows the preparation of particles with non-spherical

shapes that would otherwise be difficult to realize in conventional methods such as

emulsion or suspension polymerizations. This is due to the minimization in interfacial

energy which makes droplets acquire a spherical shape in the conventional methods.

One of the ways to tune the shapes of particles generated by the multi-phase MF

synthesis is the polymerization of droplets under confinement.20,21,72,73,101 The shape of

particles is determined by the relationship between the diameter, d, of unperturbed droplets

(d=(6V/π)1/3 where V is the volume of the droplet) and the dimensions of microchannels.

For example, when w>d and h<d where w and h are the width and the height of the

microchannel, respectively, droplets acquire a discoid shape (Figure 1.6A). On the other

15

hand, when w<d and h<d, the droplets assume a rod-like shape (Figure 1.6B).102,103

Subsequent continuous polymerization yields disk-shaped and rod-shaped polymer

particles.20,21,72,103 Geometrical confinement combined with controlled coalescence between

aqueous droplets of sodium alginate and CaCl2 yielded spherical, discoid, rod-like and

thread-like alginate gel particles.104

Figure 1.6 MF synthesis of particles with non-spherical shapes. (A,B) SEM images of poly(tripropyleneglycol diacrylate) particles with (A) disk and (B) rod shapes. Insets show the relationship between the particle shape and the channel dimensions. Adapted with permission from references 20 and 103. Copyright 2005, Wiley-VCH. Copyright 2005, American Chemical Society. (C) SEM image of poly(tripropyleneglycol diacrylate) particles with bowl-like shapes. The inset shows the precursor droplet with a small fraction of nonpolymerizable liquid (indicated by the arrow). Scale bar: 40 μm. Adapted with permission from reference 85. Copyright 2005, American Chemical Society. (D–F) SEM images of poly(ethylene glycol diacrylate) particles with various shapes synthesized in one-phase MF synthesis using continuous-flow lithography. Insets show the corresponding photomasks. Scale bars: 10 μm. Adapted with permission from reference 15. Copyright 2006, Macmillan.

In another MF approach, semi-spherical and bowl-like polymer particles were

generated from the pre-cursor multi-phase droplets containing a monomer and a non-

polymerizable liquid (Figure 1.6C).78,85 The droplets were formed by breaking a coaxial jet

of silicone oil and a photo-curable monomer to form multi-phase droplets with a range of

16

morphologies. Following the polymerization of the monomer in the droplets, the oil phase

was removed. Since the volume fraction of each liquid was determined by its relative flow

rate, the morphologies of the precursor multi-phase droplets and the corresponding particles

were accurately described by a phase-like diagram.85

Toroidal particles were generated by generating droplets compartmentalizing

solutions of different polymers (e.g. poly(ether sulfone), polysulfone, or poly(methyl

methacrylate)) and removing the solvent from the droplets.105 The diffusion of the solvent

proceeded at spatially different rates across the surface of droplets. The difference in the

solvent diffusion rate led to a non-uniform solidification between the circumferential and

central regions of the droplets and resulted in the formation of the donut-shaped toroidal

particles.

Single-phase MF synthesis of polymer particles employed continuous flow projection

photolithography.15 Site-specific photomask-limited UV-irradiation of the poly(ethylene

glycol diacrylate) pre-polymer solution flowing through a microchannel led to the site-

specific photo-initiated polymerization, thereby creating polymer particles. The shapes of

the particles (e.g., triangles, rings, cubes and polygons) were precisely determined by the

features of the photomasks (Figure 1.6D-F). A similar approach has been applied to

synthesize long flattened particles encoded with multiple probes for molecular

identification.106 Amphiphilic non-spherical microparticles were also created using

multiphase stratified flows composed of a hydrophilic monomer and a hydrophobic

monomer stream.107 The particles showed the ability to self-assemble in water or at the oil-

water interface. Furthermore, three-dimensional and multifunctional particles were

synthesized by controlling fluid flows, channel topologies and intensity of UV

irradiation.108-110

17

1.3.5. Microfluidic Synthesis of Bubble-templated Polymer Particles

Micrometer-size bubbles have numerous biomedical applications such as ultrasound

contrast agents,111,112 targeted drug delivery vehicles,113,114 and tumor/thrombus demolition

materials.115-116 Bubbles are also used in the preparation of various porous materials, which

have a range of applications including the pharmaceutical, food and the cosmetic

industries.117 Currently, it is a challenge to generate and store bubbles with high uniformity

in size and long-term usability. Conventional methods of preparation of bubbles involve

sonication, mechanical agitation, or shear mixing of liquids containing amphiphilic

molecules like lipids, proteins, or surfactants.118-120 Bubbles produced by these methods

tend to have a broad distribution of sizes. In addition, bubbles stabilized by amphiphilic

molecules are prone to dissolution and coarsening via Ostwald ripening: excess Laplace

pressure across the air-water interface hampers the long-term stability of bubbles.121

MF approaches enable the production of monodisperse bubbles with highly controlled

dimensions.31,122,123 The polydispersity of these bubbles is typically less than 5%, however,

the long-term stability of bubbles remains a problem. Recently, it has been shown that the

stability of bubbles can be significantly enhanced by forming close-packed layers of

colloidal particles at the gas-water interface124,125 or by choosing appropriate

surfactants.126,127 MF production of particle-stabilized bubbles was obtained by

hydrodynamic shear flows92 and by chemical means.128 A mixture of lipids was chosen as a

shell material to improve the stability of bubbles.127 Depending on the type of materials

dispersed in the oil phase, gas-in-oil-in-water emulsions generated using a MF platform

could be used as templates for bubbles with a particle shell and drug-encapsulated

bubbles.129,130 For example, hydrophobic silica or magnetic nanoparticles were dispersed in

18

a volatile organic solvent. Upon the evaporation of solvent, the nanoparticles formed a stiff

multilayer shell at the gas-water interface, which protected the bubbles against dissolution

and coarsening.129 Consecutive adsorption of protein and polysaccharide layers on the

surface of bubbles produced using a MF device created monodisperse bubbles with long-

term stability.131 Furthermore, bubbles with a hydrogel shell were produced by

photopolymerizing gas-in-water-soluble monomer-in-oil emulsions.86

1.3.6 Productivity of Continuous Microfluidic Reactors

Although continuous MF synthesis of polymer particles is an efficient way for the

production of high value particles, future application of this method depends on the ability

to increase the productivity of MF synthesis. Typically, the productivity of a single planar

MF reactor does not exceed several mL/h.132 The simplest way to overcome this limitation

is to increase the number of reactors working in parallel in the approach called “numbering

out”.133 To avoid high cost and practical complications associated with linking many tubes,

connectors, and pumps, it is imperative to supply liquids in multiple parallel reactors using

only a few pumps.134

The integration of multiple droplet generators in a single chip causes complex fluid

behaviors, which originate from the feedback between adjacent individual generators

sharing the same liquid supply.135 Another problem originates from the lack of fabricating

multiple MF reactors with identical geometries.136 Consequently, the size distribution of

droplets broadens and polydispersity of the resulting particles increases.

The fabrication of a MF droplet generator using a silicon membrane containing a few

hundred thousands microchannels increases the efficiency of MF emulsification.137 A high

19

throughput of 60–70 mL/h was achieved; however, it was not as simple as in a common

MF droplet generator to control the size of droplets and create droplets with different

shapes and morphologies. A different type of MF reactor was implemented in a monolithic

module combining up to 256 cross-junction droplet generators.79,134 The device generated

monodisperse droplets (CV<4%) with the productivity of up to 320 mL/h. The generation

of Janus droplet was also feasible in the same type of reactor. The production of polymer

particles in this device was achieved by photopolymerization in the drain tubing or by

thermally initiated polymerization in a batch reactor.

Although the productivity of droplet and particle formation greatly increased by using

the above-mentioned approaches, the synthesis lacked the ability to control the dimensions

of the droplets137 thereby necessitating post-fractionation of the microbeads.79,134

Furthermore, device failures, e.g. clogging of a particular microchannel, could result in the

re-fabrication of the entire MF reactor.

Recently, a multiple modular microfluidic (M3) reactor for the synthesis of polymer

particles was proposed, which contained 8 modules accommodating 128 parallel

reactors.136 Several important features were implemented in the M3 reactor. First, one could

easily replace one of the fouled modules with the new one if the component failure took

place. Second, cross-talk between adjacent reactors was suppressed by elongating the

hydrodynamic path for the droplet phase prior to its entrance to the droplet generator. These

features allowed high throughput in droplet and particle production of up to 50 mL/h and

the generation of particles with polydispersity not exceeding 5 %. Third, the integration of a

polymerization compartment into each module allowed in situ particle synthesis, thereby

suppressing coalescence and/or Ostwald ripening of the precursor droplets.

20

1.4 Outlook

Future MF reactors have the potential to differentiate themselves from conventional

batch reactors through a number of beneficial features. In order to accomplish this goal, MF

platforms will need to become robust, well-integrated, scalable, stand-alone units (ideally,

without external pumps) and will require virtually no infrastructure investment such as

fume hoods, or glove boxes. These systems will be modular, computer-controlled and will

be interfaced with standard laboratory equipment and preparatory procedures. Currently,

continuous MF synthesis of a variety of colloidal particles has been successfully

demonstrated at a proof-of concept stage. The next major directions in the development of

MF synthesis of particulate materials include the followings.

i) MF synthesis produces high-value particles. Although the properties and “proof-of-

concept” applications of these particles have been demonstrated, to the best of our

knowledge, no application has been realized beyond the lab bench. Future work should be

directed towards the synthesis of materials with truly unique features that are critically

important in specific applications. Search for such applications is vital for biological

research. Currently, the encapsulation of cells appears to be the most promising application

of the particles generated by MF processes.

ii) A lot of work should be done to make MF synthesis cost-effective, robust and

reliable. For example, MF reactors have to be fabricated from materials that can

accommodate multi-hour MF synthesis. An appropriate selection of reactor material is one

of the most significant factors. Along with chemical stability, surface properties and ease of

fabrication of the device have to be taken in account. Other considerations are pertinent to a

particular mechanism of particle synthesis. For example, photoinitiated MF synthesis of

21

polymer particles requires transparency of the rector material in the wavelength range used

for photoinitiation.

iii) MF synthesis allows the high throughput screening of formulations used for the

synthesis of polymer colloids, which is achieved by varying the flow rates of the liquid

reagents supplied to the reactor. More work is needed in this field in order to fully realize

the exploratory side of continuous MF synthesis of particulate materials.

iv) The location and time of chemical reactions can be defined in MF reactor due to

time-to-distance transformation characteristics of MF synthesis. This feature enables multi-

step reactions to be conducted in a highly controlled manner. Such reactions triggered “on

demand” can benefit the synthesis of colloidal particles.

v) MF synthesis will greatly benefit from in-line chemical and physical

characterization of reaction products. Since microliter volumes are often sufficient for

analysis, MF synthesis is particularly advantageous for rapidly screening different reaction

conditions. As a result, the variables of reactions can be much more rapidly explored.

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30

Chapter 2

Materials and Methods

This chapter describes the materials and methods used in the present research from

Chapter 3 to 6.

2.1 Materials

Gaseous N2 (purity 99.99 %) and CO2 (purity 99.8 %) were purchased from BOC

Canada. Sodium hydroxide (NaOH, EMD Canada) and hydrochloric acid (HCl, EMD

Canada) were used to adjust the pH. Deionized water was purified by using a Mlli-Q Plus

purification system (Millipore Corp.). Ethanol was received from Commercial Alcohols

Inc.. Hexadecane, tri(propylene glycol) diacrylate (TPGDA), poly (ethylene glycol)

diacrylate (PEGDA, Mn~700 g/mol), divinylbenzene (DVB, technical grade, 80%), styrene,

acrylic acid (AA), methacrylic acid (MAA, 99%), methyl methacrylate (MMA, 99%) and

4-vinylpyridine (4VP, 95%) were purchased from Sigma-Aldrich, Canada. 2-(6-

methacryloyloxyhexyl)-thioxantheno[2,1,9-dej]isoquinoline-1,3-dione (Hostasol Yellow,

HY) dye monomer1 was synthesized and provided by Dr. Hung Pham. Initiators, hydroxy-

2-methylpropiophenone (HMPP), 2,2-dimethoxy-2-phenyl-acetophenone (DMPA), and

2,2′-azobis(2-methylpropionamidine) dihydrochloride (V50) were purchased from Sigma-

Aldrich, Canada. 2,2’-azobis(2-methylbutyronitrile) (AMBN) was purchased from Wako

Pure Chemical Industries Ltd.. Water soluble stabilizers, polyvinylpyrrolidone (PVP360,

Mw~360,000 g/mol), Triton X-305 (TX-305, 70% solution in water) and Triton X-100 (TX-

31

100) were purchased from Sigma-Aldrich, Canada. Biopolymers, alginic acid sodium salt

(alginate), hen egg white lysozyme (lysozyme) and bovine albumin serum were purchased

from Sigma-Aldrich, Canada. Fluorescein isothiocyanate (FITC)-conjugated bovine

albumin serum2 was provided by Dr. Raheem Peerani. Thioflavin-T (ThT), gold (III)

chloride trihydrate and sodium citrate (Sigma-Aldrich, Canada) were used as received.

Poly(dimethylsiloxane) (PDMS, Sylgard® 184) and photoresist resin (SU-8 50, 25) were

purchased from Dow Corning (USA), and Microchem Co. (MA, USA), respectively.

Carboxylated 3 μm-diameter SiO2 particles were purchased from Micromod

Partikeltechnologie GmbH, Germany. 100 nm-diameter Fe3O4 particles were purchased

from Chemicell, Germany. The synthesis of 3.5 µm-diameter poly(styrene-co-AA)

particles was carried out by Mr. Ahmed I. Abdelrahman.3,4 Cationic poly(MMA-co-4PV)

particles with a diameter of 0.7 µm were synthesized using a multi-stage surfactant-free

emulsion polymerization by Dr. Alla Petukhova.5 Carboxylated SiO2-encapsulated

CdSe/ZnS particles were synthesized and provided by Mr. Ivan Gorelikov.6 Poly(DVB-co-

MAA) particles with a diameter of 3.5 µm-diameter were synthesized by precipitation

polymerization.7 Gold nanoparticles with a diameter of 12 nm-diameter were synthesized

by a citrate reduction method.8

2.2 Methods

2.2.1 Mask Design

We designed masks using FreeHand® (Adobe Systems Inc., USA) software, and

printed the designed pattern on transparencies using laser printers with a resolution of 2500

dpi.

32

2.2.2 Microfabrication of Negative Masters

Masters were prepared in the cleanroom facility at Bahen Center, University of

Toronto. We fabricated the negative masters with microchannels of SU-8 photoresist (SU-8

25 or 50) on 3-inch silicon (Si) wafer substrates using photolithography.9,10 Briefly, the Si

wafers were washed several times with acetone and methanol. The surface of the wafers

was dried by blowing dry N2 gas. We spin-coated the Si wafers with SU-8 photoresist at a

typical spin rate of 1200 rpm in order to achieve 120 µm height features. After the spin-

coating process, the photoresist layer was thermally baked to evaporate the solvent, γ-

butyrolactone. The photoresist layer covered with a mask was exposed to and cured by UV-

light (Karl Suss MA6 mask aligner, λ=365~405 nm) to create the microchannel. Typical

exposure time was approximately 50 sec. Following UV-exposure, a post-bake process was

conducted to enhance the crosslinking of the UV-exposed areas of the photoresist. The

post-baked Si wafers with the crosslinked patterns of microchannel were immersed for ca.

10 min in the developer solution (1-methoxy-2-propanol acetate) to remove the non-

crosslinked regions of the photoresist. The Si wafers with the microchannel pattern were

rinsed several times with isopropanol and methanol and dried under a gentle stream of

nitrogen.

2.2.3 Fabrication of Microfluidic Devices

Microfluidic (MF) devices were fabricated in poly(dimethylsiloxane) (PDMS)

elastomer using a standard soft-lithographic procedure.11 The PDMS elastomer was

prepared from Sylgard® 184 (Dow Corning Cop., USA). The Sylgard® 184 base polymer

33

contains vinyl-terminated dimethylsiloxane oligomers, platinum catalyst, and silica filler

(dimethylvinylated and trimethylated silica).12 The base polymer was mixed with the curing

agent containing a cross-linker (dimethyl-methylhydrogen siloxane) and an inhibitor

(tetramethyltetravinyl cyclotetrasiloxane) at 10:1 (w/w) ratio. Air was removed from the

mixture under vacuum for 20 min. The mixture of the prepolymer mixture was poured onto

the master and baked at 75 °C in an oven for more than 12 hours. After curing, the replica

was peeled from the master, and holes were created by flat-tipped needle at the designated

positions corresponding to the inlets and outlets. The replica and a substrate (a plane PDMS

sheet) were oxidized in a plasma cleaner chamber (PDC-3XG, Harrick, USA) for 120 sec.

The plasma-treated replica and the substrate were brought in contact and sealed

immediately. Polytetrafluoroethylene tubings (Small Parts, USA) were placed into the

holes and sealed with epoxy glue (Lepage 12, Henkel Canada Co., Brampton, ON).

2.2.4 Microfluidic Experiments

Typically, gases (CO2 and N2) were supplied to the MF device via

polytetrafluoroethylene tubing (Small Parts, USA) attached to a Bellofram pressure

regulator. The applied pressure was from 27.6 to 82.7 kPa. Syringe pumps (Harvard

Apparatus, USA, PHD 2000 series) at flow rates ranging from 4 to 24 mL/h were used to

introduce liquids into the microchannel. To ensure stable bubble or droplet generation, after

introducing the liquids or gases in the microchannel, the MF device was equilibrated for at

least, 3 minutes.

34

2.2.5 Photopolymerization Experiments

To image the cross-section of bubbles coated with poly(styrene-co-AA) particles, 0.5

mL of the diluted bubble dispersion were mixed with 0.2 mL of 70 wt. % aqueous solution

of PEGDA containing the photoinitiator, HMPP. The dispersion was exposed to a UV-lamp

for 20-30 sec (UVAPRINT 40C/CE, Dr. K. Hönle GmbH UV-Technologie, Germany, λ =

330 ~ 380 nm, intensity of 200 mW/cm2). The UV-cured polymer embedded with the

armoured bubbles was microtomed (Leica, UCT Ultramicrotome) and mounted on a sample

grid before imaging.

Photopolymerization of monomer droplets of TPGDA loaded with the photoinitiator,

DMPA, and HY-labelled poly(DVB-co-MAA) particles was carried out immediately after

the release of droplets from the MF device. The droplets were collected in a vial and

exposed to UV irradiation for 1 min (UVAPRINT 40C/CE, Dr. K. Hönle GmbH UV-

Technologie, Germany, λ = 330 ~ 380 nm, intensity of 200 mW/cm2).

2.3. Characterization

2.3.1 Optical and Fluorescence Microscopy Imaging

An Olympus BX51 microscope (Olympus, USA) with a high-speed camera

(Photometrics CoolSNAR ES) was used to image the generation of bubbles, droplets and

particles in the MF device. We also used the same setup to image bubbles and droplets on a

glass slide. The fluorescence mode was used to acquire images of bubbles, droplets and

particles coated with fluorescence dye-labelled polymer beads.

35

2.3.2 Size Distribution of Bubbles, Droplets and Particles

The dimensions of bubbles, droplets and particles were analyzed using Image Pro Plus

(Media Cybernetics, USA) software. The polydispersity of bubbles or droplets was

characterized as the coefficient of variance (CV) as CV (%) = (σ/D)x100 where σ is the

standard deviation of the size of bubbles or droplets and D is the mean diameter of bubbles

or droplets. The volume, V, of spherical bubbles was calculated as V = (4/3)π(D/2)3. When

the value of D exceeded the height, h, of the microchannels, the bubbles formed discoids.

Their volume was approximated as V = (π /12)[2D3-(D-h)2(2D + h)].14

2.3.3 Scanning and Transmission Electron Microscopy imaging

We used scanning electron microscopy (SEM, Hitachi S-570 or Hitachi S-3400N) and

scanning transmission electron microscopy (STEM, Hitachi S-5200) to image the surface

and internal morphologies of bubbles and polyTPGDA particles coated with polymer beads.

The same microscopes were used to image the surface structure of the bubbles encapsulated

with a mixture of lysozyme, alginate and nanoparticles. The samples were washed with

deionized water five times and dried or frozen at -20 oC on the SEM or TEM grids prior to

imaging.

2.3.4 Laser Confocal Fluorescence Microscopy imaging

Laser confocal fluorescence microscopy (LCFM, Zeiss LSM 510 Meta microscope

equipped with an Axiovert 200M microscope, λex= 364 or 380 nm) was used to image

36

bubbles encapsulated with HY-labelled polymer beads, or a mixture of lyzoyme and

alginate carrying SiO2-encapsulated CdSe/ZnS nanoparticles. We also used LCFM to

image poly TPGDA particles loaded with HY-labelled polymer beads.

2.3.5 Contact angle measurement

A Drop Shape Analysis System (DSA100, KRÜSS, USA) was used to examine the

pH-dependent wetting properties of the polymer particles. The contact angles between

poly(styrene-co-AA) film on a glass substrate and an aqueous solution of NaOH at various

pHs were recorded. We also measured the contact angles between poly(DVB-co-MAA)

film and a mixture of water and ethanol (85:15, v/v ratio) or TPGDA monomer. A 3 μL-

droplet was deposited on the surface of the polymer film where advancing contact angles

were acquired.

2.3.6 Circular Dichroism Spectroscopy

Circular dichroism (CD) spectra were obtained using a JASCO J-710

spectropolarimeter in the range 200-250 nm, in order to examine the conformational

changes for lysozyme following their deposition on the bubble surface. The bubbles

encapsulated with a mixture of lysoyzme and alginate were washed with water five times.

The bubble dispersion was further diluted approximately 30 times with a 50 mM of

trishydroxymethylaminomethane-HCl buffer (pH=8) and transferred to a quartz cuvette

with 1 cm path length.

37

2.3.7 Fluorescence Spectroscopy

A fluorescence spectrophotometer (Cary Eclipse, Varian) was used to carry out ThT

binding tests. Aliquots of biopolymer-encapsulated bubble dispersions were added to 2 mL

of 50 μM ThT solution at pH=8. The intensity of fluorescence emission was recorded at

λex=412 nm. In corresponding control experiments, we examined fluorescence intensity of

freshly prepared a 50 μM ThT solution and a 0.003 wt. % of lysozyme or alginate dissolved

in 50 μM ThT solution at pH=8. In addition, the intensity profiles of photoluminescence

emission were measured at λex=364 nm for dispersions of biopolymer-engulfed

nanoparticle-free bubbles and biopolymer-engulfed bubbles functionalized with SiO2-

encapsulated CdSe/ZnS nanoparticles.

References

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Chem. 2003, 41, 766.

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(4) J. S. Song, F. Tronc, M. A. Winnik, Polymer 2006, 47, 817.

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Kumacheva, Canadian Journal of Chemistry-Revue Canadienne De Chimie 2010, 88, 298.

(6) I. Gorelikov, N. Matsuura, Nano Letters 2008, 8, 369.

(7) W. H. Li, H. D. H. Stover, J. Polym. Sci. Pol. Chem. 1999, 37, 2899.

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(10) http://www.microchem.com/products/su_eight.htm.

(11) Y. N. Xia, G. M. Whitesides, Annual Review of Materials Science 1998, 28, 153.

(12) A. Olah, H. Hillborg, G. J. Vancso, Applied Surface Science 2005, 239, 410.

40

Chapter 3

Microfluidic Generation and Dissolution of CO2 bubbles

Reproduced with permission from Soft Matter, 2010, 6, 630. Copyright 2010 RSC

Publishing.

This chapter describes a microfluidic strategy for the generation of small (<10 μm)

microbubbles with a narrow size distribution. The proposed approach exploits the

following features: (i) the generation of bubbles from gaseous CO2 and (ii) the controlled

dissolution of these bubbles until they reach a desired size. We investigate the role of

various factors on the formation and the extent of bubble dissolution and show that the final

dimensions of bubbles are determined by the flow rate of the continuous aqueous phase and

the acid-base equilibria established in the continuous phase flowing within the

microchannels.

3.1 Introduction

Bubbles with dimensions smaller than 10 μm have a broad range of biomedical

applications, including ultrasound imaging, gene therapy and targeted site-specific drug

delivery.1-3 Control of the size and the size distribution of bubbles is critical in their ability

to safely circulate in blood vessels and their reflectivity to ultrasound wave.4 Generally,

bubbles with dimensions in the size range specified above are generated by sonication and

high shear emulsification.5-7 These methods simply dispersing gas or liquid in a suspension

41

containing a suitable coating material by applying high intensity ultrasound or shear stirring.

Since the experimental variables cannot be precisely managed, these methods provide

insufficient control over bubble dimension and size distribution.

Microfluidic (MF) generation of bubbles has recently emerged as a means for

producing bubbles with extremely narrow size distribution.8-12 Bubbles were generated in

three types of MF devices, namely, a T-junction,11,12 a planar flow-focusing device9 and a

capillary flow-focusing device.8 Although the mechanism of bubble generation in these MF

devices was different depending on the geometry of the MF device, the size of bubbles was

precisely controlled by the periodic breakup of the gaseous thread and it was tuned by

varying the flow rate of the continuous liquid phase and the pressure of the gas.9

Typically, the size of bubbles produced in MF devices was in the range from 5 to 200

μm.9-12 Smaller bubbles were generated in a capillary flow-focusing device8 or in a planar

flow-focusing device by reducing the dimensions of the nozzle.13 For example, bubbles

with diameters of ca. 5 μm were produced by using an orifice with a width of 7 μm and a

height of 25 μm. It is noted that coaxial electrohydrodynamic atomisation has been lately

developed for the controlled production of small bubbles (ca. 6 μm). In this technique, a

coaxial jet of gas and liquid mixed with a surfactant is formed and then atomised to

generate uniform bubbles by applying the difference in an electrical potential.1 The

dimension of bubbles is determined by the flow rate ratio of gas and liquid, and the applied

voltage.

In this chapter, a different approach to the generation of small (<10 μm) bubbles is

described. Our strategy includes (i) MF generation of bubbles of CO2 mixed with a minute

amount of low-soluble impurity gases in an aqueous phase and (ii) controllable in situ

dissolution of CO2 under conditions of varying acidity of the continuous medium while the

42

size of bubbles is stabilized. In this chapter, we show that the final dimensions of bubbles

are determined by their initial dimensions (controlled by the gas pressure, the flow rate of

the continuous phase, and the initial pH of the liquid phase) and the acid-base equilibria

established in the continuous phase in the microfluidic device.

3.2 Results and Discussions

The materials and methods described in the present chapter are provided in Chapter 2.

3.2.1 Generation of CO2 bubbles

Figure 3.1 shows a schematic of the MF device used in the present work. The device

contains a flow-focusing bubble generator9 and a serpentine downstream channel with a

length of 230 mm. A study of the formation and dissolution of CO2 bubbles was performed

in the device with the orifice length, width, and height of 30, 33, and 115 μm, respectively.

Small (<10 μm in diameter) bubbles were generated in the MF device with the orifice

length, width, and height of 60, 22, and 40 μm, respectively.

43

Figure 3.1 Schematic of the MF device and the dissolution of CO2 bubbles. Inset shows a zoomed in schematic of the orifice and the generation of bubbles. CO2 gas (99.8% purity) and an aqueous continuous phase (2 wt. % Triton X-100) were introduced into the central and the outer channels of the MF flow-focusing device, respectively. The pressure of the gas (PCO2) was fixed as 27.6 kPa (for the generation of small bubbles it was 55.2 kPa). The flow rate (QL) of the continuous aqueous phase varied from 5 to 12 mL/h. The value of pH of the continuous phase was controlled by varying the concentrations of HCl or NaOH in the range 1.5≤pH≤13.2 At the exit of the microchannel, the bubbles were exposed to an ambient pressure of 101.3 kPa.

Figure 3.2 schematically shows the shrinkage of CO2 bubbles. The diffusion of CO2

from the bubbles into the continuous phase was followed by the chemical reaction with

H2O or OH− ions.14 The process of CO2 dissolution is described by Henry’s law (eq. 3.1)

and pH-dependent chemical reactions (eq. 3.2-4):15

[CO2]l kH PCO2 (eq. 3.1)

pH<10: CO2 + H2O HCO3- + H+ (K = 4.4 x 10-7) (eq. 3.2)

pH>10: CO2 + OH- HCO3- (K = 3.2x 107) (eq. 3.3)

HCO3- + OH- CO3

2- + H2O (K = 3.5x 103) (eq. 3.4)

44

Figure 3.2 Schematic of pH-dependent dissolution of CO2 bubble leading to bubble shrinkage.

where [CO2]l, kH, and PCO2 are the concentration of molecularly dissolved CO2, Henry’s

law constant (for CO2 at 25oC kH=3.2x10-4 mol/(L kPa)), and the applied inlet gas pressure,

respectively, and K is the equilibrium constant. We note the important role of the acidity of

the medium: the equilibrium constant at pH>10 is 14 orders of magnitude larger than that at

pH<10. Thus, the change in the acidity of the continuous phase can be used to control the

extent of CO2 dissolution and hence the ultimate dimensions of bubbles flowing through

the MF device.

Figure 3.3A,B shows the representative optical microscopy images of the CO2 bubbles

generated in the MF device in acidic (pH=1.5) and basic (pH=13.2) aqueous solutions,

respectively. The bubbles formed in the geometrically controlled regime. In this regime, the

formation of bubble is determined by blockage of the orifice and the downstream channel

by the gas thread, which subsequently causes the increase of pressure behind the orifice and

break up of the bubble. Since the unperturbed dimension of the bubbles was larger than the

height of the microchannel, they had a disc-like shape.9,16 Polydispersity (CV) of the

bubbles was maintained below 5%.

45

Two features are clearly shown from Figure 3.2A,A’,B,B’: (i) in the basic solution the

bubbles underwent a stronger reduction in size than in the acidic solution and (ii) upon the

formation, the bubbles had a significantly larger initial size at higher pH values. The former

effect was expected: the reduction in the size of the bubbles was caused by the dissolution

of CO2 in the aqueous continuous phase, accompanied by the reaction between CO2 and

H2O (at pH=1.5) or between CO2 and OH- ions (at pH=13.2).15 A greater extent of

shrinkage of the bubbles at higher pH values occurred due to the stronger mass transfer of

CO2 into an aqueous phase.14 However, we expected that a larger initial size of the bubbles

in the basic solution was counter-intuitive: at higher pH values, the initial size of CO2

bubbles would rapidly decrease with increasing pH values.

Figure 3.3 Representative optical microscopy images taken immediately after the generation of CO2 bubbles (A,B) and N2 bubbles (C,D) and in the downstream channel 107 mm (A’-D’) away from the orifice. The initial pH value of the continuous phase was 1.5 (A,A’,C,C’) and 13.2 (B,B’,D,D’). The bubbles were generated at PCO2=27.6 kPa and QL=6 mL/h. Scale bar is 200 μm.

46

We hypothesized that a pH-dependent formation of CO2 bubbles was affected by their

dissolution in the downstream channel and the related change in resistance to flow. Thus

the size of a newly formed ‘‘successor’’ bubble was affected by the pH-dependent

dissolution of its predecessors. Such coupling or ‘‘memory’’ was earlier reported for the

generation and flow of foams in a MF device, at which at constant input parameters (the

flow rate of the continuous phase and the pressure of the gas) the inflow gas flow rate and

bubble volume showed periodic oscillations affected by the presence of pre-formed bubbles

flowing in the downstream channel.17

To verify this hypothesis, we examined the formation of N2 bubbles in the same MF

bubble generator and under the same hydrodynamic conditions as those used for producing

CO2 bubbles: we note that compared to the solubility in water of CO2 of ca. 0.15 wt%, the

solubility of N2 in water is only ca. 0.005 wt% at 101.3 kPa and 25 oC.18 Figure 3.3C,D

illustrates the generation of N2 bubbles under acidic (pH=1.5) and basic (pH=13.2)

conditions; Figure 3.3C’,D’ shows the corresponding N2 bubbles in the downstream

channel. Since the pressure in the microchannel reduced along its length,19,20 the bubbles

slightly expanded. Thus, we conclude that no noticeable effect of the acidity of the medium

on the size of N2 bubbles was observed.

Furthermore, we examined the effect of pH of the continuous phase on the initial and

final (stabilized) volume of CO2 and N2 bubbles at various pH values is shown in Figure

3.4. For CO2 bubbles, the changes in their initial and final volumes were in anti-correlation:

a higher pH value led to a stronger increase in the initial volume and a stronger reduction in

the final bubble size. On the other hand, both the initial and final volumes of N2 bubbles

showed no significant change at 1.5≤pH≤13.2.

47

Figure 3.4 Variation in initial (●, ), and final (○,) volumes of CO2 (●,○) and N2 (,) bubbles plotted as a function of the initial pH of the continuous aqueous phase.

Based on the results presented in Figure 3.3 and 3.4, we attributed the effect of pH of

the continuous phase on the original dimensions of CO2 bubbles to their pH-dependent

dissolution in the downstream channel. The dissolution of CO2 led to the reduction in

pressure in the MF device: it increased the inflow rate of the gaseous CO2 to compensate

for the drop in pressure. Thus a stronger dissolution of bubbles in the downstream channel

at high pH values led to the formation of larger bubbles. These results were qualitatively

agreed to the observations by other research groups on increasing initial volume of bubbles

when the hydrodynamic pressure in the microchannel was reduced.20

In addition, we found the effect of pH on the frequency (f) of formation of CO2 bubbles

by the ratio f/fpH=13.2 where f and fpH=13.2 are the frequencies of bubble formation at a

particular pH and at pH=13.2, respectively (the value of pH=13.2 was the most basic

condition used in the experiments). The frequency of the formation of bubbles was

estimated using experimental results as follows.

48

where Pc is the pressure drop in the downstream channel, Vc is the volume of the channel, a

is a dimensionless parameter, μ is the viscosity of the continuous phase, L, w, and h are the

length, the width, and the height of the channel, respectively, Lnb is the length of the

channel excluding the dimensions of the CO2 bubbles, and Di is the diameter of the i-th

bubble.19,20 Figure 3.5 shows that the frequency of the formation of CO2 bubbles increased

with increasing pH. In contrast, for N2 bubbles, the frequency of bubble generation was

practically constant regardless of the value of pH of the continuous phase owing to the

invariance in the volume of bubbles with the changing acidity of the medium.

Figure 3.5 pH-dependence of the relative frequency of bubble generation with respect to the frequency of the formation of bubbles at pH=13.2 for CO2(■) and N2() bubbles. The bubbles were generated at P= 27.6 kPa, QL=6 mL/h.

2

2

2

2

CO

CCO

CCO

CCO )()(P

PPVP

PPQf L −

∝−

∝

31

L

3nbL

C

)(

wh

DLQa

whLQaP

ni

ii∑

=

=

−==

µµ

(eq. 3.5)

(eq. 3.6)

49

3.2.2 Dissolution of CO2 Bubbles

In the downstream channel, CO2 bubbles underwent uniform, pH-dependent

dissolution, until their dimensions were stabilized. Figure 3.6A-C shows the snapshots of

the individual bubbles flowing in the continuous phase with varying initial pH values at the

distance, d, of 0.05, 12 and 107 mm from the orifice, respectively. The dissolution of CO2

led to the decrease in the initial value from pH=13.2 to 12.5. When the initial value of pH

was 11 the value of pH decreased to 9 with CO2 dissolution. Similarly, at initial pH ranging

from 5 to 9 the value of pH reduced to 4.5. The dissolution of CO2 bubbles did not alter the

initial value of pH=1.5. The polydispersity (CV<5%) was similar to that of the initial

bubbles. Due to the narrow distribution in bubble sizes, the difference in the Laplace

pressure was reduced, thereby leading to the uniform dissolution of all the bubbles.21 In

addition, Ostwald ripening during the dissolution of bubbles was suppressed due to the

presence of low-soluble gases in the gas phase, which provided an osmotic stabilizing

effect.22

Figure 3.6 (A-C) Optical microscopy images of CO2 bubbles taken at varying distances away from the orifice in the device at (A) pH=1.5, (B) pH=9, and (C) pH=13.2. Scale bar is 50 μm. (D) Variation in the volume of CO2 bubbles examined at different pH values, plotted vs the distance, d, away from the orifice.

50

Although larger initial CO2 bubbles formed at high pH values, they shrank to smaller

size than the bubbles shrunken at low pH values. This trend is shown in Figure 3.6D, where

the variation in bubble volume is plotted as a function of the distance, d, from the orifice.

For example, the initial volume of bubbles generated at pH=13.2 was 1.7-fold larger than

the volume of bubbles produced at pH=1.5, however the final volume of bubbles generated

at pH=13.2 was only 0.2 of the stabilized volume of bubbles formed at pH=1.5. Change in

pH also affected the kinetics of bubble dissolution. Increase in pH resulted in more rapid

shrinkage of the bubbles and the stabilization of their size within a shorter distance from the

orifice. However, at d~107 mm, the dimensions of bubbles were stabilized irrespective of

the pH of the continuous phase.

In principle, the stabilization of bubble size could be attributed to one of two

mechanisms: the saturation of the aqueous continuous phase with dissolved CO2, or the

increase in the concentration of impurities of low water soluble gases in the bubbles due to

CO2 dissolution; the latter effect could suppress further CO2 dissolution owing to the low

partitioning parameter between gaseous and aqueous phases.22

In order to determine the mechanism controlling the final dimension of bubbles, we

determined the experimental and theoretical amount of dissolved CO2 in the continuous

phase. The concentration of dissolved CO2 was estimated by using Henry’s law (eq. 3.1)

and acid-base equilibria reactions (eq. 3.2-3).14,15 In [CO2]l = kH PCO2 (eq. 1), the value of

kH varies on the concentration of the ionic species in water and it is estimated as

kH=kH010-0.138I where kH0 is Henry’s law constant for CO2 in pure water (kH0=3.3 x 10-4

mol/(L kPa)) and I is the ionic strength, I=0.5∑i

ii zC 2 and Ci is the concentration of ions

with charge z.15

51

At pH<5 the total concentration of dissolved CO2 is dominated by Henry’s law (eq. 1)

because the effect of reactions are negligible.23 At pH ≥5 the total concentration of

dissolved CO2 was estimated by adding the unreacted amount of CO2 (determined by

Henry's law(eq. 1)) and reacted CO2 (from the chemical reactions (eq. 3.2-3)).15 Below we

show the detailed calculation of the total concentration of dissolved CO2 for pH=13.2,

kH=3.2 x 10-4 mol/(L kPa) and PCO2=27.6 kPa. The calculations of the amount of dissolved

CO2 for other pH values are given in Appendix. Based on (eq. 3.1), we calculated [CO2]l

~0.009 mol/L. Then, using the reaction (eq. 3.3), we determined the concentration of

reacted CO2 by writing acid-base equilibria as follows.

CO2(l) + OH- HCO3- K = 3.2 x 107

Initial concentration 0.009 0.16

Reacted concentration -x -x x

Final concentration 0.009-x 0.16-x x

x~0.009 mol/L

The total amount of dissolved CO2 (both unreacted and reacted) is [CO2]l + x, that is,

0.018 mol/L. Using the same approach we found that for pH=11, the concentration of

dissolved CO2 was ca. 0.01 mol/L. For 5≤pH<10, by combining Henry’s law (eq. 3.1) and

reaction (eq. 3.2), we determined the total concentration of dissolved CO2 to be

approximately 0.009 mol/L. For pH=1.5, we used Henry’s law (eq. 3.1) and found the total

concentration of dissolved CO2 to be ca. 0.009 mol/L. Experimentally, to find the actual

concentration of dissolved CO2 we defined the volume of ca. 6x10-6 mL as a unit volume

containing a single initial CO2 bubble (imaged immediately after the orifice). By applying

52

the same unit volume to the position of the bubble the downstream channel at which the

bubble size was stabilized, we used the relative change in bubble volume to find the amount

of dissolved CO2 per unit volume, and obtained the actual total concentration of dissolved

CO2.

Figure 3.7A shows that in the range 1.5≤pH≤13.2 the estimated and experimentally

found concentrations of dissolved CO2 were very close. Such agreement indicated that the

dissolution of CO2 bubbles and stabilization of the dimension of bubbles were controlled by

the saturation of the aqueous continuous phase. Figure 3.7B shows the fractional change in

the volume of bubbles, ΔV/V0, plotted as a function of initial pH of the continuous phase,

where ΔV is the difference between the initial and final (stabilized) volumes of bubbles and

V0 is the initial volume of bubbles. With increasing pH values the extent of shrinking of

bubbles increased, featuring the change in the slope at pH~10. Such change was in

agreement with the difference in the equilibrium constants for reactions (eq. 3.2-3): the

equilibrium constants for reaction (eq. 3.3) (pH>10) are over 10 orders of magnitude larger

than that for reaction (eq. 3.2). At pH=13.2, the strong shift of equilibrium towards the

products led to the 97.5 % volume loss of CO2 from the bubbles, whereas at pH=1.5 it was

81.6 %.

53

Figure 3.7 (A) Comparison of theoretical (■) and experimental () amounts of dissolved CO2 (mol/L), (B) Relative change in bubble volume plotted as a function of pH of the continuous phase. PCO2=27.6 kPa, QL=6 mL/h.

Next, we examined the effect of flow rate, QL, of the continuous phase on the initial

and final volume of CO2 bubbles and the relative change in bubble volume (Figure 3.8). In

Figure 3.8A, at a particular pH value, with increasing QL the initial volume of bubbles

decreased in agreement with the earlier results on the formation of bubbles from gases with

low solubility.9 The final volume of bubbles also decreased with increasing value of QL,

presumably, due to the stronger flux of HCO3- or CO3

2- ions from the surface of CO2

bubbles occurring at higher flow rate of the continuous phase (Figure 3.8B).14 We note a

relatively narrow range of flow rates studied for PCO2=27.6 kPa: at QL<5, the gas thread

did not break up into bubbles, whereas at QL>7 mL/h the gas thread was completely

retracted in the upstream channel.

54

Figure 3.8 Effect of the flow rate of the continuous phase, QL, on (A) the initial and (B) final volume of CO2 bubbles generated at different pH values of the continuous phase. pH=1.5 (,), pH=5(,), pH=7 (,), pH=9 (,), pH=11 (,), and pH=13.2 (,). PCO2=27.6 kPa. The lines are given for eye guidance.

To provide guidance in generating small bubbles by the “dissolution” approach, the

results of dissolution experiments were summarized as a three-dimensional graph (Figure

3.9). The final stabilized bubble diameter was controlled by varying the pH value and the

flow rate of the continuous phase (at constant PCO2). The initial size of the bubbles was not

used as a variable: it was controlled by tuning the values of pH and QL. The smallest, 47

μm-diameter bubbles were produced at a flow rate of 7 mL/h and pH=13.2, by 97.5%

fractional dissolution of CO2. It should be emphasized that this graph presents the results

obtained in the microfluidic device with particular dimensions used in the present work.

55

Figure 3.9 Variation in the final diameter of CO2 bubbles, plotted as a function of pH and QL (each represents the QL range from 5 to 7 mL/h, respectively).

In addition, we estimated the diffusion time, t, of CO2 at the scale of the initial bubble

size using the relation t ~D2/DCO2, D is the bubble diameter and DCO2 is the diffusion

coefficient of CO2 in water (DCO2~1.94x104 μm2 s-1).24 For pH=13.2, PCO2=27.6 kPa,

QL=6 mL/h, and the average initial bubble diameter of 200 μm, the diffusion time was ca. 2

sec.

3.2.3 Generation of Small Bubbles

Finally, we exploited the strong, uniform, and rapid dissolution of CO2 achieved in

microfluidic channels for the preparation of small, monodisperse bubbles with dimensions

below 10 μm.

56

Following guidance provided in Figure 3.8, we generated CO2 bubbles at pH=13.2 and

flow rate of the aqueous solution of QL=12 mL/h. Following the dissolution of bubbles with

an initial diameter D0=30 μm, we achieved a bubble size of 8 μm at a polydispersity of

4±1%. The estimated frequency of bubble generation was ca. 3x104 bubbles/sec.

Figure 3.10 (A) Generation of 30 μm-diameter CO2 bubbles. (B) Bubbles with a stabilized diameter of 8 μm in the downstream channel. The bubbles were generated in the microfluidic device with the length, width, and height of the orifice of 60, 22 and 40 μm, respectively, at PCO2=55.2 kPa, QL=12 mL/h, and pH=13.2.

3.3 Conclusion

The approach based on the MF formation of CO2 bubbles mixed with minute amounts

of low water soluble gases, followed by their uniform and controllable dissolution provides

a route to generation of small, monodisperse bubbles with controllable size. In the present

work, by generating 30 μm-size CO2 bubbles we produced bubbles with dimensions of 8

μm. Sub-micron size CO2 bubbles can be generated by reducing the dimensions of

microchannels and exploiting higher flow rates of the continuous aqueous phase, which

would allow for the generation of smaller initial bubbles and a stronger flux of HCO3- and

CO3-2 ions from the bubble surface. Under the latter conditions, following complete

dissolution of CO2 the ultimate size of bubbles will be determined by the minute

57

concentration of weakly-soluble gases, e.g. perfluorocarbon, that are either exist as

impurities, or are intentionally introduced in with CO2.22

In the present work, in order to focus on the effect of pH and hydrodynamic

conditions on bubble dimensions, bubbles were not stabilized against dissolution and

coalescence. However, one can improve their stability by encapsulating them with suitable

polymers,25,26 colloidal particles,27-29 or surfactants.30

This work has several other important implications. Gases with high solubility in the

continuous phase, e.g., ammonia or CO2 have strong environmental impact, due to their

high solubility and reactivity in water.31 They are also used in heterogeneous gas-liquid

chemical reactions.32 It can be expected that microfluidic generation of monodisperse

bubbles of highly soluble, reactive gases, accompanied with a study of the variation in

bubble dimensions would enable detailed studies of gas dissolution, as well as the kinetics

of gas-liquid reactions.

Secondly, microbubbles of CO2 are used for enzyme inactivation,33 as ultrasound

contrast agents,34 and as templates for the synthesis of inorganic microspheres or porous

materials.35 Current methods of the production of CO2 bubbles provide poor control of

bubble dimensions.33-35 Controlled generation and dissolution of CO2 bubbles could solve

the problem associated with bubble disparity in size and enable the study of interactions of

CO2 with the surrounding liquid medium.

References


58

(2) K. Ferrara, R. Pollard, M. Borden, Annual Review of Biomedical Engineering 2007,

9, 415.

(3) S. R. Sirsi, M. A. Borden, Bubble Science, Engineering and Technology 2009, 1, 3.

(4) I. Lentacker, S. C. De Smedt, N. N. Sanders, Soft Matter 2009, 5, 2161.

(5) Y. Z. Zhao, H. D. Liang, X. G. Mei, M. Halliwell, Ultrasound in Medicine and

Biology 2005, 31, 1237.

(6) W. L. Nyborg, Ultrasound in Medicine and Biology 2001, 27, 301.

(7) B. B. Jiang, C. Y. Gao, J. C. Shen, Colloid and Polymer Science 2006, 284, 513.

(8) A. M. Ganan-Calvo, J. M. Gordillo, Physical Review Letters 2001, 87.



(10) M. Yasuno, S. Sugiura, S. Iwamoto, M. Nakajima, A. Shono, Aiche Journal 2004,

50, 3227.

(11) J. H. Xu, S. Li, G. G. Chen, G. S. Luo, Aiche Journal 2006, 52, 2254.

(12) P. Garstecki, M. J. Fuerstman, H. A. Stone, G. M. Whitesides, Lab on a Chip 2006,

6, 437.

(13) K. Hettiarachchi, E. Talu, M. L. Longo, P. A. Dayton, A. P. Lee, Lab on a Chip

2007, 7, 463.

(14) T. Madhavi, A. K. Golder, A. N. Samanta, S. Ray, Chemical Engineering Journal

2007, 128, 95.

(15) P. V. Danckwerts, Gas-liquid Reactions, McGraw-Hill Book Company, New York

1970.

(16) P. Garstecki, M. J. Fuerstman, G. M. Whitesides, Physical Review Letters 2005, 94.

(17) J. P. Raven, P. Marmottant, Physical Review Letters 2006, 97.

(18) D. W. Oxtoby, N. H. Nachtrieb, Principles of Modern Chemistry, Harcourt Brace

Jovanovich College Publishers, Orlando 1990.

(19) M. J. Fuerstman, A. Lai, M. E. Thurlow, S. S. Shevkoplyas, H. A. Stone, G. M.

Whitesides, Lab on a Chip 2007, 7, 1479.

(20) M. T. Sullivan, H. A. Stone, Philosophical Transactions of the Royal Society a-

Mathematical Physical and Engineering Sciences 2008, 366, 2131.

59

(21) E. Lorenceau, Y. Y. C. Sang, R. Hohler, S. Cohen-Addad, Physics of Fluids 2006,

18.



(23) J. N. Butler, Carbon dioxide equilibria and their applications, Lewis Publisher,

Michigan 1981.

(24) A. Tamimi, E. B. Rinker, O. C. Sandall, Journal of Chemical and Engineering Data

1994, 39, 330.

(25) F. Cavalieri, A. El Hamassi, E. Chiessi, G. Paradossi, R. Villa, N. Zaffaroni,

Biomacromolecules 2006, 7, 604.

(26) D. G. Shchukin, K. Kohler, H. Mohvald, G. B. Sukhorukov, Angewandte Chemie-




(28) B. P. Binks, R. Murakami, Nature Materials 2006, 5, 865.




(31) R. Pierantozzi, Kirk-Othmer Encyclopedia of Chemical Technology 2003, 4, 803.

(32) C. D. Cooper, Kirk-Othmer Encyclopedia of Chemical Technology 2007, 26, 667.

(33) M. Shimoda, Y. Yamamoto, J. Cocunubo-Castellanos, H. Tonoike, T. Kawano, H.

Ishikawa, Y. Osajima, Journal of Food Science 1998, 63, 709.

(34) P. Redondo, J. Cabrera, Seminars in Cutaneous Medicine and Surgery 2005, 24,

175.

(35) G. Hadiko, Y. S. Han, M. Fuji, M. Takahashi, Materials Letters 2005, 59, 2519.

60

Chapter 4

Assembly of Colloidal Particles at Gas-Water and Water-Oil

Interfaces

Reproduced with permission from Angewandte Chemie-International Edition 2009, 48,

5300. Copyright 2009, Wiley-VCH, and Journal of the American Chemical Society 2008,

130, 16508. Copyright 2008, American Chemical Society.

Small colloidal particles with size range from a few nanometers to micrometers and

appropriate wettability strongly adsorb to gas–liquid and liquid-liquid interfaces. Such

colloidal particles can be used as stabilizers of bubbles and droplets. Interesting materials,

including colloidosomes and anisotropic and porous particles, have been prepared by

assembling particles at fluid interfaces. In this chapter, we describe a microfluidic strategy

for the continuous, single-step production of particle-coated bubbles with a predetermined

size and narrow size distribution. In addition, this chapter demonstrates a microfluidic

“inside-out” approach for the generation of monodisperse water-in-oil and oil-in-water

Pickering emulsions in which the coverage of droplets with particles can be precisely

controlled by hydrodynamic means.

4.1 Introduction

Particle-stabilized bubbles and droplets (Pickering emulsion) have recently seen a

surge in interest owing to their high stability against coalescences and Ostwald ripening.

61

These systems had a broad range of applications in the fabrication of functional materials, e.

g., hollow permeable capsules,1 anisotropic structures2 and hybrid supracolloidal

assemblies.3 Adsorption of particles at a particular fluid interface occurs when they are not

completely wet by any of the adjacent phases.4 The wettability of the surface of particles

determines their location at the fluid interface. The hydrophilic particles largely reside in

water. On the other hand, they favourably stay in gas or oil when the particles are

hydrophobic. More importantly, the particles attached to the interface build up the energy

barrier required to remove them into one of the fluid phases. 4,5 This results in irreversible

adsorption of particles at the interface in comparison with conventional surfactants which

adsorb and desorb on the time scale of a few tens of miliseconds.6 Consequently, particle-

stabilized bubbles and droplets are stable against coalescence. In addition, Ostwald ripening

is suppressed by reducing the Laplace pressure-induced dissolution of bubbles and

droplets.4 This effect is at maximum when the ratio of the radius of particle to that of

bubble or droplet is approximately 0.1, which allows bubble or droplet to adopt faceted

polyhedral shape. At this shape, the Laplace pressure is eliminated by reducing the mean

surface curvature close to zero.7

Current production of particle-stabilized bubbles and droplets relies on injection

methods, or shear of a mixture of two immiscible fluids.8-10 These methods generate

polydisperse bubbles/droplets in an uncontrollable manner, thus hampering the fabrication

of materials with hierarchical periodic structures and the related interesting optical and

mechanical properties. Recently, hydrodynamic flow was used for the controlled assembly

of colloidal particles at gas-liquid and liquid-liquid interfaces.11 The strategy relied on the

shear-assisted delivery of colloidal particles to the surface of bubbles or droplets. The

62

produced bubbles or droplets with colloidal armour had a narrow size distribution, however

the method had a low productivity of ca. 10 bubbles (droplets)/s.

In this chapter, we present a microfluidic (MF) method for the controlled production of

particles-coated bubbles12and droplets.13 First, we describe a chemically mediated MF-

based approach for the continuous production of bubbles encapsulated with a shell of

colloidal particles (armoured bubbles) (Chapter 4.2.1).12 The strategy relies on several

events, occurring in succession: i) a MF generation of monodisperse CO2 bubbles in a

dispersion of anionic particles, ii) dissolution of CO2, resulting in the shrinkage of the

bubbles and a local increase in the acidity of the solution around the bubbles, and iii)

adsorption of the particles to the gas-liquid interface, driven by the chemically induced

change in the surface energy of the particles. The dimensions and shapes of bubbles with

colloidal armours are precisely controlled by hydrodynamic and chemical means. In

addition, the generality of the MF strategy is demonstrated by encapsulating bubbles with a

variety of particles and their mixtures.

Secondly, we describe a MF “inside-out” approach to the generation of monodisperse

water-in-oil and oil-in-water Pickering emulsions. The emulsions were used for the

generation of supracolloidal polymer microspheres (Chapter 4.2.2).13 This method

addresses the challenges frequently occurring in conventional methods of the preparation of

Pickering emulsions, that is, an insufficient control of droplet dimensions and an excess of

particles in the continuous media. The proposed method minimizes waste of particles, due

to their introduction in the droplet phase, and allows control of the coverage of droplets

with particles by tuning the concentration of particles or the flow rates of the liquids.

63

4.2 Results and Discussion

The materials and methods described in the present chapter are provided in Chapter 2.

4.2.1 Synthesis of Colloidal Particle Armoured Bubbles

4.2.1.1 Experimental Design

We used a planar MF T-junction device to produce plugs of CO2 bubbles in an aqueous

dispersion of anionic polymer particles (Figure 4.1A).14 A dispersion of poly(styrene-co-

acrylic acid) (PS-co-PAA) particles15 in an aqueous NaOH solution was supplied to the

microchannel at a typical flow rate, QL, from 7.5 to 24 mL/h. Gaseous CO2 comprising 0.2

vol.% of inert gases was introduced into the orthogonal channel by applying CO2 pressure

(PCO2) as 34.5 kPa.

Figure 4.1 (A) A schematic of a microfluidic (MF) T-junction bubble generator. The widths of the main and the side channels are 220 and 40 μm, respectively. The height of the channels is 130 μm. (B) A schematic of the formation of colloidal particle shell during the dissolution of CO2 bubbles.

64

In this experimental design, we hypothesized the dissolution of CO2 bubble would increase

the acidity of the medium adjacent to the bubbles. This would result in the formation of

bubbles armoured with PS-co-PAA particles due to the reduction in the surface energy of

the particles (Figure 4.1B, see below).

4.2.1.2 Generation of Armoured Bubbles

Figure 4.2 illustrates the formation of CO2 bubbles, their time-dependent dissolution

and adsorption of the particles to the gas-water interface. The thread of CO2 periodically

broke up to release gaseous plugs with a polydispersity of 2-5% (Figure 4.2A). Owing to

the rapid dissolution of CO2, the bubbles underwent a dramatic decrease in volume and

acquired a spherical shape (Figure 4.2B). While the bubbles were shrinking, the particles in

the continuous phase adsorbed to the gas-water interface, due to the local increase in acidity

in the area adjacent to the bubbles (Figure 4.2C). The bubbles were completely covered

with the particles in the downstream channel (Figure 4.2D). The entire process took place

around 2 sec.

Figure 4.2 Progression of the plugs to spherical armoured bubbles. Optical microscopy images of bubbles at a distance of 0 (A), 30 (B), 80 (C) and 150 (D) mm from the T-junction. Scale bars are 200 μm. Bubbles generated at pH=14, PCO2=34.5 kPa, QL=10.5 mL/h, initial particle concentration, Cp=1.5 wt. %, and 28.8oC.

65

Figure 4.3A shows a typical image of the bubbles collected at the outlet of the MF

device. A uniform dissolution and mass transfer achieved in the microchannels yielded

armoured bubbles with a narrow size distribution. On the bubble surface the particles

formed a close-packed 2D crystalline shell (Figure 4.3B). Imaging of microtomed bubbles

using Scanning Electron Microscopy (SEM) confirmed that the particles formed a

monolayer-thick shell (Figure 4.3C). Labeling of the polymer particles with a fluorescent

dye Hostasol Yellow (HY) allowed visualization of the colloidal shell using Laser Confocal

Fluorescence Microscopy (LCFM) (Figure 4.3C, inset).

Figure 4.3 (A) Optical microscopy image of armoured bubbles generated as in Figure 4.2 and collected at the outlet of the MF device. Scale bar is 200 μm (B) Optical microscopy image of close-packed crystalline shell of armoured bubble. Scale bar is 25 μm (C) SEM image (side view) of the shell of the microtomed armored bubble infiltrated with poly(ethylene glycol) diacrylate. Scale bar is 10 μm. The inset shows an LCFM image of the bubble coated with HY-labeled PS-co-PAA particles. Scale bar is 25 μm.

The armoured bubbles were stable to coalescence and Ostwald ripening, and had

narrow polydispersity of 2-5%, similar to the initially generated bubbles. The frequency of

the generation of armoured bubbles was up to ca. 700 bubbles/s; however the productivity

of the method was increased up to ca. 3000 bubbles/s by using a MF flow-focusing bubble

generator (Appendix, Figure A4.1).16

We conducted a series of control experiments. In a particle-free environment (Figure

4.4A) the shrinkage of CO2 plugs was similar to that in the particle suspension (Figure 4.2).

66

This indicated that at this pH value the size of bubbles was determined by the dissolution of

CO2 and not by the formation of the colloidal shell. The role of CO2 and carboxylated

particles was confirmed by showing that the armoured bubbles did not form in experiments

conducted with PS-co-PAA particles and N2 bubbles (Figure 4.4B), or with PS-co-PAA

particles and CO2 bubbles in the presence of surfactants (Figure 4.4C). Surfactants changed

the delicate balance in the surface energy of the particles at the gas-liquid interface. In

another series of control experiments, N2 and CO2 bubbles were not fully covered with

cationic poly(methyl methacrylate-co-4-vinyl pyridine) (PMMA-co-P4VP) beads (Figure

4.4D-E, respectively). These bubbles were prone to coalescence and they acquired a broad

size distribution when collected at the outlet of the MF device (Figure 4.4F-G).

Figure 4.4 Optical microscopy images of bubbles and plugs flowing through the microchannels. (A) Dissolution of CO2 plugs in particle-free at pH=14. (B) Plugs of gaseous N2 formed in a dispersion of anionic PS-co-PAA particles. (C) Bubbles of CO2 generated in a dispersion of PS-co-PAA particles containing 2 wt. % of the non-ionic surfactant (Triton X-100). (D) Plugs of N2 formed in a dispersion of 700 nm diameter cationic PMMA-co-P4VP particles. (E) Bubbles of CO2 dispersed in an aqueous dispersion of 700 nm diameter PMMA-co-P4VP particles. In all experiments, the bubbles were generated at 28.8oC, pH=14, PCO2/N2=34.5 kPa, QL=10.5 mL/h, and Cp=1.5 wt. %. Optical microscope images of collected bubbles (F),(G) from (D),(E), respectively. Scale bars: 200 μm.

67

We explained the formation of armoured bubbles as follows. The dissolution of CO2 in

an aqueous phase was followed by the reactions.17

pH<10: CO2 + H2O HCO3- + H+ (K = 4.4 x 10-7) (eq. 4.1)

pH>10: CO2 + OH- HCO3- (K = 3.2x 107) (eq. 4.2)

These reactions (eq. 4.1-2) led to a local decrease in the pH of the liquid adjacent to

the surface of bubbles, the protonation of the carboxylic groups on the surface of PS-co-

PAA particles and an increase of the contact angle (θ) of water on the polymer surface, as

illustrated in Figure 4.5.

Figure 4.5 Variation in contact angle (θ) of an aqueous solution of NaOH in air on the PS-co-PAA film, measured at different pH values.

The protonation of the surface carboxylic groups on the particles in the region of the

reduced pH favoured adsorption of the microbeads to the gas-water interface. The energy

barrier required for the removal of particles from the interface was given by

( )2LG,2 cos1 θγπ ±= paE (eq. 4.3)

where γG,L is the interfacial tension of the gas-liquid interface and ap is the radius of the

particles. The sign inside the bracket is negative for removal of particles into the water

68

phase, and positive for removal of particles into the gas phase.4 We stress that the particles

were hydrophobized only in the vicinity of the bubbles. In the rest of the continuous phase,

the particles retained their colloidal stability.18

4.2.1.3 Effect of Flow Rate on the Dimensions of Armoured Bubbles

Figure 4.6 shows the effect of the flow rate of the continuous phase, QL, on the final

diameter, Df, of the armoured bubbles at initial pH=14. At the largest values of QL of 10.5

mL/h, the fractional reduction in volume, ΔV/V0 (V0 and ΔV are the initial volume and the

change in volume of the plugs, respectively), of the plugs was up to 99.5%. This indicated

almost all the CO2 left the gaseous plugs (Figure 4.6A). Upon increasing QL, the Df of the

armoured bubbles decreased (Figure 4.6B,C). This effect occurred due to the generation of

smaller initial gaseous plugs16 (Figure 4.6B,C insets) and a more efficient dissolution of

CO2 achieved at high values of QL.19

Figure 4.6 (A) Effect of the flow rate, QL, of the continuous phase on the fractional volume change (ΔV/V0) of CO2 plugs and the final diameter Df of armoured bubbles in the microchannel. The produced armoured bubbles at QL= 8.5 (B) and 10.5 mL/h (C). Scale bars are 50 μm. The insets show the initial plugs of CO2 bubbles. Scale bars are 200 μm. Bubbles were generated at pH=14, PCO2=34.5 kPa and Cp=1.5 wt. %.

69

4.2.1.4 Effect of pH and Particle Concentration on the Dimension and

Morphologies of Armoured Bubbles

Figure 4.7A shows the effect of the initial pH of the continuous phase on the Df of the

bubbles and the fractional change in volume ΔV/V0, of gaseous plugs at an initial particle

concentration, CP, of 1.5 wt %. With increasing pH a more rapid dissolution of CO2 yielded

smaller bubbles that were uniformly covered with particles (Figure 4.6B-D). At pH=14 the

value of ΔV/V0 reached 99.5% while it was 40 % at pH=5 (Figure 4.7A). In the

experiments conducted at pH=14 in a particle-free environment, the CO2 plugs underwent a

similar change in volume, suggesting that at this pH value the size of bubbles was not

affected by adsorption of particles. Thus, we conclude that the final volume of the bubbles

was determined by the volume of the remaining inert gas.

Figure 4.7 (A) Effect of the initial pH of the continuous phase on the fractional volume reduction (ΔV/V0) of CO2 plugs and the final diameter, Df, of armoured bubbles in the microchannel. The produced armoured bubbles at initial pH=14 (B) 8 (C) and 5 (D). Scale bars are 100 μm. The insets show the bubbles before the exit of the microchannel. Scale bars are 200 μm. Bubbles were generated at PCO2=34.5 kPa, QL=9.0 mL/h and Cp=1.5 wt. %.

70

For pH<10, the bubbles retained their plug-like shape and were only partly covered

with particles (Figure 4.7C,D, insets). This effect occurred because the weaker dissolution

of CO2 at this range of pHs did not produce small enough surface area of bubbles to be

fully covered with the particles.19,20 Owing to the incomplete coverage with the particles,

the plugs were prone to coalescence. When collected at the outlet, they formed large, non-

spherical armoured bubbles due to the jamming of the particles at the interface (Figure

4.7C,D).

Figure 4.8 shows that with increasing value of Cp the final size of the armoured

bubbles increased (the initial dimensions of the CO2 plugs did not depend on CP). Rapid

formation of the colloidal shell counteracted the shrinkage of the plugs, thereby providing

additional control over the dimensions of armoured bubbles (Figure 4.8A). At CP <1 wt. %

the bubbles were poorly covered with particles and at the outlet of the MF device they

coalesced to form large armoured bubbles with a broad size distribution (Figure 4.8B). For

the concentration of particles in the range 1.5≤CP /wt. %≤5.0 (1 wt. % ~ 4.3 x 108

particles/mL) the colloidal armour suppressed coalescence of the bubbles, whose

polydispersity remained below 5% (Figure 4.8C,D).

71

))()(4()(

))()(4()(

22

223

tnatRqdt

tdn

tnatRqdt

tdR

ppbpp

ppbbb

αππ

βαππ

−=

−−=

Figure 4.8 (A) Effect of the initial concentration of particles, Cp, of the continuous phase on the fractional volume reduction, ΔV/V0, of CO2 plugs and the final diameter, Df, of armoured bubbles in the microchannel. The produced armoured bubbles at initial Cp =0.1 (B) 1.5 (C) and 5 wt. % (D). Scale bars are 100 μm. The insets show the bubbles before the exit of the microchannel. Scale bars are 200 μm. Bubbles were generated at PCO2=34.5 kPa, QL=10.0 mL/h and pH=14.

We developed a model that rationalized our observations and accounted for the

shrinkage of bubbles and the adsorption of particles to the gas-liquid interface. These two

processes were coupled: dissolution of CO2 occurred only through uncovered regions of the

gas-liquid interface.

For a bubble of radius Rb(t) and the number of adsorbed particles np(t), the uncovered

bubble area was described as (4πRb(t)2−απap2np(t)) where α depends on the contact angle.

The two equations have the form

(eq. 4.4)

(eq. 4.5)

72

BtdntdR

p

b −=)()( 3

22

330 4

peq

eq

aB

R

RR=

−

where the constant β≈1 accounts for the arrest of CO2 dissolution once the particles are

close-packed.7 The fluxes of gas and particles, qb and qp respectively, depend on the speed

U and the radius (a) of the bubble. For large Peclet numbers, URb(t)/D>>1, where D is the

diffusion coefficient of dissolved CO2, we expect qb and qp to scale similarly with U and

Rb(t). For such transport processes qp∝ Cp, analogous to Fick’s law. The bubbles shrink

faster under high pH conditions, since the diffusion of dissolved CO2 in the liquid boundary

layer is accompanied by the reaction with OH- ions. Thus, we expect qb to increase with

increasing pH. Neglecting the weak dependence of qb on β, the ratio of the two equations

(eq. 4.4-5) yields

where B is constant at a fixed pH or Cp. It decreases as Cp increases and it increases as the

pH increases. Thus, for the initial conditions Rb(0)=R0 and np(0)=0, the equilibrium bubble

radius Req is given by

Thus, the value of Req decreases monotonically with increasing B. Since the value of B

increases with high pH values, it can be expected that the bubble radius will decrease as the

pH increases, which is in agreement with the experimental trends shown in Figure 4.7A.

Further, based on eq. 4.7, the bubble radius should increase as the Cp increases (decreasing

B), which is consistent with experimental observations (Figure 4.8A).

(eq. 4.7)

(eq. 4.6)

73

4.2.1.5 Generality of the Assembly of Colloidal Particles at Gas-Water

Interface

The generality of our approach for the production of particle-coated bubbles was

demonstrated by coating bubbles with different types of anionic particles and mixtures of

anionic particles. Figure 4.9A shows bubbles encapsulated with a mixture of HY dye-

labelled and dye-free PS-co-PAA microspheres with similar dimensions. Figure 4.9B

illustrates bubbles with a shell of carboxylated silica particles and a mixture of

carboxylated silica particles and HY dye-labelled PS-co-PAA (Figure 4.9B inset). Bubbles

encapsulated with 20 nm-diameter carboxylated silica nanoparticles capped CdSe/ZnS

quantum dots (QDs) are shown in Figure 4.9C. We also encapsulated bubbles with the

protein bovine serum albumin labelled with fluorescein isothiocyanate (FITC-BSA), which

was dissolved in an aqueous phase at pH=7. Following the dissolution of CO2, the bubbles

were coated with protein particles, which were 1-2 μm in size (Figure 4.9D). We believe

that this observation indicates that in the acidic environment adjacent to the shrinking

bubbles the protein molecules reached their isoelectric point of pH=4.8 and aggregated to

form clusters precipitating on the bubble surface.21

74

Figure 4.9 Generation of bubbles with various types of colloidal armour. (A) Optical fluorescence microscopy image of bubbles coated with a shell of 2.8 μm diameter HY-labelled PS-co-PAA particles (Cp=0.5 wt. %) and 3.5 μm diameter PS-co-PAA particles (Cp=1.0 wt. %), PCO2=41.4 kPa, QL=12 mL/h, pH=14. Scale bar is 50 μm. (B) Optical microscopy image of bubbles coated with 3 μm diameter carboxylated silica particles. PCO2=82.7 kPa, QL =24 mL/h, pH=14, and Cp=1.5 wt. %. Scale bar is 100 μm. The inset shows a fluorescence microscopy image of the surface of an armoured bubble coated with 3 μm diameter carboxylated silica particles and 2.8 μm diameter HY-labelled PS-co-PAA polymer particles in the continuous phase in the weight ratio 3:1, respectively. Scale bar is 25 μm. (C) LCFM image of bubbles encapsulated with 20 nm diameter carboxylated silica nanoparticles loaded with CdSe/ZnS QDs, λex=480 nm. PCO2=55.2 kPa, QL=23 mL/h, pH= 10, and Cp =0.12 wt. %. Scale bar is 100 μm. (D) Fluorescence optical microscopy image of armoured bubbles engulfed with the FITC–BSA shell, λex=495 nm. The bubbles were generated at PCO2=44.8 kPa, QL=13 mL/, pH=7, and a protein concentration of 0.02 wt. %. Scale bar is 100 μm.

4.2.2 Synthesis of Colloidal Particle-Coated Droplets

Research activities in the field of Pickering emulsions have led to the production of a

wide range of supracolloidal materials.1-3 For a particular system, the concentration of

particles required for the efficient stabilization of droplets depends on the ratio between the

dimensions of droplets and particles, and the number of particles at the fluid interface.20,26

75

Current methods for producing particle-coated emulsions generate droplets with a broad

distribution in sizes,8-10 which hampers the precise rationalization of the amount of particles

required for the effective system. We demonstrate a MF “inside-out” approach to the

production of monodisperse water-in-oil and oil-in-water Pickering emulsions, as well as

the supracolloidal polymer microspheres.13 The MF emulsification of a dispersion of

colloidal particles in the particle-free continuous phase allowed the control of the coverage

of the droplets with a layer of solid particles in a pre-determined way.

4.2.2.1 Experimental Design

The generation of particle-coated droplets was conducted in a MF flow-focusing

device (Figure 4.10A).16 Droplets of a water-ethanol (85/15 v/v) mixture containing 3.5

μm-diameter poly(divinylbenzene-co-methacrylic acid) (poly(DVB-co-MAA)) particles22

was emulsified in hexadecane as the continuous phase. The generation of droplets occurred

at a flow-focusing regime, where the dispersed thread was focused and broken up in the

orifice by the continuous phase.16 The polydispersity of produced droplets was below 5 %.

The value of the contact angle (θ) between the polymer film derived from the particles and

the water-ethanol mixture was 82.2±2.1o. Thus, the microbeads migrated from the interior

of the droplets to the surface of droplets. Figure 4.10B shows the typical microscope image

of the collected particle-coated droplets at the exit of the outlet.

76

Figure 4.10 (A) Generation of droplets from the water-ethanol mixture (85/15, v/v) containing 4 wt. % of poly (DVB-co-MAA) particles. The flow rates of the droplet and continuous phases are 0.5 and 3.5 mL/h, respectively. Scale bar is 200 μm. (B) Collected particle-coated droplets at the exit of the microchannel. Scale bar is 150 μm.

4.2.2.2 Microfluidic Control of Particle Coverage at Water-Oil Interfaces

Since the diameter of particles used in the present work was significantly smaller than

the diameter of droplets, we estimated the surface coverage of the droplets,δ, was

simplified as

δ = Ap/Ad = Cp⋅ρd⋅ad/(4ρp⋅ap) (eq. 4.8)

where Ad and Ap are the surface areas of the droplet and of the droplet coated with particles,

respectively and ρd and ρp are the densities of the droplet and the particles, respectively.

In Figure 4.11, the dashed line shows the estimated variation in the concentration of

particles required to achieve complete coverage of the droplets with different sizes. We

77

assumed that (i) all particles migrate from the droplet interior to the droplet surface and (ii)

at the interface the particles form a hexagonal lattice with a packing density of 0.906. The

surface area of the droplet coated with particles, Ap, is proportional to the volume of the

droplet, ad3. On the other hand, the surface area of the droplet, Ad, increases with ad

2. Thus,

droplets with larger dimensions require a lower concentration of particles to achieve the

complete coverage of particles. In Figure 4.11, below the dashed line, the amount of

particles is not sufficient for the complete coverage of the droplets; whereas above the line,

the particles exist in excess to form a multilayer shell or to remain in the droplet interior.

Figure 4.11 A diagram of the surface coverage of water-ethanol droplets with 3.5 μm-diameter poly(DVB-co-MAA) particles. The dashed line shows the theoretical conditions for the complete coverage of the droplets with a monolayer of 2D hexagonally packed particles. Filled circles show the experimental data points.

Experimentally, we controlled the surface coverage of the droplets by varying

independently the value of Cp from 4 to 16 wt. % and the values of ad from 40 to 80 μm (by

tuning the ratio of flow rates of the droplet-to-continuous phases).23 Filled symbols in

Figure 4.11 show the experimental results. Droplets with the surface coverage of δ ≥0.7

78

(ad>63 μm, Cp=8 wt. %) were stable to coalescence, whereas droplets with δ <0.7 (ad≤63

μm, Cp=8 wt. %) were prone to coalescence when collected at the exit of the MF device.

The attachment of particles from the droplet phase to the liquid-liquid interface and the

formation of the close-packed crystalline shell occurred rapidly, being assisted by the

hydrodynamic flow.11 Owing to the very rapid particle jamming at the interface at high

values of Cp, we were able to produce non-spherical particle-coated droplets with the shape

determined by the geometry of the microchannel (Figure 4.12).24,25

Figure 4.12 Plug-shaped particle-covered droplets. Scale bar is 100 µm. Inset shows the corresponding elliptical droplets flowing in the microchannel. Scale bar is 200 μm. Cp=14 wt. %. The flow rate of the droplet and continuous phases are 0.5 and 5.5 mL/h, respectively.

In Figure 4.13, representative optical microscopy (top) and LCFM (bottom) images

show that for Cp=8 wt. % and increasing the size of the droplets, a transition occurred from

the non-complete coverage to the complete coverage, and then to the excess-of-particles

regime. The non-complete coverage of the surface of droplets with polymer particles is

79

illustrated in Figure 4.13A,D. Figure 4.13 B,E shows the complete coverage of the droplet

with particles. With the values of Cp and ad predicted by the diagram in Figure 4.11, the

microbeads formed a close-to-hexagonal lattice on the droplet surface (Figure 4.13B, inset).

In the excess-of-particles regime, the microbeads were concentrated at the interior of

droplet, which was visualized by the darker concentrated region of droplet (Figure 4.13C).

In the LCFM image, this region appeared brighter in the interior of the droplets (Figure

4.13F).

Figure 4.13 (A-C) Optical and (D-F) LCFM images of the water-ethanol droplets armoured with a shell of poly(DVB-co-MAA) particles at the not-complete (A,D) and complete (B,E) surface coverage, and at the excess of particles in the droplet interior (C,F). In (C) an excess of particles appears as the large dark region on the background of the droplet coated with a monolayer of particles. In (D-F) poly(DVB-co-MAA) microbeads were labelled with anthryl methacrylate. LCFM images show the plane located in the centre of the droplets. Cp =8 wt. %. λex=380 nm. Scale bars in (A-E) and in (F) are 50 and 100 μm, respectively. Scale bars in insets are 5 μm.

The MF approach was also used for the preparation of oil-in-water Pickering emulsions

and the corresponding supracolloidal structures. The droplet phase contained a mixture of

tripropylene glycol diacrylate, TPGDA, a photoinitiator, 2,2-dimethoxy-2- phenyl-

80

acetophenone (4 wt. %), and poly(DVB-co-MAA) particles at Cp=14 wt. %. Following the

deposition of particles at the surface of droplets, they were collected at the exit of the MF

device and exposed for 1 min to UV-irradiation (400W, λ=330-380 nm).

Photopolymerization of TPDGA yielded large polymer microspheres armoured with a shell

of poly(DVB-co-MAA) particles. Figure 4.14A shows the surface of the supracolloidal

microsphere produced at Cp =14 wt% (approximately 40% higher than required to achieve

the complete surface coverage of δ of 0.906). The average diameter of poly(DVB-co-

MAA) particles protruded on the surface of microsphere was 3.0 μm (the original diameter

of the particles was 3.5 μm). The center-to-center distance between the particles was 4.2

μm of (Figure 4.14A). This indicated that (i) a larger fraction of the microbead surface

resided in the droplets than in the continuous phase and (ii) approximately 0.7 μm mean

distance existed between the surfaces of the particles immersed in the droplet phase.26

Inspection of the colloidal shell using LCFM revealed that poly(DVB-co-MAA) particles

formed a multilayer on the surface of the microspheres (Figure 4.14B), presumably due to

the long range attraction between the microparticles in the TPGDA medium.27

Figure 4.14 SEM (A) and LCFM (B) images of poly(TPGDA) particles armoured with poly(DVB-MAA) particles. Cp=14 wt. %. Scale bars are 40 μm in (A) and 60 μm in (B).

81

4.3. Conclusion

In summary, we have demonstrated a MF approach to the preparation of particle-

encapsulated bubbles and droplets. Our method yielded bubbles with high stability against

coalescence and Ostwald ripening. It increased productivity in comparison with other MF

approaches. It demonstrated the ability to form bubbles with precisely controlled

dimensions. The armoured bubbles generated by the proposed strategy have potential

applications in producing 3-D foams with hierarchical order and ultralight 2-D coatings

with precisely controlled pore sizes. Furthermore, standard methods for the generation of

small bubbles are either cost-inefficient,28 or lacks control over bubble size distribution. In

our approach, these problems are solved by (i) producing large monodisperse bubbles from

gaseous mixtures and (ii) controllably removing one of the components of the mixture to

reach the targeted bubble size. The described strategy can be extended in several ways.

First, the deposition of cationic particles can be achieved by dissolving, e.g. NH3 and

increasing the pH in the region adjacent to the surface of the bubbles. Second, an increase

in the productivity of the process can be achieved by using parallel integrated MF bubble

generators.29,30 Third, controllable transfer of one of the components of the gaseous

mixture to the continuous phase can provide the ability to control the composition of the

bubbles and of the surrounding medium and to activate a particular reaction at a particular

timing, on demand.

A MF “inside-out” approach for the preparation of particle-encapsulated droplets has

the following features: (i) high efficiency and a minimized quantity of particles needed for

the generation of Pickering emulsions , (ii) a narrow size distribution of droplets, which

provides a route for easier analysis of the dynamics of formation and buckling of Pickering

82

emulsions, as well as colloid crystallization on curved fluid interfaces, and (iii) the ease of

the control over the coverage of droplets with particles by manipulating the concentration

of particles or the hydrodynamic conditions of fluids. We anticipate that the present

approach will pave a way to the controlled preparation of colloidal materials with complex

structural hierarchy.31

References

(1) A. D. Dinsmore, M. F. Hsu, M. G. Nikolaides, M. Marquez, A. R. Bausch, D. A.

Weitz, Science 2002, 298, 1006.

(2) B. Liu, W. Wei, X. Z. Qu, Z. H. Yang, Angewandte Chemie-International Edition

2008, 47, 3973.

(3) T. Chen, P. J. Colver, S. A. F. Bon, Advanced Materials 2007, 19, 2286.

(4) B. P. Binks, Current Opinion in Colloid & Interface Science 2002, 7, 21.

(5) P. Pieranski, Physical Review Letters 1980, 45, 569.

(6) J. Eastoe, A. Rankin, R. Wat, C. D. Bain, International Reviews in Physical

Chemistry 2001, 20, 357.

(7) M. Abkarian, A. B. Subramaniam, S. H. Kim, R. J. Larsen, S. M. Yang, H. A. Stone,

Physical Review Letters 2007, 99.

(8) B. P. Binks, T. S. Horozov, Angewandte Chemie-International Edition 2005, 44,

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(10) R. G. Alargova, D. S. Warhadpande, V. N. Paunov, O. D. Velev, Langmuir 2004,

20, 10371.

(11) A. B. Subramaniam, M. Abkarian, H. A. Stone, Nature Materials 2005, 4, 553.



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(13) Z. H. Nie, J. Il Park, W. Li, S. A. F. Bon, E. Kumacheva, Journal of the American


(14) T. Thorsen, R. W. Roberts, F. H. Arnold, S. R. Quake, Physical Review Letters

2001, 86, 4163.

(15) J. S. Song, L. Chagal, M. A. Winnik, Macromolecules 2006, 39, 5729.



(17) P. V. Danckwerts, Gas-Liquid Reactions, McGraw-Hill, New York 1970.

(18) B. P. Binks, R. Murakami, S. P. Armes, S. Fujii, A. Schmid, Langmuir 2007, 23,

8691.

(19) T. Madhavi, A. K. Golder, A. N. Samanta, S. Ray, Chemical Engineering Journal

2007, 128, 95.

(20) K. Golemanov, S. Tcholakova, P. A. Kralchevsky, K. P. Ananthapadmanabhan, A.

Lips, Langmuir 2006, 22, 4968.

(21) X. M. Qi, S. J. Yao, Y. X. Guan, Biotechnology Progress 2004, 20, 1176.

(22) W. H. Li, H. D. H. Stover, Journal of Polymer Science Part a-Polymer Chemistry

1999, 37, 2899.

(23) J. D. Tice, H. Song, A. D. Lyon, R. F. Ismagilov, Langmuir 2003, 19, 9127.

(24) D. Dendukuri, K. Tsoi, T. A. Hatton, P. S. Doyle, Langmuir 2005, 21, 2113.

(25) S. Q. Xu, Z. H. Nie, M. Seo, P. Lewis, E. Kumacheva, H. A. Stone, P. Garstecki, D.

B. Weibel, I. Gitlin, G. M. Whitesides, Angewandte Chemie-International Edition 2005, 44,

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(26) T. S. Horozov, R. Aveyard, B. P. Binks, J. H. Clint, Langmuir 2005, 21, 7405.

(27) M. E. Leunissen, A. van Blaaderen, A. D. Hollingsworth, M. T. Sullivan, P. M.

Chaikin, Proceedings of the National Academy of Sciences of the United States of America

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18, 2177.

85

Chapter 5

Bubbles Encapsulated with a Shell of Biopolymers

Reproduced with permission from Macromolecular Rapid Communications, 2010, 31,

222. Copyright 2010, Wiley-VCH.

This chapter describes a microfluidic route to producing small (<10 μm) bubbles with a

narrow size distribution and a long-term stability. The bubbles were encapsulated with a

protein-polysaccharide shell. The strategy includes the following events occurring in

sequence: i) microfluidic generation of bubbles comprising a mixture of CO2 and a minute

amount of gases with limited solubility. The bubbles were dispersed in an aqueous solution

of lysozyme and alginate; (ii) the dissolution of CO2 leading to the shrinkage of bubbles

and an increase in acidity of the medium surrounding the bubbles; (iii) the deposition of

lysozyme at the gas-water interface and electrostatically-driven complexation of alginate

with lysozyme on the surface of bubbles.

5.1 Introduction

Bubbles with a diameter in the range from 1 to 100 μm that are encapsulated with

biopolymers (e.g, proteins, polysaccharides, or lipids) find applications in biomedical

research,1,2 and in the food and cosmetics industries.3,4 Recently, interesting applications in

simultaneous ultrasonic imaging and targeted drug delivery have emerged for small (<10

μm) bubbles.5 Ultrasound imaging originates from the ability of bubbles to improve the

86

scattering of applied ultrasound wave.6 The drug delivery function is achieved by

intravenously injecting bubbles carrying a drug in the shell and applying a focused

ultrasound excitation at a particular site. The disruption of the bubbles leads to the release

of the drug.7 The requirements to bubbles used for biomedical purposes include long-term

stability, biocompatibility and control over bubble dimensions and size distribution. The

most challenging requirement is the long-term stability of bubbles. Generally, bubbles

continuously change their dimensions due to the intrinsic kinetic and thermodynamic

instabilities. Coalescence of bubbles occurs when the thin film separating two adjacent

bubbles ruptures.10 Dissolution of bubbles occurs because surface tension at the gas-liquid

interface generates the excess Laplace pressure, ΔP = 2σ/r, where ΔP is the difference in

pressure inside and outside the bubble, σ is the surface tension at the gas-liquid interface,

and r is the radius of bubble. Since the value of ΔP rapidly increases as r decreases, the

chemical potential of the bubbles increases and so does the solubility of the gas. Therefore,

bubbles with smaller sizes have a higher propensity to dissolution. Furthermore, bubbles

with a broad size distribution undergo Ostwald ripening: due to the higher solubilty, gas

molecules from smaller bubbles dissolve and diffuse into larger bubbles. This leads to the

growth of larger bubbles at the expense of the shrinkage of smaller bubbles.11

Generally, the stabilization of bubbles is achieved by adsorbing surfactants to their

surface, in order to achieve electrostatic or steric repulsion between the bubbles and to

decrease the value of interfacial tension (σ). Although conventional low-molecular weight

surfactants provide a barrier to coalescence, they do not prevent bubble dissolution due to

their inability to form a robust surface layer on the bubble surface. In addition, surfactants

rapidly desorb from the gas-liquid interface.12 (we note that it was recently reported that

87

patches of sucrose stearate crystallized on the bubble surface can stop gas dissolution,

thereby providing long term bubble stability.13)

Alternatively, bubbles can be stabilized with lipids or biopolymers such as proteins or

polysaccharides.14 Typically, bubbles encapsulated with biological species are prepared by

sonication or mechanical agitation.8,9 These methods produce bubbles with dimensions in

the size range from one micrometer to tens of micrometers and a broad distribution of

size.1,8,9

Due to their amphiphilic nature, proteins rapidly adsorb to the gas-water interface

where they undergo partial unfolding and form the interfacial layer with high a mechanical

strength.14,15 In practice, however, protein layers alone do not provide a sufficiently strong

barrier to the dissolution of bubbles, unless protein unfolding is enhanced by adding

chemical agents (e.g. reducing agents) or by applying heat.11,14,16,17

Significant enhancement of bubble stability is achieved by co-adsorbing on the bubble

surface of a mixture of proteins and polysaccharides. Electrostatically driven complexation

between cationic proteins and anionic polysaccharides increases the thickness and the

strength of the adsorbed layer at the gas-water interface.18,19 In this approach, the value of

pH and ionic strength of the medium, as well as the charge distribution on the biopolymer

molecules, play an important role in bubble stabilization.20

In this chapter, a microfluidic (MF) route to the generation of small, stable,

monodisperse bubbles encapsulated with a lysozyme-alginate shell is described. The

approach relies on three events occurring in sequence. (i) A MF generation of bubbles from

gaseous CO2 containing a small amount of gas with a low solubility in water. The bubbles

are produced in an aqueous solution of lysozyme and sodium alginate. (ii) A rapid and

uniform dissolution of CO2 leading to the reduction in bubble size and the decrease in pH

88

of the aqueous medium in the vicinity of the bubbles. (iii) The deposition of positively

charged lysozyme at the gas-water interface and its complexation with anionic sodium

alginate.

We show that a MF method provides the ability to generate bubbles with a controlled

size, narrow size distribution, and a high (> 1 month) stability when stored in a sealed

container. At a constant gas pressure the ultimate size of the bubbles is determined by the

flow rate of the continuous phase and the concentration of lysozyme and alginate. The

method has the productivity of up to 4x104 bubbles/min and in order to generate small

bubbles, it does not require a significant reduction of microchannel dimensions.


The materials and methods described in the present chapter are listed in Chapter 2.

5.2.1 Preparation of Bubbles Encapsulated with a Mixture of Biopolymers

Figure 5.1 shows the schematic of the formation of monodisperse bubbles encapsulated

and stabilized with a biopolymer shell. In Step 1, bubbles are generated from the gaseous

CO2 mixed with 0.2 vol % of the gas with low solubility in water. The bubbles are

generated in an aqueous solution of lysozyme and alginate at an initial pH of 12. At this pH,

both lysozyme and alginate are negatively charged.21 In Step 2, CO2 dissolves in the

continuous aqueous phase and the pH value of the solution surrounding the bubbles reduces.

The lysozyme molecules acquire a positive net charge (for this protein the isoelectric point

pI =11)22,23 and adsorb to the negatively charged surface of the bubbles.24 In Step 3, the

89

anionic alginate molecules (pKa= 3.5)25 form a complex with the positively charged

lysozyme on the surface of bubble. The complexation yields a biopolymer shell

encapsulating the shrunken bubbles. The ultimate size of the bubbles is controlled by their

original size, the extent of CO2 dissolution, the stabilizing effect of the biopolymer shell,

and the fraction of the low-water soluble gas in the gaseous CO2.

Figure 5.1 Schematic of co-adsorption of lysozyme and alginate on the surface of CO2 bubble during its dissolution.

Figure 5.2A shows a schematic of the planar MF flow-focusing device used to form

biopolymer-encapsulated bubbles. The CO2 bubbles were generated in the geometrically

controlled regime and had a discoid-like shape since the initial diameter of unperturbed

bubble was larger than the height of microchannel. Polydispersity of the bubbles was

below 5%.26 Figure 5.2B shows typical optical microscopy images of the bubbles, which

were acquired immediately after bubble formation in the aqueous solution of lysozyme and

sodium alginate (Figure 5.2B, top) and following bubble shrinkage in the downstream

microchannel ca. 3 sec after their formation (ca. 100 mm from the orifice of the bubble

90

generator) (Figure 5.2B, bottom). At this point, the dimensions of the bubbles were

stabilized due to the saturation of the continuous aqueous phase with CO2.27 By comparing

the initial and the stabilized volume of the bubbles, we estimated that the fractional

change in bubble volume was approximately 80 vol %.

Figure 5.2 A) Schematic of a MF flow-focusing bubble generator, B) Representative optical microscopy images of the generation (top) and dissolution of CO2 bubbles in a MF channel (bottom). The image is taken 250 mm away from the orifice of the MF device. The width and height of the orifice are 50 and 120 μm, respectively. PCO2=48.3 kPa, QL=6 mL/h. Clys=Calg=0.2 wt. %, pH=12. Scale bar is 200 μm.

Under strongly basic conditions used in the present work, the dissolution of CO2 is

governed by Henry’s law (eq. 5.1) and chemical reactions (eq. 5.2-3):28

[CO2]l kH PCO2 (eq. 5.1)

pH>10: CO2 + OH- HCO3- (K = 3.2x 107) (eq. 5.2)

HCO3- + OH- CO3

2- + H2O (K = 3.5x 103) (eq. 5.3)

where K is the equilibrium constant and [CO2]l, kH, and PCO2 are the concentration of

molecularly dissolved CO2, Henry’s law constant (for CO2 at 25oC kH=3.2x10-4 mol/(L

kPa)), and the gas pressure, respectively.

91

The dissolution of CO2 led to the acidification of the liquid surrounding bubbles.29

The aqueous solution collected at the exit of the MF device acquired pH of

approximately 8. The bubbles were collected at the outlet of the MF device and stored

in a sealed container.

5.2.2. Long-term Stability of Bubbles

The dimensions of collected bubbles in a sealed container further reduced within 2

h, due to the change of CO2 pressure to ambient condition (Figure 5.3A-C). In the

microchannel, CO2 bubbles experienced a pressure of 48.3 kPa, while partial CO2

pressure at 1 atm (outside the device) was 0.04 kPa.30 At this point, the fractional

change in bubble volume was 99.8 %, which implied that all CO2 was removed from the

bubbles and their size was determined by the remaining volume of low water soluble

impurities. Following this initial shrinkage, there was no noticeable change in the

dimensions of the bubbles during up to 4 weeks of storage in a sealed contained (Figure

5.3C-F).

To explore the role of pH reduction induced by CO2 dissolution, we conducted a

control experiment by generating N2 bubbles with a narrow size distribution in the

aqueous solution of lysozyme and alginate. After 24 h, the bubbles of N2 showed a broad

distribution of sizes dominated by their coalescence (Figure 5.4). This result suggested that

the adsorption of lysozyme governed by the amphiphilic nature of this protein was not

sufficient for bubble stabilization. This indicated the importance of the change in pH caused

by the dissolution of CO2.

92

Figure 5.3 A-C) Optical microscopy images of the bubbles at the exit of the microchannel (inset shows the image of initial bubble at the orifice, scale bar: 100 μm) (A), after 0.3(B), 2 (C), 24 (D) and 720 h (E) storage. The bubbles were formed at PCO2=48.3 kPa and QL=6 mL/h. Clys=Calg=0.2 wt. %. Scale bars: 50 μm. D) Change in the diameter of bubbles plotted as a function of time; the dashed line separates on-chip (left) and off-chip (right) bubble storage.

93

Figure 5.4 N2 bubbles after 24 h storage. Scale bar: 200 μm. N2 bubbles generated at PN2=48.3 kPa, QL=6 mL/h, pH=12, Clys=Calg=0.2 wt. %.

5.2.3 Control of Bubble Dimensions

Control over bubble dimensions was achieved (i) by varying the flow rate (QL) of the

continuous aqueous phase at constant PCO2; (ii) by changing the concentration of alginate

(Calg), and (iii) by concentration of lysozyme (Clys) (Figure 5.5-7).

Figure 5.5A shows the effect of QL on the initial and final diameters (Di and Df,

respectively) of the bubbles (the value of Df was defined as the diameter of bubbles after

their 24 h storage in a sealed container). The value of Di decreased with increasing QL,

which subsequently, led to the reduction in Df (Figure 5.5B-D).31

94

Figure 5.5 A) Effect of QL of the continuous aqueous phase on the initial (Di) and final dimension (Df) of bubbles. PCO2=48.3 kPa, Clys=Calg=0.2 wt. %, B-D) Optical microscopy images of the bubbles after 24 h (insets show the image of initial bubble at the orifice, scale bars: 100 μm) at QL=4.5 (B), 5.5 (C) and 6.5 mL/h (D). Scale bars: 15 μm

Figure 5.6 shows the variation in the values of Di and Df plotted as a function of Calg.

The initial size of the CO2 bubbles decreased with increasing Calg, due to the increase in the

viscosity of the continuous phase.31.32 As expected, the smaller Di resulted in the smaller Df

(Figure 5.6B-D). We note that the encapsulation of bubbles with lysozyme alone (at Calg= 0

wt.%) did not protect the bubbles from coalescence and/or Ostwald ripening (Figure 5.6B).

Presumably, lysozyme did not form a dense layer on the surface of bubbles, owing to

repulsive interactions between the cationic protein molecules.33

95

Figure 5.6 A) Effect of Calg on the initial and final dimensions of bubbles, PCO2=48.3 kPa, QL=6 mL/h. Clys=0.2 wt. %, B-D) Optical microscopy images of the bubbles after 24h (insets show the image of initial bubble at the orifice, scale bars: 100 μm) at Calg=0 (B, scale bar: 30 μm), 0.15 (C) and 0.2 wt. % (D). Scale bars: 15 μm

The effect of the variation in Clys on the initial and final dimensions of the bubbles is

shown in Figure 5.7A. We note that the bubbles coated with alginate only (at Clys= 0 wt. %)

completely dissolved after 24 h storage (Figure 5.7B). The value Di of the bubbles

decreased when Clys increased, owing to the higher viscosity of the continuous phase.31,34

however, the final size of bubbles, Df, increased (Figure 5.7B-D). The latter effect was

caused by the higher surface concentration of lysozyme at the gas-liquid interface35 and the

formation of a more robust barrier to the dissolution of bubbles.

96

Figure 5.7 A) Effect of Clys on the initial and final dimensions of bubbles, PCO2=48.3 kPa, QL=6 mL/h. Calg=0.2 wt. %, B-D) Optical microscopy images of the bubbles after 24h (insets show the image of initial bubble at the orifice, scale bars: 100 μm) at Clys=0 (B), 0.05 (C) and 0.15 wt. % (D). Scale bars: 15 μm

Figure 5.8 The preparation of 7 μm-diameter bubbles produced at PCO2=72.4 kPa, QL=10.5 mL/h. Clys=0.05 wt. % and Calg=2 wt. %. Optical microscopy image of the bubbles after 24 (A). (inset: the initial CO2 bubble at the orifice. scale bar: 80 μm) and 720 h (B). Scale bars: 15 μm.

97

Overall, these results indicate the crucial role of both lysozyme and alginate in the

stabilization and control of the final dimension of bubbles. At the optimized concentration,

the lysozyme shell controlled partial CO2 dissolution thereby controlling the value of Df,

while alginate contributed in the formation of the robust shell protecting bubbles from

coalescence. Figure 5.8A shows 7 μm-diameter biopolymer-encapsulated bubbles

generated from the bubbles with the initial diameter of 135 μm (inset) at Clys=0.05 wt.

% and Calg=0.2 wt. %. The 7 μm-diameter bubbles retained their size distributions for

more than a month (Figure 5.8B).

5.2.4 Characteristic Properties of the Bubbles

The biopolymer-encapsulated bubbles were imaged using laser confocal fluorescence

microscope (LCFM) and scanning electron microscope (SEM). In the first series of

experiments, we used the autofluorescence of lysozyme (attributed to the presence of

tryptophan).36 A typical LCFM image of the encapsulated bubble in Figure 5.9A shows a

well-defined bright fluorescent shell with the thickness of ca. 1 μm, which surrounded a

dark gaseous core. The cryo-SEM imaging carried at -20oC revealed the surface and

internal structure of the biopolymer shell (Figure 5.9B-D). A few micrometer-thick strands

surrounding the bubble appear due to the complexation of lysozyme and alginate in the

continuous phase (Figure 5.9B). The images of both the surface of the shell and of the inner

layer, adjacent to the bubble surface, showed the presence of aggregates of lysozyme

(Figure 5.9C, D).37 In particular, the lysozyme layer was composed of 500~700 nm-size

spherical aggregates.

98

Figure 5.9 A) LCFM image of the bubble encapsulated with a lysozyme-alginate shell and stored for 10 days (PCO2=48.3 kPa, QL=6 mL/h, Clys=Calg=0.2 wt. %). The focal plane is located at 8 μm below the surface of the bubble. B) SEM images of the bubble produced under the same conditions as in A). In A) and B) the scale bar is 7 μm. C) A high magnification SEM image of the surface of the bubble shown in (B). The scale bar is 2 μm. D) SEM image of the fractured bubble surface. The bubble was stored for 28 days. The scale bar is 4 μm.

We examined the change in the secondary structure of lysozyme deposited on the

surface of a bubble. Figure 5.10 shows the CD (circular dichroism) spectra of the aqueous

solution of lysozyme and the bubbles encapsulated with a lysozyme-alginate shell. All

spectra were acquired in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)

buffer solution at pH=8. Although the intensity profile differed compared to the native

lysozyme, the dispersion of bubbles stored for 5 days showed the minimum negative peak

99

at ca. 207 nm which is characteristic of the secondary structure of lysozyme.16,38 After 28

day storage, the CD spectra showed the shift in the minimum negative peak to around 215

nm, which occurs when the conformation of the protein changes to a β-sheet rich

structure.39

Figure 5.10 Comparison of CD spectra of the native lysozyme solution (Clys=0.003 wt. %) (━) and the bubbles encapsulated with a lysozyme-alginate shells (,). The bubbles were stored for 5 days () and 28 days (). Conditions of bubble formations: PCO2=48.3 kPa, QL=5 mL/h, Clys=Calg=0.2 wt. %.

We further investigated on the organization of the β-sheet structure using cross-

polarization microscopy. The Maltese-cross pattern was observed for the lysozyme-alginate

shells following 28 day bubble storage (Figure 5.11A). This pattern was previously

observed for spherical aggregates with the radial arrangement of amyloid fibrils whose

internal structure is largely cross β-sheet structure, at low pH or elevated temperature

conditions.40 However, since in our work the dimensions of the spherical aggregates in the

bubble shell were only 500~700 nm (as in Figure 5.9D), we assumed that the spherical

aggregates consists of short fragments of amyloid fibrils.41 To verify the existence of

100

fragments of amyloid fibrils in the biopolymer shell of the bubbles stored for 28 days, we

performed a Thioflavin-T (ThT) dye binding test. The binding of the dye molecule to

amyloid fibril results in the enhancement of fluorescence intensity by restricting the free

rotation of the dye. 42 An aliquot of the bubble dispersion was added into the 50 μM ThT

solution at pH=8. In comparison with control experiments with a freshly prepared

lysozyme-ThT solution, alginate-ThT solution and a stock ThT solution, we observed the

increase in fluorescence intensity (Figure 5.11B), which confirmed the existence of

fragmented amyloid fibrils on the bubble surface.40,41

Figure 5.11 A) Polarization optical microscopy image of the bubbles coated with a lysozyme-alginate shell. The bubbles were produced under PCO2=48.3 kPa, QL=6 mL/h, Clys=Calg=0.2 wt. % and stored for 28 days. The scale bar is 50 μm. B) Fluorescence intensity profile of 50 μM ThT solution (), freshly prepared lysozyme (Clys=0.003 wt. %) dissolved in 50 μM ThT solution (), freshly prepared alginate (Calg=0.003 wt. %) dissolved in 50 μM ThT solution () and the dispersion of biopolymer-encapsulated bubbles in 50 μM ThT solution (). The bubbles were stored for 28 days.

5.3 Conclusion

The potential applications of bubbles in biomedical field are continually growing as

novel formulations and methods appear. Bubbles in an appropriate size range (below 10

μm) provide a unique range of responses to ultrasound, which makes them useful for

101

ultrasound contrast imaging, identifying molecular expression and targeting drugs to

specific tissue sites.1,2,7 Advances in the understanding of physicochemical properties has

led to the recent development of bubbles encapsulated with various shell materials

including polymer brushes, polyelectrolyte multilayers, and nanoparticles.7

In the proposed approach, a uniform dissolution of the CO2 bubbles generated by the

MF approach provides the ability to produce small (~7 μm) bubbles and encapsulate them

with a protein–polysaccharide shell. The polymers used for bubble encapsulation are

biocompatible and have good mechanical properties, which is crucial for the above-

mentioned biomedical applications.1,7 The bubbles have a controllable size, a narrow size

distribution, and a long-term stability. Further reduction in bubble size to 2-3 μm can be

achieved in the microfluidic devices with ca. 20 % smaller size of features of the MF

device. Further increase in productivity of the device can be achieved by using multiple

modular MF devices.43 Moreover, the described method will allow further functionalization

of the biololymer shell by conjugating it with e.g., peptides or incorporating in it

therapeutic agents, magnetic or fluorescent species.17,44,45

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Chapter 6

A Single-step Microfluidic Route to Producing Multifunctional

Microbubbles

This chapter describes a single-step microfluidic approach to producing small and

stable bubbles functionalized with nanoparticles (NPs). The strategy includes the following

events occurring in sequence: (i) a microfluidic generation of bubbles from a mixture of

CO2 and a minute amount of gases with low solubility in water, in an aqueous solution of a

protein, a polysaccharide and NPs; (ii) rapid dissolution of CO2 leading to the shrinkage of

bubbles and an increase in acidity of the medium in the vicinity of the bubbles; and (iii) co-

deposition of the biopolymers and NPs at the gas-liquid interface. The proposed approach

yields microbubbles with a narrow size distribution, long term stability and multiple

functions originating from the attachment of metal oxide, metal, or semiconductor NPs onto

the bubble surface. We show the potential applications of these bubbles in ultrasound and

magnetic resonance imaging.

6.1 Introduction

Ultrasound (US) imaging is non-invasive, safe and cost-effective.1 The limitation of

this method is its low contrast, in comparison with other imaging techniques such as X-ray

tomography or magnetic resonance imaging (MRI).2 To address this shortcoming,

microbubbles are utilized as US imaging contrast agents. Microbubbles enhance

backscattered acoustic signal, due to the large impedance mismatch between the bubbles

and the living tissue, and enable resonant scattering.1,3,4 Further improvement in the

105

accuracy of clinical assessment of a disease can be achieved by combining US with MRI or

fluorescence imaging.5-10 These imaging modalities are realized by immobilizing magnetic

or semiconductor nanoparticles (NPs) on the bubble surface.9,10 Attachment of NPs to the

bubble surface also increases the contrast in US imaging, owing to enhanced asymmetric

bubble oscillations.1,11 In addition to diagnostics, microbubbles carrying drug-loaded NPs

have promising therapeutic applications: bubbles could be disrupted by a localized US to

release therapeutic agents at the target site.12-15

Currently, microbubbles coated with NPs are produced in a multi-step procedure by

sonicating a mixture of NPs and oil in the presence of a gas and subsequently transferring

the resulting bubbles into an aqueous medium.9,16 Alternatively, NPs are attached to the

surface of bubbles in a layer-by-layer (LbL) deposition process.10,17 These time-consuming

processes yield bubbles with dimensions in the size range between one and tens of

micrometers and a broad distribution of sizes.18 Recently, a microfluidic (MF) strategy

enabled the synthesis of monodisperse NP-loaded bubbles via the formation of double

emulsions.19 The method required very careful tuning of the surface energies at the gas-

liquid and liquid-liquid interfaces.19,20 In addition, the dimensions of bubbles exceeded 10

µm, the size utilized in US diagnostics or therapeutics.21

This chapter decribes a new MF approach to producing NP-coated microbubbles with a

narrow size distribution and a long-term stability. The proposed approach exploits the

following events, occurring concurrently within 3 sec: (i) the MF generation of

monodisperse bubbles from a mixture of CO2 and a minute amount of a gas with a low

solubility in water, in an aqueous solution of lysozyme, alginate and anionic NPs; (ii) the

controllable dissolution of CO2 leading to bubble shrinkage and an increase in acidity of the

medium in the neighbourhood of the bubbles. The decrease in pH renders positive net

106

charge to lysozyme; and (iii) the adsorption of cationic lysozyme to the negatively charged

surface of bubbles followed by the deposition of anionic alginate and NPs onto the

lysozyme layer. This rapid and simple process generates bubbles with polydispersity not

exceeding 6 % and stability of at least, 3 months. We show the ability to produce 5 μm-

diameter bubbles, however for convenience of optical imaging we present most of the

results for bubbles with a mean diameter of approximately 10 μm. Bubbles with such

dimensions, comparable to red blood cells, can safely pass through the microvasculature

without diffusing across the endothelium.22 We demonstrate the generality of the MF

approach by attaching to the bubble surface metal oxide, metal and semiconductor NPs.

Furthermore, we show the applications of the NP-functionalized bubbles as imaging agents

in US, fluorescence and MRI.


The materials and methods described in the present chapter are listed in Chapter 2 and

Appendix.

6.2.1 Experimental Design

Figure 6.1A shows the schematic of the MF device used in the present work. A MF

flow-focusing bubble generator23 was followed by a serpentine downstream microchannel

channel (Appendix, Figure A6.1). The CO2 gas mixed with 0.2 vol % of N2, O2, He, and

Ne supplied under pressure PCO2 of 48.3 kPa to the central channel. An aqueous solution

107

containing a mixture of lysozyme, alginate and anionic NPs (Fe3O4, Au or SiO2-

encapsulated CdSe/ZnS NPs) was introduced into the two side channels as a continuous

phase using a syringe pump at the flow rate, QL, varying from 6.5 to 9.5 mL/h. The value of

pH=12 of the continuous phase was achieved by adding to it a 1M NaOH solution.

Formation of bubbles occurred via a periodic breakup of the gas thread in the orifice of

the MF device.23 Since the unperturbed diameter of the bubbles immediately after their

formation was larger than the height of the downstream channel, they acquired a discoid

shape (Figure 6.1A, top inset). In the downstream channel the dimensions of the

bubbles dramatically reduced, due to the dissolution of CO2, and acquire a spherical

shape (Figure 6.1A, bottom inset).

Figure 6.1 Schematics of the microfluidic (MF) generation of multifunctional bubbles. (A) Schematic of a MF reactor. The height of the MF device is 120 µm. The width of the orifice and the length of the downstream microchannel are 50 µm and 260 mm, respectively. The top and the bottom insets show optical microcopy images of the bubbles at the beginning and the end of the process, respectively. Bubbles were generated at PCO2=48.3 kPa and QL=9.5 mL/h. Scale bars in insets are 200 µm. (B) Schematic of the formation of NP functionalized bubbles stabilized with a mixed lysozyme-alginate layer.

108

Figure 6.1B illustrates the sequence of events leading to the generation of small and

stable NP-functionalized bubbles encapsulated with the mixture of biopolymers. Rapid and

uniform shrinkage of bubbles is driven by the dissolution of CO2 and chemical reactions

of CO2 with OH- ions (see below). The dissolution of CO2 leads to the decrease in pH of

the aqueous medium in the vicinity of the gas-liquid interface (Figure 6.1B(1)). As a result

of the change in acidity, lysozyme molecules in the neighborhood of the bubbles gain a net

positive charge and adsorb to the negatively charged bubble surface (Figure 6.1B(2)). (The

isoelectric point of lysozyme is ~11).24,25 Anionic NPs and anionic alginate molecules

deposit on the cationic lysozyme-coated bubble (Figure 6.1B(3)). The formation of the

lysozyme-alginate shell on the surface of bubbles is described in greater detail in Chapter 5.

An important feature of our work was the ability to control the dimensions of

bubbles, which was achieved by generationg bubbles with a well-defined size,

controllable dissolution of CO2 and stabilization of the bubbles with a biopolymer layer.

The dissolution of CO2 in the microchannel was governed by Henry’s law (eq. 6.1), which

was combined with chemical reactions occurring at pH>10 (eq. 6.2- 3):26

[CO2]l kH PCO2

CO2 + OH- HCO3-

HCO3- + OH- CO3

2- + H2O

where K1 = 3.2x107 and K2= 3.5x103 are the equilibrium constants and [CO2]l, kH, and

PCO2 are the concentration of molecularly dissolved CO2, Henry’s law constant (for

CO2 at 25oC kH=3.2x10-4 mol/(L kPa)), and the gas pressure, respectively. The extent of

dissolution of bubbles in the MF device was controlled by the saturation of the continuous

aqueous phase, 27 and in the present work the dissolution led to ~80% of the reduction in

K1

K2

(eq. 6.1)

(eq. 6.2)

(eq. 6.3)

109

bubble volume. Bubbles were generated at a frequency of 700 bubbles/sec. The

bioencapsulated NP-functionalized bubbles were collected in a 2 mL container, where

their dimensions further decreased due to the reduction of external pressure to 1

atm.28,29 The container was sealed for long-term bubble storage.

6.2.2 Long-term Stability of Biopolymer Encapsulated-Bubbles

Functionalized with NPs

Figure 6.2A-C shows typical optical microscopy images taken at different times

after the generation of bioecapsulated bubbles loaded with Fe3O4 NPs. The

polydispersity of the bubbles did not exceed 6%. Following bubble formation, within 3

sec their mean diameter reduced from 150 to 40 μm (on-chip), and subsequently, to 5

μm within 1 h of off-chip storage. At this point, the fractional reduction in the bubble

volume reached 99.9 %, which implied that all CO2 and 0.1% of the low-soluble gases

were removed from the bubbles. No further change in bubble dimensions was observed

after 2000 h storage.

Figure 6.2 Optical microscopy images of the bubbles functionalized with Fe3O4 NPs after different storage times: (A) 3 sec, (B) 1 h and (C) 2000 h. The scale bars in (A), (B) and (C) are 50, 15 and 15 µm, respectively. Inset in (A) shows the bubble imaged immediately after its generation in the orifice of the MF device. Scale bar is 100 µm. Bubbles were generated at PCO2=48.3 kPa and QL=9.5 mL/h.

110

6.2.3 Control of the Dimensions of Biopolymer Encapsulated-Bubbles

Functionalized with NPs

The final dimensions of bubbles - defined as the diameter of bubbles after 1 h

storage – were tuned by varying the flow rate of the continuous aqueous phase (QL),

while maintaining PCO2 as 48.3 kPa. Figure 6.3A shows the variation in the initial (Di)

and the final (Df) dimensions of NP-coated bubbles, plotted as a function of the QL of

the continuous phase. The value of QL affected the final size of bubbles in two ways.

With increasing value of QL the original bubble size reduced, thereby resulting in the

decrease of Df. Second, the extent of dissolution of CO2 increased at higher values of

QL,27,30 also leading to smaller bubble size (Figure 6.3B-D).

Figure 6.3 (A) Variations in the initial,Di, and final dimensions, Df, of microbubbles are plotted as a function of the flow rate of the continuous phase, QL. (B-D) Representative optical microscope images of the initial (insets) and final dimension of bubbles coated with the biopolymers and Fe3O4 NPs at different QL s (A) 6.5, (B) 7.5 and (C) 8.5 mL/h. Clysozyme=0.05 wt. %. Calginate=0.1 wt. %, and CFe3O4 dispersion = 1 wt. %. PCO2=48.3 kPa. Scales bars are 15 µm (100 µm, insets).

111

6.2.4 Control Experiments

In the control experiments conducted in the absence of lysozyme, the bubbles were

completely dissolved within a few minutes after their generation. Without alginate in

the continuous phase the bubbles aggregated due to the insufficient electrostatic and

steric stabilization (Figure 6.4A). The importance of local acidification of the medium

was examined by generating N2 bubbles at initial pH=12. These bubbles did not have a

dense lysozyme-alginate shell and within 1 h they underwent coalescence (Figure

6.4B).

Figure 6.4 Optical microscope images of (A) aggregated bubbles generated with lysozyme and Fe3O4 NPs. Scale bar is 15 µm and (B) coalesced N2 bubble generated with lysozyme, alginate and Fe3O4 NPs. Scale bar is 200 µm.

6.2.5 Characterization of Biopolymer Encapsulated-Bubbles

Functionalized with various NPs

The NP-coated bubbles were collected at the outlet of the MF device, washed several

times with de-ionized water, dried and imaged using scanning transmission electron

microscopy (STEM). The STEM images of the bubbles coated with Fe3O4, Au and SiO2-

encapsulated CdSe/ZnS NPs are shown in Figure 6.5A-C, respectively. We note that the

CdSe/ZnS NPs were encapsulated within a SiO2 shell, in order to suppress the cytotoxicity

of these NPs.31 Upon drying, the bubbles maintained a spherical shape and featured

112

wrinkles on their surface (see e.g., Figure 6.5A). In the high magnification images (insets in

Figure 6.5A-C), the NPs appeared bright on the dark background of the lysozyme-alginate

biopolymer shell. The Au and SiO2-encapsulated CdSe/ZnS NPs were well-separated,

however Fe3O4 NPs tend to form clusters in the biopolymer shells.

Figure 6.5 Scanning transmission electron microscopy (STEM) images of the bubbles coated with the lysozyme-alginate shell and (A) Fe3O4 NPs, (B) Au NPs, and (C) SiO2-encapsulated CdSe/ZnS NPs, Scale bars are 6 µm. Insets in (A-C) show corresponding high magnification images of the surface of the bubbles. Scale bars in insets are 150 nm. (D-F) Energy dispersive X-ray (EDX) spectrometry line scanning profiles for the system shown as a red line in (A), (B) and (C). Bubbles were generated at PCO2 = 48.3 kPa and QL = 8.5 mL/h.

In each system, the presence of NPs on the surface of bubbles was verified by energy

dispersive X-ray (EDX) analysis. Figure 6.5D-F shows the results of EDX analysis of the

surface of bubbles, based on the major constituent of the NPs. The EDX line profiles

featured strong EDX signals of Fe, Au and Si (Appendix, Figure A6.2-4).

113

6.2.6 Control over the Amount of NPs on the Bubble Surface

Control over the amount of NPs deposited on the surface of bubbles was achieved by

varying the concentration of NPs in the continuous phase. Figure 6.6A-C shows the STEM

images of the surface of bubbles coated with a different amount of Fe3O4 NPs. When the

initial concentration of the NPs in the continuous phase was 1.0x1012, 4.0x1012 or 1.0x1013

NPs/mL, their density on the surface of bubbles was 1.5x105, 6.6x105, and 1.5x107

NPs/µm2, respectively (Figure 6.6A-C).

Figure 6.6 (A-C) STEM images of the surface of bubbles coated with Fe3O4 NPs at surface density of (A) 1.5x105, (B) 6.6x105 and (C) 1.5x106 NPs/µm2. Scale bars are 300 nm. Insets show the corresponding bubbles. Scale bars are 3 µm. Bubbles were generated at PCO2=48.3 kPa and and QL=8 mL/h.

6.2.7 Properties of NP-functionalized Bubbles

The NP-functionalized bubbles had a narrow size distribution with polydispersity not

exceeding 6 %. The bubbles coated with Fe3O4 NPs were readily aligned in chains when

manipulated by an external magnetic field (Figure 6.7A).32 The chains of bubbles moved

towards a magnet at a velocity of ~20 µm/s. The bubbles did not lose their ability for

magnetic actuation for at least, 3 month-long storage (Appendix, Figure A6.5).

Figure 6.7B shows the extinction spectra of the aqueous dispersion of bubbles coated

with Au NPs and of the original citrate-stabilized Au NPs in an aqueous solution. The

114

incorporation of Au NPs within a biopolymer shell led to a ~13 nm red shift of the surface

plasmon resonance bands, due to interparticle electromagnetic coupling.33,34

Figure 6.7C shows a typical confocal fluorescence microscopy image of the bubbles

coated with SiO2-encapsulated CdSe/ZnS NPs. The shell of bubbles showed strong

fluorescence when excited at 364 nm (Appendix, Figure A6.6). Figure 6.7C, inset shows a

gaseous core (dark) and a bright fluorescent shell of an individual bubble.

Figure 6.7 Properties of NP-coated bubbles. (A) Magnetic actuation of bubbles functionalized with Fe3O4 NPs. Scale bar is 50 µm. Bubbles were generated at PCO2=48.3 kPa and and QL=8.5 mL/h. (B) Extinction spectra of Au NPs (red spectrum, top) and of the bubbles coated with these Au NPs (blue spectrum, bottom). Bubbles were generated at PCO2=48.3 kPa and and QL =8.5 mL/h. (C) Confocal fluorescence microscopy image of bubbles carrying SiO2-encapsulated CdSe/ZnS NPs. Scale bar is 30 µm. λex=364 nm. Inset shows an image of the individual bubble. Scale bar is 5 µm. Bubbles were generated at PCO2=48.3 kPa and and QL=9.5 mL/h.

6.2.8 Application of NP-functionalized Bubbles in US Imaging

Aqueous dispersions of the bubbles were introduced in the Opticell chamber (Thermo

Scientific Inc.) and imaged in a bubble-specific, nonlinear imaging mode using a clinical

US system (iU22, Philips, Appendix). The nonlinear US signal was caused by asymmetric

(compression vs. expansion) oscillations of the bubbles in response to US excitation. Figure

6.8A-D shows in-vitro US images of the dispersion of NP-free and NP-functionalized

bubbles. A stronger contrast in US images in Figure 6.8B-D signified a stronger nonlinear

signal enhancement for the NP-functionalized bubbles, in comparison with NP-free bubbles

115

in Figure 6.8A. Quantitatively, signal enhancement for the NP-coated bubbles is presented

in Figure 6.8E. The signals of the bubble dispersions were compared to the background

signal of water. The enhancement was calculated as the ratio of the integrated power

measured in the chamber filled with the dispersion of bubbles to the integrated power

measured for the chamber containing pure water. The bubbles functionalized with Fe3O4,

Au and SiO2-encapsulated CdSe/ZnS NPs showed 24.1, 32.7 and 34 dB signal

enhancements, respectively, in comparison with 21.7 dB signal enhancement measured for

the NP-free bubbles (Figures 6.8B-D). We ascribe the increase in the US signal to the

enhanced non-linearity in oscillations of the NP-coated bubbles, owing to the increased

microbubble resistance to compression. This effect is strongly influenced by the surface

coverages of bubbles with NPs.11,16,35 The stronger nonlinear US signal measured for the

bubbles carrying Au or SiO2-encapsulated CdSe/ZnS NPs, in comparison with the bubbles

carrying Fe3O4 NPs supports this explanation. Based on the STEM image analysis, the

surface coverages of the bubbles with Au and SiO2-encapsulated CdSe/ZnS NPs were

three- and five-fold higher, respectively, than that for the bubbles coated with Fe3O4 NPs.

We note that the role of different types of NPs on US signal is unknown. We conclude that

the deposition of NPs onto the bubble surface increased the detectability of bubbles with

contrast-specific US pulse sequences which were designed to reject the linear signal

components arising from the tissue and bubbles and to preserve the nonlinear components

from the bubbles.1,34

The NP-coated bubbles did not lose their echogenicity under continuous exposure to low-

power US pulses (130 kPa at 5 MHz) which suggested an excellent stability of the

lysozyme-alginate shell with embedded NPs. Under increased peak negative US pressure of

1100 kPa, the bubbles were disrupted. This result implied that the NP-loaded bubbles can

116

be controllably disrupted with US and the NPs can be released and deposited at the target

sites under US image guidance.

Figure 6.8 In-vitro US imaging of the dispersion of dispersion of biopolymer-coated bubbles at 90 % receive gain. The dispersion is placed in the Opticell chamber. (A) NP-free bubbles, (B-D) bubbles coated with Fe3O4 NPs (B), Au NPs (C) and SiO2-encapsulated CdSe/ZnS NPs (D). (E) US signal enhancement over background for the systems shown in A-D. The concentration of bubbles in all systems was 104 bubbles/mL.

6.2.9 Application of NP-functionalized Bubbles in MRI

We examined the application of the Fe3O4 NP-coated bubbles in MRI, a method

providing a high spatial resolution. The NP-coated bubbles with the surface density of 0,

1.5x105, 6.6x105 and 1.5x106/µm2 were suspended in water and placed in 1.8 mL

Eppendorf tubes. The dispersions were imaged at 3.0 Tesla with a 2D coronal fast field

echo sequence (Philips Healthcare, Andover, MA, Appendix). Figure 6.9A shows that the

bubbles carrying a higher amount of Fe3O4 NPs exhibited enhanced negative contrast,

117

reflected as the darker area in the center of the Eppendorf tubes.36,37 Figure 6.9B shows the

increase in relaxation rate (1/T2*) for dispersions containing bubbles with a higher surface

density of Fe3O4 NPs. The NPs modified the MRI signal by locally perturbing the magnetic

field and thereby leading to the negative signal enhancement by increasing the T2*

relaxation rate of the nearby water molecules.37,38 These results indicate that the Fe3O4 NP-

coated bubbles can serve as effective MRI contrast agents.

Figure 6.9 (A) In-vitro MRI images (top-view) of the dispersions of biopolymer-encapsulated bubbles coated with different amounts of Fe3O4 NPs. The images were obtained at 6.9 ms echo time. (B) Variation in T2* relaxation rate plotted as a function of surface density of Fe3O4 NPs on the surface of bubbles. The concentration of bubbles in all systems was 104 bubbles/mL.

6.3 Conclusions

In conclusion, we have developed a single-step, simple MF method for producing

microbubbles functionalized with various types of NPs. The method has the productivity of

4x104 bubbles/min. The higher productivity can be achieved by using multiple modular MF

devices. The bubbles exhibit small size, a low polydispersity and long-term stability. The

functionalization of the surface of bubbles rendered the bubbles with plasmonic,

fluorescence and magnetic properties. The NP-coated bubbles showed enhanced

performance in US imaging. We also demonstrated multiple imaging modalities for NP-

118

functionalized bubbles, such as fluorescence and MRI. We envisage that these

multifunctional bubbles will have important applications in medical diagnostics, where

multiple imaging methods using a single contrast agent will be advantageous. The proposed

method can also be used in triggered site-specific release of drugs by using magnetic

actuation and focused US exposure.

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121

Chapter 7

Conclusion and Outlook

The main goal of this thesis was to develop microfluidic (MF) methods for the

preparation of microbubble-templated materials in a continuous fashion and explore their

applications. In particular, this work pioneered a pH-regulated MF formation and

dissolution of CO2 bubbles. The MF dissolution of CO2 bubbles led to two important

results: the ability to generate small (<10 µm in diameter) bubbles and the capability to

produce bubbles encapsulated with various types of colloidal particles and biopolymers. In

the second approach we exploited a local decrease in pH at the length scale of an individual

bubble. In addition, this thesis described a MF method for the synthesis of Pickering

emulsions and polymer particles with supracolloidal structure.

In Chapter 3, we investigated a pH-dependent MF formation and dissolution of CO2

bubbles. We found the initial formation of CO2 bubbles was affected by the extent of the

bubble dissolution in the downstream channel. The degree of CO2 dissolution was precisely

controlled by the flow rate of the continuous aqueous phase and the acid-base equilibria in

the continuous phase in the microchannel. The proposed approach provided a new route to

generation of small bubbles (<10 µm in diameter) with a narrow size distribution (CV<5%).

Although our focus was limited to the CO2-water reactions, these experiments can be

extended to other soluble and reactive gases such as NH3 and SO2, which would enable

systematic kinetic studies of various gas-liquid reactions. Due to the environmental

importance of such gases, this research will have significant impact on science and industry.

122

Chapter 4 described a MF method for the preparation of bubbles and droplets with a

shell of colloidal particles (colloidal armour). First, we developed a chemically mediated

MF approach for the production of bubbles encapsulated with various colloidal armours.

The strategy utilized the following events occurring in the microchannel: i) monodisperse

CO2 bubbles were generated in a dispersion of anionic particles, ii) the dissolution of CO2

bubble led to the increase in the acidity of the solution at the periphery of the bubbles, and

iii) this increased the hydrophobicity on the particle surface and resulted in the adsorption

of particles to the gas-water interface. The size and shapes of bubbles with a shell of

colloidal particle were precisely controlled by the flow rate and pH of the continuous phase,

respectively. The generality of the MF approach was demonstrated by depositing various

particles and their mixtures on the surface of bubbles.

The armoured bubbles generated by the MF method can be used as templates for

fabricating acoustic insulators, separation membranes, light-weight structures, and scaffolds

for tissue engineering.

In Chapter 4, we also presented a MF “inside-out” method for the preparation of liquid

droplets coated with colloidal particles (Pickering emulsions). We demonstrated the

utilization of this system for the synthesis of supracolloidal polymer microparticles. The

proposed method has several advantages over the conventional methods of the preparation

of Pickering emulsions. First, it allows the precise control of the dimensions of droplets.

Second, it minimized the waste of particles by introducing them into the droplet phase.

Third, it provides the ability to control the surface coverage of droplets with particles.

The proposed method for the generation of Pickering emulsion can be applied for the

controlled encapsulation of reagents, drugs, and biological species.

123

In Chapter 5, a MF route to the generation of small, stable, and monodisperse bubbles

encapsulated with a lysozyme-alginate shell was proposed. The dissolution of CO2 bubbles

resulted in the increase of the acidity around the bubble surface. This led to the deposition

of positively charged lysozyme on the negatively charged surface of bubbles. The alginate

molecules in the continuous phase formed a complex with the positively charged lysozyme

on the bubble surface. As a result, the bubbles were coated with a biopolymer shell, which

provided a long-term stability (up to a month, at least) against bubble dissolution and

coalescence. The dimensions of bubbles were controlled by the hydrodynamic means and

by tuning the concentrations of lysozyme and alginate in the continuous phase.

In Chapter 6, we presented a MF single-step functionalization of bubbles with various

types of nanoparticles (NPs). The MF dissolution of CO2 bubbles resulted in the co-

deposition of biopolymers and NPs on the bubble surface due to the electrostatic

interactions. The presence of NPs on the surface of bubbles rendered the bubbles with

plasmonic, fluorescence and magnetic properties. The amount of NPs on the bubble surface

was controlled by tuning the initial concentration of NPs in the continuous phase.

Furthermore, we explored the applications of these NP-coated bubbles as contrast agents in

in-vitro medical imaging. First, we demonstrated the application of bubbles with a shell of

biopolymers and NPs as ultrasound (US) contrast agents. The bubbles encapsulated with

biopolymers and NPs showed improved contrast in US imaging, in comparison with the

NP-free bubbles. Second, the incorporation of Fe3O4 NPs on the surface of bubbles allowed

us to use them as effective magnetic resonance imaging (MRI) agents. The contrast in MRI

was enhanced as the concentration of Fe3O4 NPs on the surface of bubbles increased.

The bubbles prepared by the approaches presented in Chapter 5 and 6 can find

applications in medical diagnostics, drug delivery and gene therapy. For instance, drug or

124

nucleic acid-loaded NPs can be deposited on the surface of bubbles along with magnetic

NPs. This would allow the release of the loaded agents at a target site by using magnetic

actuation and focused US exposure. In addition, the localization of NPs on the bubble

surface would benefit the increase of the drug payload that is delivered at the target site.

125

Appendix

Appendix to Chapter 3

Estimation of the Amount of Dissolved CO2 and the Change of pH of the

Continuous Phase Following the Dissolution of CO2

For pH=13.2, kH=3.2 x 10-4 mol/(L kPa) and PCO2=27.6 kPa. Thus, [CO2]l kH PCO2,

[CO2]l ~0.009 mol/L (Henry’s law). The total concentration of dissolved CO2 can be

estimated by adding the dissolved unreacted amount of CO2 (determined by Henry's law)

and reacted CO2 (following eq. 3.3).

CO2 (l) + OH- HCO3- K = 3.2 x 107



Final concentration 0.009 -x 0.16- x x

x ~ 0.009 mol/L

The total amount of dissolved CO2 (both unreacted and reacted) is [CO2]l + x, that is, 0.018

mol/L.

For the estimation of the change in the value of pH after the dissolution of CO2 in the

microchannel, we consider the reaction (eq. 3.4) to determine the final concentration of OH-.

HCO3- + OH- CO3

2- + H2O K = 3.5x 103


Reacted concentration -y -y y

Final concentration 0.009-y 0.15-y y

y~0.009 mol/L

Therefore, the final [OH-] ~ 0.14 mol/L and pH=-log(10-14/[OH-]) ~ 13.1

126

For pH=11, kH=3.3 x 10-4 mol/(L kPa) and PCO2=27.6 kPa. Thus, [CO2]l = kH PCO2, [CO2]l

~0.009 mol/L (Henry’s law). The total concentration of dissolved CO2 can be estimated by

adding the dissolved unreacted amount of CO2 (determined by Henry's law) and reacted

CO2 (following eq. 3.3).

CO2 (l) + OH- HCO3- K = 3.2 x 107



Final concentration 0.009 -x 0.001- x x

x ~ 0.001 mol/L

The total amount of dissolved CO2 (both unreacted and reacted) is [CO2]l + x, that is, 0.01

mol/L.

For the estimation of the change in the value of pH after the dissolution of CO2 in the

microchannel, we consider the reaction (eq. 3.4) to determine the final concentration of OH-.

HCO3- + OH- CO3

2- + H2O K = 3.5x 103


Reacted concentration -y -y y

Final concentration 0.001-y 0.00003-y y

y~0.00002 mol/L

Therefore, the final [OH-] ~ 0.00001 mol/L and pH=-log(10-14/[OH-]) ~ 9





CO2 + H2O HCO3- + H+ K = 4.4 x 10-7

Initial concentration 0.009

Reacted concentration -x x x

Final concentration 0.009 -x x x

x ~ 0.00006 mol/L

127


0.00906 mol/L.

Here, the final concentration of [H+] ~ x ~ 0.00906. Therefore, the final pH=-log[H+] ~ 4.2.





CO2 + H2O HCO3- + H+ K = 4.4 x 10-7




x ~ 0.00006 mol/L


0.00906 mol/L.






CO2 + H2O HCO3- + H+ K = 4.4 x 10-7




x ~ 0.00006 mol/L


0.00906 mol/L.


128

For pH=1.5, kH=3.3 x 10-4 mol/(L kPa) and PCO2=27.6 kPa. The total concentration of

dissolved CO2 is dominated by Henry’s law, [CO2]l = kH PCO2 ~0.009 mol/L. For the

estimation of the change in the value of pH after the dissolution of CO2 in the microchannel,

we consider the reaction (eq. 3.2)

CO2 + H2O HCO3- + H+ K = 4.4 x 10-7



Final concentration 0.009 x 0.032+x

x ~ 0.0000001 mol/L


129


A4.1 Generation of Armoured Bubbles in a Microfluidic Flow-Focusing

Bubble Generator

Figure A4.1 Production of armoured bubbles in a microfluidic (MF) flow-focusing bubble

generator. Generation of the CO2 plugs (A), their dissolution (B), and armouring of the

shrunken bubbles with PS-co-PAA particles in the downstream channel (C). In (A-C), scale

bars are 200 μm. (D) Armoured bubbles collected at the outlet of the MF device. The scale

bar is 100 μm. (E) Magnified image of the bubbles in (D), showing the close-packed PS-co-

PAA particles on the bubble surface. Scale bar is 50 μm. Bubbles generated at PCO2=48.5

kPa, QL=10.4 mL/h, Cp=1.0 wt% and pH=14.

130


A6.1 Experimental Design

The dimensions of the MF bubble generator are shown in Figure S1. The continuous

phase contained 0.05 wt % of lysozyme, 0.1 wt % of alginate (the mixture of lysozyme and

alginate is referred as biopolymers) and 1 wt % of NP dispersion. The concentrations of

NPs were controlled by the number of centrifugation cycles of NP dispersions at 8000 rpm

for 30 min.

Figure A6.1 Schematic of the MF flow-focusing bubble generator. Wg= 105 μm, Wa=75

μm, Wo=50 μm, Wc=240 μm, and Lc=260 mm. The height of the microchannels is 120 μm.

A6.2 Inductively Coupled Plasma Atomic Emission Spectroscopy

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to

determine the concentration of Fe3O4 NPs on the surface of bubble. The amount of Fe in the

bubbles was estimated as follows: Cbubbles(Fe) = Cinitial(Fe) - Cfinal(Fe) where Cbubbles(Fe) is the

concentration of Fe in the suspension of bubbles (g/mL), Cinitial(Fe) and Cfinal(Fe) are the initial

and final concentrations of Fe in the continuous phase (g/mL), respectively. The number of

NPs per bubble was estimated as Cbubbles(Fe) x MWFe3O4 /(3 x MWFe x d x V x N) where

MWFe3O4 and MWFe are the molecular weight of Fe (55.8 g/mol) and Fe3O4 (231.5 g/mol),

131

respectively. d and V are the density (~1.25 g/mL), volume (~4.2x10-18 mL) of the NP,

respectively. N is the number concentration of bubbles (~106 bubbles/mL).

A6.3 In vitro Ultrasound Imaging

10 mL of the bubble suspension with concentration of approximately 104 bubbles/mL were

introduced into Opticell chamber (Thermo Scientific Inc.). The bubbles were exposed to a

focused ultrasound pulse (5 MHz) using L9-3 transducer and iU22 ultrasound system

(Philips). In vitro US images were obtained using a bubble-specific, nonlinear imaging

mode. The signal enhancement values were obtained by examining a 5 mm x 5 mm region

of interest and recording the average integrated power within this region for the contrast-

specific (non-linear mode) image. The enhancement was calculated as the ratio of the

integrated power measured in the chamber filled with bubbles to the integrated power

measured in the fashion when the chamber contained only water.

A6.4 In vitro Magnetic Resonance Imaging

Dispersions of bubbles with bubble concentration of approximately 104 bubbles/mL and

with varying amount of Fe3O4 NPs on the bubble surface were introduced into 2 mL

Eppendorf tubes. The tubes were placed in a plastic holder and submerged in water to

eliminate unwanted external susceptibility effects arising from air-water interfaces, which

would also cause a signal decrease on T2-weighted images. Imaging was conducted using a

Philips Achieva 3.0Tesla MRI scanner (Philips Healthcare, Andover, MA) with a 2D

coronal fast field echo sequence (parameters: 128mm field of view, 1mm x 1mm x 5mm

resolution, repetition time 100 ms, flip angle 15̊ , and four different echo times: 4.6, 6.9, 9.2,

132

and 20.7ms). T2* values were fit to this data using MatLab (The MathWorks, Inc., Natick,

MA).

A6.5 Energy Dispersive X-ray (EDX) Line Scan Results for Nanoparticle-

functionalized Bubbles

In addition to the elements of interest, in the control experiments the energy dispersive

X-ray (EDX) scans were carried out for V. Figure A6.2 shows the EDX line scan results for

bubbles encapsulated with a biopolymer shell and functionalized with Fe3O4 nanoparticles

(NPs). We note that the average intensity of EDX signal from V (run in control

experiments) is significantly weaker than that from Fe.

Figure A6.2 (A) Scanning transmission electron microscope (STEM) image of bubbles engulfed with the biopolymer shell and Fe3O4 NPs. Scale bar is 6 μm. (B-F) EDX line scan results for (B) Fe, (C) O, (D) C, (E) N, and (F) V.

133

Figure A6.3 shows the EDX line scan results for bubbles encapsulated with a

biopolymer shell and functionalized with Au NPs. The average intensity of the EDX signal

from V was substantially lower than that from Au.

Figure A6.3 (A) STEM image of bubbles encapsulated with a biopolymer shell and functionalized with Au NPs. Scale bar is 6 μm. (B-F) EDX line scan results for (B) Au, (C) O, (D) C, (E) N, and (F) V.

134

Figure A6.4 shows the EDX line scan results for bubbles engulfed with a biopolymer

shell and functionalized with SiO2-encapsulated CdSe/ZnS NPs. The average intensity of

EDX signal from V as a control was weaker than those from Si, Cd, Se, Zn and S.

Figure A6.4 (A) STEM image of bubbles coated with a biopolymer shell and SiO2-encapsulated CdSe/ZnS NPs. Scale bar is 6 μm. (B-J) EDX line scan results for (B) Si, (C) O, (D) C, (E) N, (F) Cd, (G) Se, (H) Zn, (I) S, and (J) V.

135

A6.6 Long-term Magnetic Actuation of Bubbles Encapsulated with the

Biopolymer Shell and Functionalized with Fe3O4 NPs

Bubbles maintained the ability of magnetic actuation even 3 months after the

preparation as illustrated in Figure A6.5

.

Figure A6.5 Optical microscope image of magnetically actuated bubbles encapsulated with a biopolymer shell and functionalized with Fe3O4 NPs. The image is taken 3 months after the preparation of the bubbles. The bubbles were generated at PCO2=48.3 kPa and QL=8.5 mL/h. Scale bar is 50 μm.

136

A6.7 Photoluminescence (PL) Emission Measurements for Bubbles

Encapsulated with a Biopolymer Shell and Functionalized with SiO2-

capped CdSe/ZnS NPs

Figure A6.6 shows the PL spectrum of the aqueous dispersion of bubbles engulfed with a

biopolymer shell and coated with SiO2-encapsulated CdSe/ZnS NPs. For comparison we

also show the spectra of NP-free bubbles coated with the biopolymer layer and SiO2-

capped CdSe/ZnS NPs. For the NP-coated bubbles the maximum in PL emission of

appeared at λmax=635 nm. The loading of the NPs into the biopolymer shell led to the slight

red shift (~ 5 nm) of the λmax, in comparison with the emission of SiO2-encapsulated

CdSe/ZnS NPs, due to the resonance energy transfer induced by close proximity of the NPs

on the bubble surface.1 NP-free bubbles did not show the characteristic emission peak of the

NPs at 635 nm.

Figure A6.6 FL emission intensity profiles of aqueous dispersions of SiO2-capped CdSe/ZnS NPs (red), bubbles coated with the NPs and biopolymers (blue), bubbles coated with the biopolymers (black). λex = 364 nm.

137

References (1) C. R. Kagan, C. B. Murray, M. G. Bawendi, Physical Review B 1996, 54, 8633.

microfluidic development of bubble-templated microstructured … · 2012-11-01 · this thesis...

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