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dissertation entitled

Fabrication of Nanostructures and Nanostructure based
Interfaces for Biosensor Application

presented by
Devesh Srivastava

has been accepted towards fulﬁllment
of the requirements for the

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FABRICATION OF NANOSTRUCTURES AND NANOSTRUCTURE BASED
INTERFACES FOR BIOSENSOR APPLICATION

By

Devesh Srivastava

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Materials Science

2008

ABSTRACT

FABRICATION OF NANOSTRUCTURES AND NANOSTRUCTURE BASED
INTERFACES FOR BIOSENSOR APPLICATION

By
Devesh Srivastava
Nanoparticles have applications from electronics, composites, drug-delivery, imaging and
sensors etc. Fabricating and controlling shape and size of nanoparticles and also
controlling the positioning of these particles in 1, 2 or 3-d structures is of most interest.
The underlying theme of this study is to develop simple and efﬁcient techniques to
fabricate nanoparticles from polymers, and also achieve control in shape, size and
ﬁmctionalization of nanoparticles, while applying them in biosensor applications. First
part of the dissertation studies the fabrication of nanostructures using anodized alumina
membrane as template. It discusses the fabrication design for injecting polystyrene
nanoparticles inside the pores of anodized alumina membranes and heating the membrane
to coalesce the particles into tapered nanoparticles. Various parameters like temperature
and amount of injected particles can vary the size and shape of fabricated nanoparticles.
Later it focuses on the fabrication of metallic nanostructures using the alumina
membranes without the aid of the injection system. It utilizes the difference in the
functionality of the pore edges of cleaved alumina membrane with respect to the pore
walls to ﬁrst deposit charged polymers using layer-by-layer deposition followed by
deposition of nickel. Second part of this study involves immobilization of enzymes for
biosensor applications. It describes a biosensor interface developed by immobilization of
tyrosinase using layer-by—layer (LBL) deposition process. The interface was modiﬁed

with functional nanoparticles and their inﬂuence on the response of biosensor was

studied. Tyrosinase sensor was further extended to develop a novel biosensor which was
used to study real time inhibition of NEST, a subunit of the medically relevant membrane
protein, neuropathy target esterase. The biosensor was developed to give real time
monitoring of dose dependent decrease in activity of NEST. Final part of this study
emphasizes on the inﬂuence of high shear rate mixer from PRIMIX, Japan on polymer
particle formation. This mixer can process speciﬁc volume of liquid and subject it to high
shear conditions. The mixing geometry consists of concentric cylinders, 52 mm inner
turbine diameter and 58 mm vessel diameter, with inner turbine rotating at high
peripheral speeds ranging from 10 m/s to SOm/s. The mixing is in turbulent regime at all
the speeds. Poly-lactic acid (PLA) nanoparticles were fabricated by nanoprecipitation and
emulsion diffusion process. Nanoprecipitation process was independent of shear rate at
low mixing speeds and particle size went up at high speeds due to coalescence of PLA
particles. Emulsion diffusion was done by making oil in water emulsion. PLA dissolved
in ethyl acetate was used as oil phase. It was followed by diffusion of ethyl acetate in
excess amount of DI water. It followed expected trend of smaller size at high mixing
speeds but was very sensitive to mixing time where particles coalesced at longer time
duration. The inﬂuence of viscosity was also studied and particles changed shape from
spherical nanoparticles to micron sized open shells at high viscosity and high mixing

speeds due to heating combined with mixing occurring in viscous turbulent regime.

COPYRIGHT BY
DEVESH SRIVASTAVA

2008

Dedicated To My Parents and Family

Acknowledgments

First of all, I would like to thank my advisor, Dr. Ilsoon Lee, who guided and mentored
me throughout the duration of my stay at Michigan State University. He provided useful
insights and tips and encouraged me to come up with new ideas. He also provided me
plenty of opportunities to travel and present my work in a professional set-up. It was a
pleasure working with him for my PhD.

I would also like to thank my committee members, especially Dr. Worden and Dr.
Ofoli for their guidance and comments during the course of my PhD especially during the
meetings for CNBI. Also like to thank Dr. Worden for letting me have uninterrupted use
of facilities in Protein Expression Laboratory. I would also like to thank Dr. Blanchard
and Dr. Hogan for agreeing to reside on my committee and mentoring and supporting me
through the course of my dissertation.

I would also like to thank PRIMIX Corporation, Japan for providing our lab with
the high shear rate mixer which led me conduct the ﬁnal part of my research. Also like to
thank them for the initial training and continuous help throughout the time period. I
would also like to thank staff at the Center of Advanced Microscopy for helping me work
on microscopes especially Ewa who was more than helpful while I worked on SEM.

Last but not the least, all the lab members of Nanobiotechnology Lab and ﬁiends
that I made during my stay at Michigan State. I would like to thank Neeraj Kohli for the
work we did together and his help initially in the lab. I would also like to thank Sachin
and Sumit for helping me in the lab and all the fun time we had outside. I would also

extend my thanks to Aaron, Troy, and Srivatsan. Also my roommates Deep C, Deep B

vi

and Ajay over the years and my friends Karuna, Shishir, Tithi, Shreya, Tamnay, Amit,
Leena, Sutty for making my 4 years of stay a truly unforgettable one.

Finally I would like to thank my family back home in India and my wife, Ankita.
They were constant source of encouragement and wishes which helped me immensely

throughout the course of my thesis.

vii

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. xi
LIST OF FIGURES ................................................................................ xii
ABBREVIATIONS ................................................................................. xix

1 INTRODUCTION ................................................................................. 1
1.1 OVERVIEW OF NANOTECHNOLOGY .......................................................... 1
1.2 FABRICATION OF NANOSTRUCTURES USING NOVEL
NANOINJ ECT ION PROCESS ....................................................................................... 5
1.3 STEP-EDGE LIKE TEMPLATED FABRICATION OF NICKEL
NANOWIRES ................................................................................................................. 6
1.4 HIGH SENSITIVITY TYROSINASE BIOSENSOR USING LBL
DEPOSITION .................................................................................................................. 6
1.5 NEUROPATHY TARGET ESTERASE BIOSENSOR ..................................... 7
1.6 FABRICATION OF POLY-LACTIC ACID NANOPARTICLES USNIG
HIGH SHEAR RATE NANOMIXER ............................................................................ 8

2 FABRICATION OF NANOSTRUCTURES USING NOVEL NANOINJECTION

PROCESS ............................................................................................ 11
2.1 INTRODUCTION ............................................................................................. 1 1
2.2 EXPERIMENTAL SE CTION .......................................................................... 13

2.2.1 Materials .................................................................................................... 13
2.2.2 Experimental Details ................................................................................. 14
2.3 RESULTS AND DISCUSSIONS ..................................................................... 17
2.4 CONCLUSIONS AND FUTURE WORK ........................................................ 26

3 STEP-EDGE LIKE TEMPLATED FABRICATION OF NICKEL NAN OWIRES ..... 27
3.1 INTRODUCTION ............................................................................................. 27
3.2 EXPERIMENTAL SECTION ........................................................................... 28
3.3 RESULTS AND DISCUSSIONS ..................................................................... 31
3.4 CONCLUSIONS AND FUTURE WORK ........................................................ 40

4 HIGH SENSITIVITY TYROSINASE BIOSENSOR USING LBL DEPOSITION ..... 41
4.1 INTRODUCTION ............................................................................................. 41
4.2 EXPERIMENTAL SECTION ........................................................................... 44

4.2.1 Materials .................................................................................................... 44
4.2.2 Preparation of Au Nanoparticles ............................................................... 44
4.2.3 Modiﬁcation of multiwalled carbon nanotubes ........................................ 44
4.2.4 Enzyme loading onto gold slides and silver membranes ........................... 45
4.2.5 Amperometric i-t curves ............................................................................ 46
4.2.6 Cyclic Voltammetry .................................................................................. 47

viii

4.2.4 Enzyme loading onto gold slides and silver membranes ........................... 45

4.2.5 Amperometric i-t curves ............................................................................ 46
4.2.6 Cyclic Voltammetry .................................................................................. 47
4.2.7 UV-vis absorbance spectroscopy ............................................................... 47
4.2.8 Microscopy ................................................................................................ 47
4.3 RESULTS AND DISCUSSIONS ..................................................................... 50
4.3.1 Layer-by-Layer of Poly-L-Lysine and Tyrosinase .................................... 50
4.3.2 Incorporation of Multiwalled Carbon Nanotubes ...................................... 52
4.3.3 Silver membrane as electrode .................................................................... 57
4.4 CONCLUSIONS AND FUTURE WORK ........................................................ 65
5 NEUROPATHY TARGET ESTERASE BIOSENSOR ................................................ 66
5.1 INTRODUCTION ............................................................................................. 66
5.2 EXPERIMENTAL SECTION ........................................................................... 68
5.2.1 Materials .................................................................................................... 68
5.2.2 NEST expression and puriﬁcation ............................................................. 68
5.2.3 Preparation of gold electrode for NEST biosensor .................................... 69
5.2.4 Preparation of phenyl valerate solution ..................................................... 71
5.2.5 Ellipsometry ............................................................................................... 71
5.2.6 Potential step voltammetry and other measurements ................................ 72
5.3 RESULTS AND DISCUSSION ........................................................................ 72
5.3.1 Ellipsometry ............................................................................................... 72
5.3.2 Dependence of current response on working potential and pH ................. 73
5.3.3 Measurement of esterase activity using NEST biosensor ......................... 75
5.3.4 Inhibition of esterase activity .................................................................... 78
5.3.5 Higher sensitivity NEST biosensor ........................................................... 82
5.3.6 Signiﬁcance of NEST biosensor ................................................................ 86
5.4 CONCLUSIONS AND FUTURE WORK ........................................................ 87
6 FABRICATION OF POLY-LACTIC ACID PARTICLES USING A HIGH SHEAR
RATE ................................................................................................. 88
6.1 INTRODUCTION ............................................................................................. 88
6.2 THEORETICAL BACKGROUND .................................................................. 91
6.2.1 Nanoprecipitation ...................................................................................... 91
6.2.2 Emulsion Diffusion .................................................................................... 92
6.3 EXPERIMENTAL SECTION ........................................................................... 98
6.3.1 Materials .................................................................................................... 98
6.3.2 Experimental Details ................................................................................. 98
6.3.3 Dynamic Light Scattering (DLS) .............................................................. 99
6.3.4 Microscopy ................................................................................................ 99
6.3.5 UV-vis absorbance spectroscopy ............................................................... 99
6.3.6 Zeta Potential ........................................................................................... 100
6.3.7 Differential Scanning Calorimetry .......................................................... 100
6.3.8 Therrnogravimetric Analysis ................................................................... 100
6.4 RESULTS AND DISCUSSION ...................................................................... 100
6.4.1 Nanoprecipitation .................................................................................... 100

ix

6.4.2 Emulsion Diffusion .................................................................................. 108

6.4.3 Effect of addition of glycerol ................................................................... 124

6.5 EFFECT OF ELECTRON BEAM ON PLA PARTICLES ............................. 138
6.5.1 Poly-Lactic Acid and Beam Damage ...................................................... 138
6.5.2 Deformation of PLA Particles By Electron Beam ................................... 139
6.5.3 Effect Of Gold Coating On The Poly-Lactic Acid Deformation ............. 143
6.5.4 Effect Of Structure Of Poly-Lactic Acid On Deformation ...................... 149

6.6 CONCLUSIONS AND FUTURE WORK ...................................................... 153

7 REFERENCES .................................................................................. 155

LIST OF TABLES

Table 3 . I ............................................................................................................................ 30
Table 4.1 Zeta potential of polyelectrolyte coated multiwalled carbon nanotubes ........... 53

Table 6.1 Effect of surfactant type on the surface charge and surface potential on
nanoparticles formed. .............................................................................................. 104

Table 6.2 Beam penetration depth for PLA and gold as a function of accelerating voltage.
................................................................................................................................. 143

Table 6.3 Fraction of beam transmitted at diﬁferent gold ﬁlm thicknesses ..................... 144

xi

LIST OF FIGURES

Images in this dissertation are presented i1 color

 

Figure 2.1: A system of solvent-aided nano-injection of nanospheres .............................. 14

Figure 2.2: Schematic illustration of solvent-aided nano-injection molding process using
alumina membranes having cylindrical nanopores. .................................................. 16

Figure 2.3: SEM images of novel tapered nanorods. A) Nanorice (small aspect-ratio (~2)
tapered nanorods). B) Higher magniﬁcation image of Nanorice. C) Nanospears
made of PS nanospheres (aspect ratio ~10). D) Nanospear bundles formed by
pumping 50 nm PS particles into a single membrane that has 20 nm and 200 nm
pore sizes at either ends. The nanospheres were pumped into 200 nm pore side. 19

Figure 2.4: Schematic representation of tapered nanorods and nanospears forming due to
partial wetting of alumina by polystyrene. ................................................................ 20

Figure 2.5: SEM images of incomplete nanorods using nanospheres. A) Incomplete
nanotubes were observed after sintering of 140 nm PS nanoparticles inside a 200 nm
pore size membranes. B) A half open nanotube formed inside a 200 nm pore size
membrane. C) A small aspect ratio nanotube. D) Broken nano-doughnuts using 140
nm particles in a single 200-20 nm pore size membrane. E) Nanodiscs formed using
100 nm PS spheres in a single 200-20 nm membrane without ultrasonication. F) An
intermediate structure with incomplete melting of the polymer nanospheres. .......... 21

Figure 2.6: Energy dispersive spectrum (EDS) of fabricated nanoparticles is shown
above. The black spectrum is the background and the line is the spectrum of the
fabricated nanoparticles. The difference in the count of carbon content shows the
presence polystyrene which is composed of carbon and rest of the elements do not
show any increase. ..................................................................................................... 23

Figure 2.7: SEM image of epoxy nanotubes prepared using A) and B) 200 nm & C) and
D) 100 nm pore sized alumina membranes. .............................................................. 24

Figure 2.8: Cross sectional SEM of alumina membrane. Its pores are decorated with the
gold nanoparticles. ..................................................................................................... 25

Figure 3.1: A) Schematic representation of the template formation process including top
and side views of alumina membranes before and aﬁer the cleavage. A side view
SEM image after the ﬂuorosilane treatment and cleavage is shown, the scale bar is
500nm. The actual membranes have quasi-hexagonally packed nanopores, but
hexagonally packed nanopores were assumed for the determination of the cleaved
edges in which nickel nanowires grow. B) Schematic illustration of each step of the
nanowire formation process, deposition of PEM layers selectively on the freshly
cleaved hydrophilic edge area and then selective nickel deposition followed. ......... 32

xii

Figure 3.2: of nickel nanowires after dissolution of alumina membrane. C) Fuzzy
nanostructure showing polyelectrolyte base as support for nickel growth. D) A
nickel nanowire with spreading ends showing possible deposition of polyelectrolytes
on the top surface of alumina membranes. ................................................................ 35

Figure 3.3: Rate of nickel deposition determined from Quartz crystal microbalance ....... 36

Figure 3.4: SEM image showing the two regions where EDS spectra were obtained, on
the sample and on the background. The sample spectrum as shown in next page has
nickel and carbon peaks, showing presence of nickel from electroless deposition and
carbon as polyelectrolytes, which were absent in background spectrum (continued).
................................................................................................................................... 38

Figure 3.4 continued: Spectrum of nickel nanowires and background. Ni peak are visible
in sample but are absent in background spectrum ....................................... 39

Figure 4.1: Different architectures for catechol sensor: (a) Thioctic acid modiﬁed gold
electrode and layer-by-layer (LBL) deposition of tyrosinase and poly-lysine, (b)
same as (a) but functionalized multiwalled carbon nanotubes instead of poly-lysine
(continued). ................................................................................................................ 48

Figure 4.1: (c) Polymer nanospears drop coated and cross-linked onto cystarnine
modiﬁed electrode and then a layer of tyrosinase adsorbed over it, and ((1) Scanning
electron micrograph of silver membranes used for tyrosinase and poly-lysine
deposition using LBL deposition ............................................................ 49

Figure 4.2: Tyrosinase biosensor for catechol as substrate, the electrode was maintained
at -O.1V vs Ag/AgCl as reference electrode. Each step is aliquot of catechol added to
achieve ﬁnal concentration of 8 uM in bulk. ............................................................ 50

Figure 4.3: Bilayer versus Current sensitivity plot for PLL-Tyr biosensor. The sensitivity
with different number of bilayers increased till 6 bilayers and then dropped and
stabilized. ................................................................................................................... 51

Figure 4.4: FTIR spectra after each layer: 1665 cm"1 (C=O stretching of amide group),
1540 cm'1 for N-H bending mode and 3300 cm"1 for N-H stretching mode ............. 52

Figure 4.5 a) TEM of pure multiwalled carbon nanotube and b) polyelectrolyte coated
MWCNTs. ................................................................................................................. 54

Figure 4.6: Comparative plot of sensitivity for PLL coated MWCNTs and pure PLL
tyrosinase LBL system. ............................................................................................. 55

Figure 4.7: (a) Comparative response curve for 3 bilayers of PDAC coated

MWCNTs/Tyrosinase and pure PDAC/Tyrosinase (Control) system, the difference
in sensitivity was signiﬁcant, (b) SEM image of LBL fMWCNTs on electrode. ..... 56

xiii

Figure 4.8: (a) Cyclic voltammogram of a silver planar as a control electrode and porous
silver membrane as electrode in 0.5M Na2804 purged with nitrogen at scan rate of
100 mV/s from -0.35V to +0.15V. (b) Amount of equivalent active enzyme loading
on planar electrodes per unit area until 8 bilayers and on porous silver membrane.
The amount of enzyme increased as number of bilayers increased. ......................... 59

Figure 4.9: Comparative response curve for 6 Bilayer PLL-Tyr and catechol biosensor on
porous silver membrane ............................................................................................. 61

Figure 4.10: Comparative sensitivity plot for different architectures. .............................. 61

Figure 4.11: (a) Diffusion of quinone (P) through the enzyme layer on a planar electrode.
The distance for quinone to diffuse to the electrode surface is much larger. (b) As
compared to the other electrode, which has higher enzyme loading due the higher

surface area while keeping the diffusion distance small (b) ...................................... 62
Figure 4.12: Sensitivity plots for a) 6 bilayers of PLL-Tyr on planar gold and b) 1 layer of

PLL-Tyr on porous silver electrode ........................................................................... 64
Figure 5.1: Molecular architecture of NEST biosensor ..................................................... 71

Figure 5.2: Ellipsometric thicknesses after the successive addition of following layers:
thioctic acid (point a), PLL-Tyr ﬁrst bilayer (point b), PLL-Tyr second bilayer (point
c), PLL-Tyr third bilayer (point (1), and PLL and NEST ﬁnal bilayer (point e) ........ 73

Figure 5.3: (a) Effect of working potential on the response current of the enzyme
electrode in 0.1 M phosphate buffer (pH 7.0) with (i) and without (ii) 12 pM phenyl
valerate solution, in 0.1 M phosphate buffer at an applied potential of -0.1 V (vs
Ag/AgCl). (b) Effect of pH on the response current of the electrode, in the presence
of 12 p.M phenyl valerate solution, in 0.1 M phosphate buffer at an applied potential
of -0.1V (vs Ag/AgCl). .............................................................................................. 74

Figure 5.4: (a) Current time response of the NEST biosensor to the addition of aliquots of
4 pM phenyl valerate, in 0.1 M phosphate buffer, pH 7.0, at an applied potential of -
0.1V (vs Ag/AgCl). (b) Calibration plot .................................................................... 76

Figure 5.5: Control experiment: Current time response on an electrode containing only
tyrosinase. The electrode was assembled in exactly the same way as NEST
biosensor, except that the ﬁnal NEST layer was not deposited ................................. 77

Figure 5.6: Current time response of NEST biosensor to the addition of phenyl valerate in
phosphate buffer (0.1 M, pH 7.0) to obtain a ﬁnal phenyl valerate concentration of 8
uM followed by the addition of NEST inhibitor PMSF to obtain a ﬁnal PMSF

concentration of 10 uM. ............................................................................................ 79

Figure 5.7: Current time response of NEST biosensor to the addition of phenyl valerate in
phosphate buffer (0.1 M, pH 7.0) to obtain a ﬁnal phenyl valerate concentration of 8

xiv

uM followed by the addition of NEST inhibitor PMSF to obtain a ﬁnal PMSF
concentration of 100 uM. .......................................................................................... 80

Figure 5.8: Current time response of NEST biosensor to the addition of phenyl valerate in
phosphate buffer (0.1 M , pH 7.0) to obtain a ﬁnal phenyl valerate concentration of 8
uM followed by the addition of NEST inhibitor PMSF to obtain a ﬁnal PMSF
concentration of 1000 uM. ........................................................................................ 81

Figure 5.9: Current time response of NEST biosensor with 6 bilayers of PLL-Tyr
underneath the NEST layer. The sensor was tested in phosphate buffer (0.1M) with
electrode maintained -0.1V vs Ag/AgCl reference electrode. In control the electrode
was prepared in exactly the same manner but NEST layer was not added. .............. 83

Figure 5.10: Current response of NEST biosensor with 6 layers of PLL-Tyr and followed
by inhibition of NEST by addition of 0.1 mM PMSF. First phenyl valerate was
added to get the ﬁnal concentration of 8 uM in bulk solution. The current response
was allowed to achieve steady state before addition of an aliquot of PMSF to get
ﬁnal concentration of 0.1 mM. .................................................................................. 84

Figure 5.11: Current response of NEST biosensor with 6 layers of PLL-Tyr and followed
by inhibition of NEST by addition of 1 mM PMSF. First phenyl valerate was added
to get the ﬁnal concentration of 8 uM in bulk solution. The current response was
allowed to achieve steady state before addition of aliquot of PMSF to get ﬁnal
concentration of 1 mM. ............................................................................................. 85

Figure 6.1: (a) Schematic of mixing chamber of the nanomixer. It has cylindrical turbine
which rotates at peripheral speed of 10m/s to SOm/s. (b) A cross-sectional cartoon, it
can have one or two injection port with continuous processing. ............................... 90

Figure 6.2: Turbine peripheral speed and equivalent Reynolds number for Couette-Taylor
geometry ﬂow. ........................................................................................................... 91

Figure 6.3: Emulsiﬁcation is carried out in the nanomixer by adding water with surfactant
and polymer in oil (solvent). This was followed by diffusion step in which emulsion
was added to excess amount of water for solvent phase to diffuse out. .................... 93

Figure 6.4: (a) Rate of energy dissipation as a function of peripheral speed and (b) Plot of
eddy diameter, A and droplet diameter for inertial turbulent regime dKH, for each
mixing speed assuming ethyl acetate and water system. ........................................... 97

Figure 6.5: SEM image of PLA particles prepared at 50m/s mixing speed. ................... 101

Figure 6.6: As can be seen in the SEM image the larger particles are heated above glass
transition temperature and colliding and coalescing into larger particles. .............. 102

Figure 6.7: Zeta potential of PLA particles (a) CTAB as surfactant; (b) Pluronic F68 as
surfactant; as a function of varying ionic strength in DI water. Increasing the ionic
strength decreases the zeta potential of the particles. .............................................. 105

XV

Figure 6.8: Mean particle size of PLA particles with Pluronic F68 and CTAB as
surfactant. It also shows the temperature in the vessel at each mixing speed. ........ 106

Figure 6.9: Decrease in the peak intensity at 540 nm after mixing gold nanoparticles
inside the nanomixer ............ 107

Figure 6.10: It shows the signature shift in the peak intensity from 540nm to 700 nm after
agglomeration of gold nanoparticles. ...................................................................... 107

Figure 6.11: Mean particle size as obtained by dynamic light scattering for particles
prepared at different mixing speeds in Nanomixer. ................................................ 110

Figure 6.12: Emulsions were made at different speeds and then added to excess amount
of water for particle formation ................................................................................. 110

Figure 6.13: Scanning electron micrographs at 7500X of PLA nanoparticles obtained by
emulsifying a) 10 m/s and b) 20m/s for 10 minutes. ............................................... 112

Figure 6.14: Higher magniﬁcation (15000X)scanning electron micrographs of PLA
nanoparticles obtained by emulsifying a) 10 m/s and b) 20m/s for 10 minutes.
Illustrates PLA particles at IOm/s are more polydisperse than particles at 20m/s. .113

Figure 6.15: Scanning electron micrographs (15000X) of PLA nanoparticles obtained by
emulsifying at a) 30 m/s for 10 minutes and b) 40m/s for 2 minutes. ..................... 115

Figure 6.16: Scanning electron micrographs (65000X) of PLA nanoparticles obtained by
emulsifying a) 30 m/s for 10 minutes and b) 40m/s for 2 minutes. ......................... 116

Figure 6.17: TEM (a) and SEM image (b) of PLA nanoparticles obtained by emulsifying
at 50m/s for 1 minute. .............................................................................................. 117

Figure 6.18: Mean diameter of the particles as obtained from DLS and as calculated from
SEM images ............................................................................................................. l 18

Figure 6.19: The particle sizes can be divided into two categories as large and small. The
mean diameter of each categories is plotted at mixing speeds of 10m/s to 30m/s..119

Figure 6.20: The mean diameter of particles at the different mixing times. Hollow
legends are used to mark the large particle diameter and small particle diameter as
observed in SEM images. ........................................................................................ 121

Figure 6.21: Illustrates the ﬂuid motion inside the mixer and how emulsion droplet can
encounter different turbulent conditions giving rise to difference in stable droplet
size. .......................................................................................................................... 121

Figure 6.22: a) Optical image of ﬂuorescent particles: PLA emulsion prepared at SOm/s

for 1 minute followed by addition of 1 micron ﬂuorescently tagged pure polystyrene
particles and further mixing for 2 minutes at 50m/s. b) Pure 1 micron ﬂuorescently

xvi

tagged pure polystyrene particles mixed at 50m/s for 2 minutes. The length of scale
bar in both the images is 20 um ............................................................................... 123

Figure 6.23 :The mean particle size as a function of glycerol volume fraction at mixing
speed of 50 m/s. ....................................................................................................... 124

Figure 6.24: The above ﬁgure illustrates how the eddy diameter increases and is greater
than both dim and de at glycerol volume percentage greater than 35%. The mean
diameter of particles for emulsion prepared at 50m/s for 1 minute also increased
beyond 40%. ............................................................................................................ 126

Figure 6.25: Mean particle size as a ﬁrnction of glycerol percentage. The mixing time at
50%: glycerol was brought down to 20 seconds to avoid heating of the emulsion to
78 C ......................................................................................................................... 127

Figure 6.26: Effect of time on the particle size and shape at 50% glycerol at 50rn/s
mixing speed. Particles changed from solid nanospheres to micron sized hollow
hemispherical shells ................................................................................................. 128

Figure 6.27: a) SEM image of (particles formed at 40% glycerol when the internal
temperature was raised to 78 C. b) Particles formed by adding emulsion prepared at
20m/s for 10 minutes to water/ glycerol (50% v/v) mixed in a beaker at 78 C ....... 130

Figure 6.28: a. TEM image of PLA nanoparticles showing that the particles are not
hollow. b) Illustrating the mixing condition in inertial and viscous turbulent regimes,
in inertial turbulent regime droplets are outside eddies and in viscous turbulent
regime they are trapped inside the eddies ................................................................ 131

Figure 6.29: Scheme of the fabrication of bowl-shaped polymer particles in a turbulent
eddy. At a short mixing time emulsion droplets caught in an eddy. Flow induced
self-assembly of nanoparticles in an eddy. (B) At a long mixing time, emulsion
droplets start coalescing in an eddy. (C) Solid form after loss of solvent. (D) Bowl
shaped polymer particle. .......................................................................................... 133

Figure 6.30: Scanning electron micrograph of poly-lactic acid open shells obtained by
mixing at 40m/s at 60% glycerol by volume for 4 minutes ..................................... 134

Figure 6.31: Effect of mixing speed on particle size at 50% glycerol volume percent. At
all the speeds it is in viscous turbulent regime and the droplet size achieves a stable
size at 30m/s. The mean particle size also reached minima at 30m/s and slightly
increased at SOm/s. .................................................................................................. 135

Figure 6.32: TEM images of PLA nanoparticles prepared at 30 m/s with 50% glycerol. A)
Shows the particles images with relatively larger particles. B) PLA nanoparticles
with mean diameter around 30 nm. The scale bar in both the images is 100 nm....137

Figure 6.33. Polylactic acid (a) before and (b) after exposure to lOKeV electron beam.
................................................................................................................................. 140

xvii

Figure 6.34: The effect electron beam damage on PLA ﬁlm dried on a glass slide coated
with 7 nm gold ﬁlm. (a) and (b) is an image of PLA ﬁlm before damage and (c) and
(d) are the images of same area after electron beam damage. The length of scale bar
in each image is 5 pm. ............................................................................................. 142

Figure 6.35: SEM image of PLA particles coated with 7 nm of gold ﬁlm (a) before
damage and (b) after damage by lOKeV electron beam .......................................... 145

Figure 6.36: SEM image of PLA particles coated with 35 nm of gold ﬁlm (a) before
damage and (b) after damage by lOKeV electron beam. ........................................ 146

Figure 6.37: SEM image of PLA particles coated with 70 nm of gold ﬁlm (a) before
damage and (b) after damage by lOKeV electron beam. ........................................ 147

Figure 6.38: a) Illustrates how beam damage is reduced with increasing thickness of gold
ﬁlm over polylactic acid particle. b) Illustrates the shrinking of particles during
electron beam damage. ............................................................................................ 148

Figure 6.39: DSC curves for poly-lactic acid: a) Crystalline and b) Amorphous. .......... 150

Figure 6.40: SEM images of PLLA particles coated with 7 nm of gold ﬁlm a) before and
b) after damage. ....................................................................................................... 151

Figure 6.41: Thermogravimetric Analysis of crystalline and amorphous poly-lactic acid
revealed that there was not signiﬁcant mass loss in the PLA particles fabricated in
the nanomixer and the shape change is due to the beam damage caused by the
electron beam ........................................................................................................... 152

xviii

ABBREVIATIONS

AF M: Atomic Force Microscopy

AuNPs: Gold Nanoparticles

CHAPS: 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
CNTs: Carbon Nanotubes

CTAB: Cetyl Trimethylammoniurn Bromide
Cys: Cystamine

DI: De-ionized

DLS: Dynamic Light Scattering

DMAB: Dimethylamine Borane

DMF: Dirnethylformamide

DSC: Differential Scanning Calorimetry

EDS: Energy Dispersive X-ray Spectroscopy
MWCNTs: Functionalized Multi-Walled Carbon Nanotubes
FTIR: Fourier Transform Infra-Red Spectroscopy
IPTG: Isopropyl Thiogalactoside

LB: Langmuir Blodgett

LBL: Layer-by-Layer

MWCNTs: Multi-Walled Carbon Nanotubes
NTE: Neuropathy Target Esterase

OPs: Organophosphorus Esters

OPIDNs: (OP)-induced delayed neuropathy

PAH: Polyallyl amine Hydrochloride

xix

PDAC: Polly(Diallyl) Dimethyl Ammonium Chloride
PEM: Polyelectrolyte Multilayers

PLA: Poly-Lactic Acid

PLL: Poly-l-Lysine

PMSF: Phenyl Methyl Sulfonyl F loride
PSNPs: Polystyrene Spherical Nanoparticles
PV: Phenyl Valerate

QCM: Quartz Crystal Microbalance

SAM: Self-assembled Monolayer

SEM: Scanning Electron Microscopy

SPR: Surface Plasmon Resonance

SPS: Sulfonated Polystyrne

STM: Scanning Tunneling Microscopy
TEM: Transmission Electron Microscopy
TGA: Therrnogravimetric Analysis

Tyr: Tyrosinase

UV: Ultra-Violet

XX

SYMBOLS

S: Catechol

P: Quinone

AH: Heat of vaporization

R: Gas Constant

C: Zeta Potential

k: Boltzmann Constant

‘I’: Surface Potential

K: Reciprocal thickness of diffuse layer
T: Temperature

so , 8,: Permittivity

00; Surface Charge Density

A: Distance to the plane of zeta-potential from the surface
n: number concentration of each ion in the bulk
5: Solubility Parameter

J: Joules

K: Kelvin (Temperature Unit)

m: Length (Meter)

3: Second (Time)

kg: Kilogram

Pa: Pascal

N: Newton

mol: Mole of substance

xxi

A: “Kolmogorov Scale” Smallest Eddy Diameter
8: Rate of Energy Dissipation

no: Viscosity of Continuous Phase

pc: Density of Continuous Phase

0: Interfacial Tension

(1m: Inertial Regime Droplet Diameter

dxv: Viscous Regime Stable Droplet Diameter
AU: Difference in rotational speeds of inner cylinder and outer cylinder in Couette-
Taylor ﬂow

Ar: Difference in radii of inner and outer diameter
1: Surface tension

Z: Atomic Number

A: Atomic Weight

Eo: Electron Beam Energy

2:: Thickness of Material

fA: Fraction of electron beam absorbed
f3: Fraction of electron beam back scattered

fT: Fraction of electron beam transmitted

xxii

1 INTRODUCTION

1.] OVERVIEW OF NAN OTECHNOLOGY

Nanotechnology is emerging as one of the greatest tool in advancement of ﬁelds
from engineering, chemistry, physics and biology. Nanotechnology deﬁnes the
fabrication and control of particles and molecules on nanoscale. One of the biggest
challenges scientists face today is control over shape, size and placement of these
particles for fabrication of large scale structures [1, 2]. Till now, most commercial
nanotechnology based devices are made ﬁ'om “top—down approach”, but these
technologies are soon expected to encounter fabrication size limitations. Hence bottom up
approaches is gaining widespread interest and is considered to be a fore-runner in
developing nanoscale fabrication tools which can either replace or assist existing
nanofabrication tools.

“Bottom-up” methods for fabrication of nanoparticles vary from, spontaneous
without constraints to directed or templated self-assembly processes. Spherical polymer
nanoparticles can be prepared in solution by precipitation and solvent evaporation [3, 4].
Polymer spherical nanoparticles are readily fabricated and are commercially available.
Metallic nanoparticles can also be readily produced by reduction of metal salts in solution
phase chemistry and also the assembly of metal atoms in solution can be controlled to
fabricate anisotropic structures. Various kinds of nanostructures using organic, inorganic
and metals are prepared by researchers [5, 6]. Gold and silver nanoparticles are very
popular because color of light they scatter can vary from visible to near infrared by

varying their aspect ratio [7, 8].

In template assisted fabrication using the membranes, the pores can be ﬁlled by
electrodeposition, sol-gel materials, polyelectrolytes or biomolecules to form wide range
of multi-functional nanorods and nanotubes [9, 10]. Other fabrication techniques involve
step-edge like fabrication or using biomolecules as templates. Allotropes of carbon like
fullerenes, carbon nanotubes, and exfoliated graphite nanoplatelets have also been studied
widely for possible applications in composites, biotechnology and electronics. Carbon
nanotubes are considered to be one the strongest and highly conducting form of matter
[1 1]. Graphite nanoplatelets can achieve very high surface area to weight ratio and are
studied widely for enhancement in polymer composites. They also offer advantage to
being electrically and thermally conducting [12, 13].

Carbon nanotubes also offer semi-conductor properties to be exploited in
electronics and also for using them as biosensors [14]. Their small size also makes them a
possible candidate for as a carrier for therapeutics. One of their biggest drawbacks in
biological applications is lack of solubility in water. There has been a considerable effort
to functionalize fullerenes, carbon nanotubes and graphite nanoplatelets, which can be
essentially classiﬁed in two categories. One is using non-covalent attachments using
surfactants, polymers, nucleic acids, peptides etc [15]. Other is by covalently
functionalizing the surface by oxidizing surface carbon atoms to carboxylic groups and
attaching other molecules or by directly attaching the moieties on the surface.

Other biggest challenge in the ﬁeld of nanotechnology is arranging these
nanostructures in a ordered form or creating nanopattems for various applications.
Conventional methodology for fabricating nanopattems involves photolithography [16],

scanning beam lithography [l7]. Researchers have developed techniques based on

scanning probe methods where surface patterns can be manipulated and imaged
simultaneously at atomic scale precision. It essentially comprises of using scanning
tunneling microscopy [18] (STM) and atomic force microscopy (AFM) [18]. A number
of different other procedure have also been developed to obtain nanoscale patterns using
molding, printing and embossing. The techniques that are developed using these are
microcontact printing [19, 20], replica molding [21], and nanoirnprint lithography [22-24]
etc.

Self-assembly is considered as one of the greatest tools in arranging these
nanostructures and also achieving nanopattems. Self-assembly is based on co-operative
efforts of small molecules and nanostructures to organize themselves in predeﬁned
manner to form large scale 2 or 3 dimensional structures. Templated self-assembly is
developing as a major tool that can fabricate periodic or patterned nanosize features.
Assembly of colloids and other particles using magnetic or electric ﬁeld has been used to
pattern or arrange these nanoparticles and nanorods. Block co-polymers are also a well-
known type of self-assembling polymers where immiscible blocks form micro—domains
so as to minimize interfacial energy and maximize chain conformational entropy. This
self-assembly can be control by template to obtained ordered orientation of the micro-
domains to form nanopattems [25, 26].

Other important self-assembly technique, that was deve10ped in early 903, is
known as layer-by-layer (LBL) self assembly process [27]. It was initially developed
using charged polyelectrolytes that can be adsorbed on a charged substrate and a charge
reversal takes place. Then a layer to oppositely charged polyelectrolyte can be adsorbed

to form a polyelectrolyte bilayer. This process can be repeated to form nanoscale thin

polymer ﬁlms. Previous techniques like Langmuir-Blodgett (LB) ﬁlms and self-
assembled monolayer (SAM) can also form ultra thin ﬁlms. But LB, though an elegant
technique, requires expensive equipments and is not applicable to a wide range of
molecules. SAMs can be formed only on limited substrates. LBL self-assembly process
offers a much simpler and inexpensive technique for deposition of multilayer as well as
allows incorporating different kinds of materials in those thin ﬁlms [28]. LBL is not just
limited to charged polymers, like polyelectrolytes. It has been shown that LBL self-
assembly process can be extended to proteins, viruses, DNA and charged
oligosaccharides. They are also been used to deposit inorganic materials like
functionalized nanoparticles like carbon nanotubes, charged colloidal nanoparticles,
nanosheets, and modiﬁed zeolite crystals [29].

Polymer nanoparticles are also gaining widespread interest in various ﬁelds of
photonics, semi-conductors, optoelectronics, biocatalysis and drug delivery. Fabrication
polymer nanoparticles in quick and efﬁcient way are really important especially for
targeted drug delivery. Nanoprecipitation and emulsion diffusion are some of the
commonly used methods to fabricate polymer nanoparticles using various biodegradable
nanoparticles for drug delivery applications.

The study of this thesis is based on two aspects of nanotechnology. One is ﬁnding
simple and efﬁcient ways to fabrication of polymer-based nanoparticles and controlling
shape and size of the nanoparticles fabricated. Nanoparticles were prepared by using
commercially available alumina membrane as templates. In other development poly-
lactic acid nanoparticles where prepared by nanoprecipitation and emulsion diffusion

process in a high shear rate nanomixer obtained from PRIMIX, Inc, Japan. This mixer

has a turbine rotating inside a cylinder and the particles are subjected to high shear rates.
The affect of high shear rate on particles was studied using the nanomixer. Another
aspect of this thesis involves assembling nanoparticles like carbon nanotubes along with
proteins onto an interface to develop electrochemical biosensors to have real time
detection of analyte of interest and also to couple two enzymes, namely Tyrosinase and
NEST, onto an interface to study the inhibition of NEST which is a medically relevant
protein.

1.2 FABRICATION OF NAN OSTRUCTURES USING NOVEL
NANOINJECTION PROCESS

Chapter 2 discusses a novel fabrication technique to make tapered nanorods from
polymeric nanospheres [30]. Polystyrene spherical nanoparticles (PSNPs) are
commercially available and were purchased from Polysciences, Inc. These spherical
nanoparticles were used as the starting material for fabrication of nanorice and
nanospears. Alumina membranes with cylindrical pores were used as the template for
fabrication of nanorice and nanospears. An injection system was built to inject PSNPs
suspended in DI water into the pores of the alumina membranes and once the required
amount was injected. The membranes were taken out and heated to temperature slightly
above the glass transition temperature of polystyrene. At that temperature the polystyrene
partial wets the alumina membrane and as PSNPs are sitting adjacent to each other the
combine to form nanorice or nanospears. Incomplete polystyrene nanotubes were also
fabricated by heating membranes to temperature well above the glass transition
temperature where polystyrene completely wets the alumina surface. Complete nanotubes
can also be readily prepared and was demonstrated by fabricating epoxy nanotubes by

complete wetting of pores of alumina membranes.

The injection system could also be used to decorate the inside of alumina
membranes with gold nanoparticles. This was done by ﬁrst ﬁmctionalizing the inside of
alumina membrane with silane—amines and then injecting colloidal gold nanoparticle
solution. Gold nanoparticles are known to attach themselves to amine groups by
chemisorption. Hence the charmels inside the membranes can be ﬁmctionalized using the
same injection system.

1.3 STEP-EDGE LIKE TEMPLATED FABRICATION OF NICKEL
NANOWIRES

Chapter 3 discusses a novel and simple method to form polyelectrolyte supported
nickel nanowires [31]. Again alumina membranes were used as a template and instead of
using the pores as the template pore wall edges were used as the template. In the method,
ﬁrst the whole membranes were treated with ﬂuorosilanes. F luorosilanes makes the
whole membrane hydrophobic. Then these membranes where cleaved and in the process
exposing the freshly cleaved pore edges. These edges are hydrophilic in nature and
alumina membranes are positively charged at the pH of 7. These edges were used as
templates to grow polyelectrolyte multilayers using LBL deposition. Then these ﬁlms on
the edges were used to deposit nickel using an electroless deposition bath. The thickness
of nickel nanowires can be controlled by varying the deposition time. Hence a simple
technique to make polyelectrolyte supported nickel nanowires is demonstrated.

1.4 HIGH SENSITIVITY TYROSINASE BIOSENSOR USING LBL
DEPOSITION

Chapter 4 discusses the fabrication of catechol biosensor using a LBL deposition
technique to immobilize enzymes onto an interface. LBL deposition is done on gold
electrode or silver electrode by ﬁrst functionalizing the surface of electrode by forming a

self-assembled monolayer of thiol terminated compounds. This gives a surface charge on

the electrodes that can be ﬁrrther used to immobilize tyrosinase. Tyrosinase catalyzes the
reaction of oxidizing catechol to quinone. The electrode is maintained at the potential of -
0.1 V vs Ag/AgCl electrode and the quinone produced is reduced back to catechol at this
potential.

Tyrosinase has isoelectric point of pH=4.7-5 and is negatively charged at a pH of
7. LBL is done using tyrosinase as one charged species and positively charged species
used were different types of polyelectrolytes like poly-l-lysine (PLL) and
poly(diallyl)dimethyl ammonium chloride (PDAC). Instead of polyelectrolytes
functionalized nanoparticles like carbon nanotubes and polystyrene nanospears were also
used. The affect of different types of polyelectrolytes and functionalized nanoparticles on
biosensor response was also studied. Addition of nanoparticles was advantageous in
PDAC system; whereas it did not inﬂuence the biosensor response in PLL system. It
showed the response of biosensor is a function of enzyme loading as well as substrate
diffusion through the enzyme layer. Silver membrane were also used which increased the

surface area to much greater extend and hence increased the sensitivity of the biosensor.

1.5 NEUROPATHY TARGET ESTERASE BIOSENSOR

In the chapter 5 a new design for fabrication of NEST biosensor is proposed [32].
NEST is a subdomain of a medically relevant protein called Neuropathy Target Esterase
(NTE). NTE is a membrane protein which plays a critical role in nervous system of
vertebrates and inhibition of this protein can cause delayed neuropathy and other
motomeuron diseases. NTE is a difﬁcult protein to express and purify, so the research is

focused on a subdomian of NTE called NEST which maintains the same catalytic activity

as NTE. NEST when active catalyzes the hydrolysis of phenyl valerate (PV) to give
phenol. Tyrosinase which is a poly-phenol oxidase oxidizes phenol to catechol and then
catechol to quinone. Quinone is detected electrochemically at the electrode interface
which is maintained at -0.1 V vs Ag/AgCl reference electrode.

NEST is immobilized on the electrode by LBL deposition along with tyrosinase.
Gold electrode is ﬁrst functionalized with thioctic acid which gives it a negative charge.
PLL and tyrosinase bilayers were ﬁrst added followed by addition of a layer of NEST.
NEST is also negatively charged at the pH=7.0 and can be used as a negatively charged
species in LBL deposition. The electrode with both the enzymes was maintained at a
potential of -0.1 V and the aliquots of PV were added and the response of the NEST
biosensor was measured. As the concentration of PV was increased there was increase in
the current measured at the electrode.

The inhibition of NEST was studied by addition of chemical species like phenyl
methyl sulfonyl ﬂoride (PMSF) to the bulk solution and the magnitude of current
measured from the NEST biosensor went down as the concentration of PMSF was
increased. Hence it demonstrated the real time detection of inhibition of NEST
electrochemically.

1.6 FABRICATION OF POLY-LACTIC ACID NANOPARTICLES USNIG
HIGH SHEAR RATE NAN OMIXER

Chapter 5 studies the effect of high shear rate nanorrrixer on the formation of
poly-lactic acid (PLA) particles formed by nanoprecipitation and emulsion diffusion.
Nanoprecipitation requires a solvent to dissolve poly-lactic acid and a non-solvent of
polymer. It also requires the solvent phase and non-solvent phase for polymer to have

high miscibility that causes polymer to precipitate out readily as solvent diffuses into

non-solvent case. A surfactant can also be added to stabilize the polymer particles once
they are formed and further prevent them from agglomerating or aggregating.

Nanoprecipitation was carried out by dissolving poly-lactic acid in acetone and a
surfactant, Pluronic F68 in de-ionized (DI) water. Pluronic F68 is co-polymer of
polyethylene and polypropylene glycol and it stabilizes the nanoparticles formed by
precipitation. It was followed by injection of PLA solution in DI water with surfactant in
the nanomixer. The affect of mixing speed was studied by adding same amount of PLA
solution in ﬁxed amount of DI water with surfactant. The affect of a different surfactant
was also studied by using cetyl trimethylarnmoniurn bromide (CTAB) which has a
positive charge on it. Zeta-potential was measured for both kinds of particles. CTAB
stabilized particles had stronger charge in magnitude as compared with Pluronic F68
stabilized particles. CTAB stabilized particles showed similar trend as in the case of
Pluronic F68.

Emulsion diffusion was carried out by using immiscible solvent-non-solvent
system of ethyl acetate and water. Poly-lactic acid was dissolved in ethyl acetate and
Pluronic F 68 in DI water. 20 ml of PLA solution was added along with 60 ml of Pluronic
F68 solution and emulsiﬁed in nanomixer at different mixing speeds and different time
duration. The emulsion was collected and added to excess amount of pure DI water for
ethyl acetate to diffuse into water. This causes PLA particles to form and they were
stabilized by the surfactant present in surrounding water. The particles size went down
with increasing mixing speeds but it was also a function of mixing time. The effect of
viscosity of continuous phase was also studied by using water-glycerol mix. The

viscosity also inﬂuenced the particle shape as higher viscosity changes the mixing regime

in the nanomixer. Finally it discusses the inﬂuence of electron beam while imaging the
nanoparticles. As the beam has strong energy the PLA particles deformed under the
inﬂuence of electron beam. It also describes the effect of thickness of gold ﬁlm on the
polymer particle deformation and how electron beam used of imaging can damage

particles.

10

2 FABRICATION OF NAN OSTRUCTURES USING NOVEL
NANOINJECTION PROCESS

2.1 INTRODUCTION

Nanosized functional particles are attractive for optical, electrical, magnetic, and
biological applications. Recently, in addition to nanosize, the shape of nanoparticle is
reported to be crucial, for example, in how it interacts with light by Halas and coworkers
[33]. They found that rice-shaped nanoparticles made of gold and iron oxide is the most
sensitive surface plasmon resonance (SPR) nanosensor yet devised. They hope to get a
far clearer picture of proteins and unmapped features on the surface cells by attaching
them to scanning probe microscopes.

Although there are tremendous potential advantages of using anisotropic
nanoparticles like nanorice instead of conventional spherical nanoparticles, the
development of controlling such shape on the nanoscale is in its early stage. Some
controlled growths of the end shape of nanorods with speciﬁc equipment at speciﬁc
conditions have been reported for inorganic or metallic nanoparticles [34, ‘35]. Even
though polymer nanorods and nanotubes have commonly been produced, since the
fabrication was introduced by Wehrspohn and coworkers [3 6], terminal contour control
of polymer nanoparticles like nanorice or nanospears has remained as one of challenging
tasks. If functional polymer nanoparticles can be shaped in the desired forms on the
nanoscale, they can easily be functionalized to have far enhanced multifunctional
properties using them as templates or substrates. In the fabrication of nanorods and
nanotubes, template-assisted fabrication is gaining widespread interest because of its

simplicity. Such novel nanostructures are expected to provide new functions in

11

optoelectronic and biological applications that can not be attained with conventional
spherical nanoparticles. Researches have used various kinds of membranes such as
polycarbonates and anodized alumina membranes as templates for the fabrication of
nanotubes and nanorods [37-41]. However, no report of polymer nanorods has shown
control of both aspect ratio and terminal contour. Additionally, no polymer nanospheres
have been incorporated into the production of nanorods. Mostly, monomers, polymer
melts or solutions are introduced into the nanopores for the production of nanorods and
nanotubes. Other template assisted techniques use the step-edge [42] and other methods
involving template molecules in solution [43]. Other nanorods production techniques
which do not require templates include the electrospining of nanoﬁbers [44, 45] and also
using biomolecules and self-assembly processes [46, 47].

The techniques that use membranes as templates include electrodeposition [48],
layer-by-layer deposition [10, 49, 50] and methods using commercially available metal
plating solutions [51, 52]. Zheng et. al. have fabricated copolymer nanotubes and
nanowires by having polymerizing copolymers inside the pores of alumina membranes
[53]. Anisotropic metallic nanoparticles like conical nanotubes rather than cylindrical
nanotubes and nanorods have also been fabricated. Such anisotropic nanoparticles were
fabricated using either a complex chemical vapor deposition set-up [54] or a tapered pore
that is coated with gold [55]. However, in this work for the ﬁrst time we report the
fabrication of tapered nanorods of varying aspect ratio and controlled terminal-contour
(e. g., nanorice to nanospears) using a simple method. First, our method exploits the
injection of a controlled amount and size of nanospheres into the cylindrical nanopores of

an alumina substrate. Second, the resulting nanostructures are controlled during the non-

12

uniform heating of the polymer nanospheres by capillary forces, and wetting (or
dewetting) onto the cylindrical alumina walls.

To achieve faster production and also obtain subtle variation in the shape, size,
and aspect ratio of nanoparticles, we have created a fabrication method using polymer
nanospheres and anodized alumina membrane templates that control the structure of the
resulting nanoparticles. Our system for fabricating novel nanorice and nanospears is easy
to set-up and perform on a laboratory bench top. Nanoscale fabrication was made without
the hassle of an elaborate set-up involving vacuum chambers or expensive equipment that
are normally required for lithography based fabrication. The cost of running the
experiment was very low and the fabrication of different types of nanostructures was
easily performed by changing key parameters, such as the particle size, template pore
size, length of ultrasonication and changing temperature or heating time. The same step is
also used to illustrate the versatility and the pores of alumina membranes where

decorated with gold nanoparticles by using the same system.

2.2 EXPERIMENTAL SECTION

2.2.1 Materials

Alumina membranes where purchased from Whatman with different pore sizes
20nm, 100nm and 200m. Polystyrene nanoparticles ﬁ'om Polysciences with mean
diameter as 140 nm, 50 nm and 100 nm. Sodium Hydroxide, 3 methoxy aminosilanes,

gold chloride trihydrate, NaOH, hydroxylamine and sodium citrate from Sigma Aldrich.

l3

2.2.2 Experimental Details

In this work, we used commercially available alumina membranes (Whatman
Anodized Alumina Membrane) as templates and polystyrene (PS) nanospheres (neutral,
carboxylated, or sulfated polystyrenes from Polysciences, Inc.) for the fabrication of
controlled aspect-ratio and terminal contour controlled nanoparticles (i.e., nanorice and
nanospears). The pores of these membranes were ﬁlled with nanospheres by a solvent
aided injection. Subsequent heat treatments then allowed the nanoparticles to coalesce
and wet (or dewet) from the alumina nanopore walls and form nanoparticles with a
controlled aspect-ratio and an interesting terminal curvature (i.e., round to sharp or
pointed). We assembled a small solvent-aided nanoinjection unit for injecting materials

into the cylindrical alumina nanopore membranes as shown in Figure 2.1.

Membrane

Holder
\

   
 

Flow Inlet Flow Outlet

 

 

Reservoir

 
 

Figure 2.1: A system of solvent-aided nano-injection of nanospheres.

l4

Figure 2.2 illustrates the overall process. A speciﬁc amount of PS nanosphere
suspension was put in the reservoir and then it was pumped through the membranes with
desired pore sizes in series (ﬁrst large and then small pores) or a membrane having both
large and small pores at each side. The size of nanospheres was in between the large and
the small pores so that the nanospheres are trapped only in the large pores. Once the large
pore membrane was ﬁlled with the desired amount of nanospheres it was carefully taken
out, heated at 1200C (above the glass transition temperature of the polymer) for various
amount of time, and then placed in a 3 M NaOH aqueous solution where the alumina
membranes dissolved. Then the remaining polymer nanoparticles were ﬁltered using a
centrifuge and washed several times in deionized (DI) water to remove any residual
NaOH. To image the resulting structure of nanoparticles, a drop of the sample suspension
was put on a glass slide and then dried. The dried samples on a glass slide were sputtered
with gold (around 5 nm thick) for scamring electron microscopy (SEM) analysis. SEM

used for high resolution imaging was J EOL 6300F with ﬁeld emission.

15

Nanoparticles in _

Solution b =L Time
0 Z 0 0

O ..

_.
_.
0°_.OO -'
e _.
—.
—o
_.
_.

 

.0

o ..00
.-*
.-

Dissolution and
Puriﬁcation

<1:

 

m
_
m
—
3 Heat
—
_
—
_
—
_

Figure 2.2: Schematic illustration of solvent-aided nano—injection molding process
using alumina membranes having cylindrical nanopores.

Gold Nanoparticles were prepared by the standard procedure of reduction of gold
salt by sodium citrate. 500 mL of 1 mM HAuCl4 was brought to a rolling boil with
vigorous stirring. Rapid addition of 50 mL of 38.8 mM sodium citrate to the vortex of the
solution resulted in a color change from pale yellow to burgundy. Boiling was continued
for 10 min; the heating mantle was then removed, and stirring was continued for an
additional 15 min [56]. The gold nanoparticle suspension was cooled to room
temperature and then stored at 40C for further use.

The membranes were decorated with gold nanoparticles using the same injection
molding system. Alumina membrane of 200nm pore size diameter was placed inside the

membrane holder. Instead of pumping polystyrene nanoparticles, ethanolic solution of

SmM amino-silanes was ﬁrst injected through the membrane for 20 mins. Then pure
ethanol was injected for 10 mins to ﬂush the membranes. The membranes where
carefully taken out from the holder and where placed in oven preheated to 60°C for 4
hours. This would cause the silanes to crosslink with alumina membrane which has
hydroxyl groups on its surface. Once crosslinked, the membrane was taken out and
placed back in the membrane holder. Gold nanoparticle suspension, prepared as
described in previous section, was injected through the membranes. Gold nanoparticles
are known to chemisorb on amine terminated self-assembled monolayers [57]. The
membranes then where ﬂushed with pure water, taken out and cleaved for imaging under

SEM.

2.3 RESULTS AND DISCUSSIONS

Figure 2.3A shows tapered nanorods of smaller aspect-ratio (i.e., nanorice) that
were fabricated by limiting the amount of nanospheres injected through the membrane.
Figure 2.3B shows a higher magniﬁcation image of the nanorice. Figure 2.3C shows the
nanorods tapered at the ends were made of (8.1 mg of 140 nm PS nanoparticles in 200 ml
of water) PS nanospheres in 200 nm pore-size alumina membrane stacked against 20 nm
pore-size membrane. A unique and peculiar terminal contour was observed of these
polymer nanorods (i.e., nanospears). As far as we know, there has been no report on such
sharp-pointed polymer nanorods that were fabricated with a uniform size at the
nanometer scale. In addition, we used smaller nanospheres and a smaller membrane.
This membrane has two pore sizes at each end, 20 nm and 200 nm. Both pores are

cylindrical and they meet at around 2 pm from the 20mm end. The concentration for 50

17

nm PS nanospheres was 2.3 mg in 200 ml of DI water. 100 ml of this suspension was
injected ﬁom the large pore side to the small side and then was heated at 120 °C for 2
hours. Since these nanospheres (50 nm) were much smaller than the large pore (200 nm)
in size they must have been closely packed and this case we observed much longer and
sharper tapered nanowires, as shown in Figure 2.3D.

A possible explanation for the formation of the tapered tip is illustrated in Figure
2.4. When the cylindrical pore diameter of the membrane was 200 nm and the PS
nanospheres were 140 nm in diameter, nanospheres align themselves in the cylindrical
pores. Pore diameter was larger than the nanosphere diameter. Hence all the nanospheres
(140 nm case) had only a small contact area with the pore wall as shown in the left side
of Figure 2.4. This illustrates an exaggerated effect of non-uniforrn heating and resulting
on the coalescence of nanospheres, and the shape change caused by wetting (or
dewetting) and capillary force.

As the nanospheres-ﬁlled membrane was heated above the glass transition
temperature of the polymer used, there would be two main heating mechanisms,
conduction through the alumina wall and convection through air. The radiation would be

negligible because of the low temperature.

18

 

Figure 2.3: SEM images of novel tapered nanorods. A) Nanorice (small aspect-ratio
(~2) tapered nanorods). B) Higher magnification image of Nanorice. C) Nanospears
made of PS nanospheres (aspect ratio ~10). D) Nanospear bundles formed by
pumping 50 nm PS particles into a single membrane that has 20 nm and 200 nm
pore sizes at either ends. The nanospheres were pumped into 200 nm pore side.

As the pore wall is a better heating source than air. There would be more heat
transfer through the small contact area between the wall and nanospheres than through
air. Hence the nanospheres would start to partially deform and wet at the pore wall. As
polymers are being soften in the cylindrical nanodomain, capillary forces come to play an
important role in moving and shaping the soften polymer parts [36, 58].

Capillary force can allow these softened polymers to ﬂow slowly onto the

nanopore walls, thus adjacent nanospheres coalesce to form continuous shape. The

nanosphere at the end just stretches further along the cylindrical nanopores. Because the
end nanosphere did not have another nanosphere to coalesce with, it forms a tapered end
as illustrated in Figure 2.4.

During the solvent-aided nanoinjection process, the nanospheres tend to stay
separated from each other rather than sitting adjacently. Intermediate ultrasonication of
the membrane is required to tightly pack the nanospheres into the membrane pores. This
intermediate step of ultrasonication after pumping the certain amount nanosphere
suspension also helped remove excess particles which tend to stay on the membrane
surface. These particles could prevent further nanospheres from being delivered to the

membrane pores and reduce the aspect ratio of nanorods.

Heated Above
Ts

E >

 

 

 

   

Figure 2.4: Schematic representation of tapered nanorods and nanospears forming
due to partial wetting of alumina by polystyrene.

20

 

Figure 2.5: SEM images of incomplete nanorods using nanospheres. A) Incomplete
nanotubes were observed after sintering of 140 nm PS nanoparticles inside a 200 nm
pore size membranes. B) A half open nanotube formed inside a 200 nm pore size
membrane. C) A small aspect ratio nanotube. D) Broken nano-doughnuts using 140
nm particles in a single 200—20 um pore size membrane. E) Nanodiscs formed using
100 nm PS spheres in a single 200-20 nm membrane without ultrasonication. F) An
intermediate structure with incomplete melting of the polymer nanospheres.

As shown in Figure 2.5, we also observed various other nanostructures like
incomplete nanotubes (i.e., perforated nanotubes) (A, B, and C), broken nanodoughnuts
(D), and nanodiscs (B). These nanostructures show evidence for the thermal wetting of
PS onto the cylindrical alumina nanopore walls as heat transfers to PS nanospheres
through the wall. As the temperature is increased well above the glass transition
temperature the PS completely wets the surface of alumina nanopores and due to limited
supply of polymers forms incomplete nanotubes [59] . It is expected that further variation
of the PS nanospheres packing, heating temperature, and time would allow the fabrication

of novel nanostructures. Figure 2.5F illustrates the intermediate step in the coalescence

21

of the particles. Due to a short heating time, the spherical particles have not completely
lost there structure. The nanospheres towards the middle can be seen merging together
whereas the nanospheres at the end are tending to form a slightly tapered shape.

The energy dispersive spectroscopy was also carried out for the nanospears and
nanorice. It showed that the particles comprise of mainly carbon. EDS is shown in Figure
2.6 with small peak for carbon which was absent in the spectrum obtained for
background. This con ﬁrms the presence of carbon as main constituent of nanoparticles.
Complete nanotubes can be readily prepared by using a polymer that completely wets the
surface was demonstrated by using commercially available epoxy-resin. The epoxy was
mixed with the cross-linking agent in the ratio of 1:1 as speciﬁed by the manufacturer.
Alumina membranes where smeared with the epoxy and the epoxy which completely
wets the membrane went inside to form nanotubes. Epoxy was allowed to cure inside
oven at 800C for 1 hour. The alumina membranes were dissolved in 3M sodium
hydroxide and the nanotubes were imaged under SEM as shown in Figure 2.7.

The same system was used to decorate the pore walls of anodized alumina
membranes. The membranes where functionalized by crosslinking silanes which have
amine functional groups. The gold nanoparticles pumped through the membranes
chemisorbed on the amines to form a ﬁne dispersion of gold nanoparticles along the
length as shown in Figure 2.8. It is an SEM image of gold nanoparticle decorated pore of

cleaved alumina membrane.

22

ES

 

Am]
009' 9

009';

endures :aumno
punorﬁrpug :rpurg

009' e

 

UZ

000'6

"2

p
0

Figure 2.6: Energy dispersive spectrum (EDS) of fabricated nanoparticles is shown
above. The black spectrum is the background and the line is the spectrum of the
fabricated nanoparticles. The difference in the count of carbon content shows the

presence polystyrene which is composed of carbon and rest of the elements do not
show any increase.

23

 

Figure 2.7: SEM image of epoxy nanotubes prepared using A) and B) 200 nm & C)
and D) 100 nm pore sized alumina membranes.

24

 

Figure 2.8: Cross sectional SEM of alumina membrane. Its pores are decorated with
the gold nanoparticles.

25

2.4 CONCLUSIONS AND FUTURE WORK

In conclusion, we present a very versatile and effective approach for shaping
nanoscale structures using ordered nanopored membranes and a simple solvent-aided
nano-injection molding process of polymer nanospheres. By exploiting non-uniform
heating and resulting wetting or capillary forces of nanospheres ﬁlled inside the
cylindrical nanopores of membranes we obtained novel nanostructured, anisotropic
nanoparticles such as nanorice and nanospears. As far as we know, these nanostructures
are unique and have not been prepared using other methods. This method is very easy to
implement without any complicated chemistry or expensive equipments. Therefore, after
obtaining spherical nanoparticles of any materials, this method can easily change the
symmetrical nanoparticle into other geometries with less symmetry. The same system
was used to functionalize the alumina membrane with amino-silanes and was further
decorated with gold nanoparticles.

The same system can be used to pump other kind of solutions like enzymes and
polyelectrolytes to form nanotubes. It can also be used to make chains of nanoparticles by

stacking them inside the pore and then cross-linking them to form nanochains.

26

3 STEP-EDGE LIKE TEMPLATED FABRICATION OF NICKEL
NAN OWIRES

3.1 INTRODUCTION

One of the biggest challenges in the ﬁeld of nanotechnology is the cost-effective
fabrication of nanowires. To date there are many methods that use top-down approaches
or involving elaborate equipments to form nanowires like dip-pen [60] and electron beam
lithography [61], but both are expensive and slow. Cost effective methods such as soft-
lithography [62, 63] and nanoimprint lithography [22-24] are also being developed. The
bottom-up approach involves the self-assembly of molecules based on various
interactions like electrostatic, hydrogen bonding or covalent bonding. It offers a more
cost effective method for the fabrication of nanostructures. It also provides more control
over the variety of nanostructures that can be fabricated. Nanowires and nanotubes of
various materials have also been fabricated using template assisted techniques like ﬁlling
the membrane pores of anodized alumina [10, 30, 36, 38, 48, 58], track etched polymer
membranes [49, 55] and decoration of step-edges [42, 64, 65]. Most methods using
membranes as a template involve electrodeposition. This is done by coating one side with
a metal to make it conductive, followed by electrodeposition of the metal inside the
pores. Electroless methods are slightly more difficult, especially in depositing metal
inside the pores, as the process is hindered by the slow diffusion of metal ions into pores.
There are a variety of metals that are being used to make nanowires such as gold [40] ,
copper [66, 67] and silver [68, 69]. Nickel nanowires are also ﬁnding great interest
among researchers due to there unique magnetic properties. Also at the nanometer scale,

quantum conﬁnement leads to unique properties. For example, the band gap of nanowires

27

varies as the inverse square of its diameter [70]. Biological molecules can also be
attached to nanowires to form nano-machines [71]. Most fabrication methods for nickel
nanowires and nanotubes involve ﬁlling pores of membranes [72, 73], step-edge [74] and
also using biomolecules like DNA [75].

In this work, we report a step-edge like methodology for the fabrication of
polyelectrolyte supported nanowires. Polyelectrolytes provide ﬂexible support to metallic
nanowires and prevent them from falling apart. Our method is simple, can be performed
on lab-bench top and does not require any equipment for metal deposition. In our method
an alumina membrane is functionalized with hydrophobic molecules and then broken to
expose ﬁ'eshly cleaved hydrophilic edges along the broken pore walls. Then,
polyelectrolyte multilayers (PEMs) were built on the hydrophilic edge of the pore
membrane and an electroless nickel bath was used for the deposition of nickel onto the
multilayers to form nickel nanowires. After dissolving the membrane, ﬁ'ee standing

nickel nanowires were obtained.

3.2 EXPERIMENTAL SECTION

Anodized alumina membranes of 200 nm pore size were purchased from
Whatman. The ﬂurosilanes (tridecaﬂuoro-l ,1 ,2,2-tetrahydrooctyl)-1-
methyldichlorosilane) were purchased from United Chemical Technologies Inc. 16-
mercaptohexadecanoic, positively charged polyelectrolyte,
poly(diallyldimethylarnmoniurn chloride) (PDAC), (Mw~100,000-200,000) and
negatively charged polyelectrolyte, poly(styrene) sulfonate (SPS) (Mw~70,000 g/mol),

were purchased ﬁ'om Sigma Aldrich. The bath for electroless deposition was prepared

28

using nickel sulfate, sodium hydroxide, sodium citrate, lactic acid and dirnethylarnine
borane (DMAB); all purchased from Sigma Aldrich. The palladium catalyst (N a2[PdCl4])

was purchased from Strem Chemicals, More details on electroless nickel deposition can
be found from our previous work in which selective nickel plating was made on the
particle arrays deposited on patterned polyelectrolyte templates [76].

In this work, alumina membranes were ﬁrst treated with ﬂuorosilanes by chemical
vapor deposition. Both the ﬂuorosilanes and the membranes were placed under vacuum
for 30 mins. This was followed by two hours of heating at 120°C which allows the
silanes to cross-link with alumina membrane both inside the pores and on outer surface.
The ﬂuorosilane treated surfaces are extremely hydrophobic. This was followed by
breaking the membrane which exposes the freshly cleaved alumina surface that is
hydrophilic [32], unlike the other surfaces of the membrane.

These fresh hydrophilic edges now act as templates for deposition of PEM and
nickel. The alumina membrane is positively charged at a pH of 7 because the isoelectric
point of alumina is at around pH 8-9 [77]. Polyelectrolyte multilayers were selectively
built on the hydrophilic edges by dipping broken membrane in (1) SPS and (2) PDAC
solutions for 30 minutes each. Intermediate washing steps were carried out using de-
ionized (DI) water. Two bilayers of (SPS/PDAC); were built leaving a positively charged
polyelectrolyte (PDAC) at the t0p.

The PEM coated membranes were then immersed in a negatively charged
palladium catalyst for 15 seconds and rinsed with DI water at pH of 3.0. The palladium
catalyst is necessary to reduce Ni2+ to metallic Ni. With out the catalyst, nickel will not

plate on any surface. The Pd catalyst was adsorbed onto the PEM surface through

29

electrostatic interactions and is the location where the reduction of Ni ions to Ni metal
occurs. This gives rise to the nanowires structure supported by polyelectrolytes. The
nickel reduction occurred when samples were placed in a 100 mL nickel bath whose
composition is summarized in Table 3.1. Before use, the pH of the nickel bath was
adjusted to 6.5 i 0.1 by adding small amounts of 1.0 M NaOH. Electroless deposition

times of up to 10 minutes were used.

Table 3.1: Chemical composition of a 100 ml nickel bath used for electroless deposition
Nickel Sulfate 4.0 g

 

 

Sodium Citrate 2.0 g

 

Lactic Acid 1.0 g

 

DMAB 0.2 g

 

 

 

 

Once nickel was deposited, the membrane was dissolved in 3M NaOH to extract
the nickel nanowires. Nickel nanowires were ﬁltered and washed in DI water using a
centrifuge. Nickel nanowires were imaged using a scanning electron microscope (SEM,
J EOL 6300F) by drop coating a glass slide with a suspension containing nickel nanowires
which were sputter coated with 7 nm of gold before imaging. Energy dispersive
spectroscopy (EDS) was also done to analyze the composition of the nanowires.

Quartz crystal microbalance (QCM) crystals (5 MHz, Maxtek, Inc., Santa Fe
Springs, CA) with gold electrodes were cleaned in piranha solution (7 parts concentrated
sulﬁrric acid and 3 parts 30% hydrogen peroxide) for thirty seconds followed by rinsing
with an excess of DI water and drying by a steam of nitrogen. A self-assembled

monolayer (SAM) of l6-mercaptohexadecanoic acid was formed on the surface of the

30

QCM crystals by immersion in a 5 mM ethanol solution for one hour followed by rinsing

with ethanol and drying with nitrogen. Then ten and a half bilayers of (PDAC/SPS)10,5

were formed on the —COOH terminated SAM. The QCM crystals were then immersed

into a palladium catalyst solution for 303, rinsed with DI water and dried with nitrogen.

3.3 RESULTS AND DISCUSSIONS

Figure 3.1 illustrates the scheme for the formation of the nickel nanowires. Here
the fabricated nanowires have two different materials interwoven along their length. The
side of the nanotubes that was in contact with the alumina membrane is polyelectrolyte
whereas the other side is metal. Hence this simple technique can impart two different
ﬁrnctionalities on the same wire. The thickness of each side can be controlled by
changing the number of PEM bilayers and the electroless deposition time.

The initial width of the edges can be estimated theoretically from the data given

by the manufacturer. The surface density of the 200 nm diameter pores in the membrane

is approximately 109 pores cm'z. The linear pore density can be approximated to 3.1 X
104 pores cm'l. Hence, the average spacing between the pores can be estimated as:

_ —7
davg= (1 3192012330 x 10 )Cm =125 nm ...................... (3.1)

 

31

v \
'1
‘. t
.

  

 

 

Top view of Fluorosilane treatment & Side view of
hexagonally packed cleaving along black line cleaved membrane
membrane

(A)

  

 

Freshly Cleaved
Hydrophilic Surface
SPS
A:
t
H d h b‘ E ZBilayers
y rop o rc _,_,-..____,-_._._.=
Surface h” [1 PDAC

Nickel
<=
Deposition

(B)

   

Figure 3.1: A) Schematic representation of the template formation process
including top and side views of alumina membranes before and after the cleavage.
A side view SEM image after the ﬂuorosilane treatment and cleavage is shown, the
scale bar is 500nm. The actual membranes have quasi-hexagonally packed
nanopores, but hexagonally packed nanopores were assumed for the determination
of the cleaved edges in which nickel nanowires grow. B) Schematic illustration of
each step of the nanowire formation process, deposition of PEM layers selectively on
the freshly cleaved hydrophilic edge area and then selective nickel deposition
followed.

32

We also observed the cross-sectional of the alumina membrane using a SEM.
From these images, we estimated edge size which to be approximately 101.5 i 32.4 nm.
This observed value is consistent with the estimated value.

The PEMs act as adhesion layers between the membrane and the metal. Without
the PEM layers the nickel would not grow. Also the polyelectrolytes are hydrophilic
molecules so after treating membranes with ﬂuorosilanes, the treated part repels the
adsorption of polyelectrolytes. Hence, once the membrane is broken, the polyelectrolytes
are selectively deposited on the new exposed region which is hydrophilic.

The purpose of polyelectrolytes is two fold. First it provides a surface adhesion
layer for the negatively charged catalyst to deposit followed by the growth of metal. It
also helps to form a continuous support on the edge of the membrane. Since
polyelectrolytes are fuzzy structures (i.e., layers of polymer which are intertwined), it can
compensate for the irregularities on the edges, hence providing a smoother support for
metal wire. Also the electrostatic interactions among the polyelectrolyte layers provide a
strong adhesion layer for metal growth. The polyelectrolytes used were strong
polyelectrolytes hence the charge on them is independent of the surrounding pH
environment. So these bilayers maintain their structure and strength in the low pH
condition, while washing of catalyst, which it encounters during the growth of the metal.

We did not deposit more than two bilayers since a larger number of bilayers may
cause multilayers on adjacent edges to merge as the diameter of nanowires for 2 bilayers
of polyelectrolytes was approx. 200 nm. The thickness of the multilayers grows with the

number of bilayers and in this case the polyelectrolytes are growing on an edge. Our

33

previous work on this system shows that each bilayer of PDAC/SPS is 3.4 nm in
thickness [78].

We investigated whether the PEM were required to form nanowires. The role of
polyelectrolytes was conﬁrmed by a control experiment. As before the alumina
membrane was treated with ﬂuorosilanes and then broken. However the PEM were not
deposited. The membrane was then dipped into the negative catalyst solution and nickel
was electrolessly deposited directly on the edges of the alumina membrane. This was
followed by ﬁltering and washing. This resulted in no nanowires structures being formed.
A second control experiment was performed where the membrane was not treated with
ﬂuorosilanes and broken. This time nickel growth occurred at the top and bottom
surfaces as well and not exclusively on the pore edge of the membrane. Again no
nanowires were obtained.

Nickel nanowires were obtained by the dissolution of the alumina membrane and
further puriﬁed by repeated washing and ﬁltering using a centrifuge and DI water. The
samples were imaged using SEM. The images obtained are shown in Figure 3.2. The
length of the resulting nanowires was 2-4 pm though the membranes are 60m in
thickness. The shorter than expected length could be due to breaking of wires during
repeated ﬁltering process. Figure 3.2A shows nickel nanowires growing on the pore
edges in an alumina membrane. Figure 3.2B is an image showing a collection of nickel

nanowires.

34

 

Figure 3.2: of nickel nanowires after dissolution of alumina membrane. C) Fuzzy
nanostructure showing polyelectrolyte base as support for nickel growth. D) A
nickel nanowire with spreading ends showing possible deposition of polyelectrolytes
on the top surface of alumina membranes.

of the nanowire. This fuzzy surface structure is caused by the presence of the supporting
polyelectrolytes layers. Hence the fabricated nanostructures can be considered more like
metal nanocactuses (desert plants) supported by a nanoﬁlm of polyelectrolytes. Figure
3.2D shows the end of a nanowire where the metal end is spreading out. This is probably
due to the metal ﬁlm forming at the end of a pore where polyelectrolytes might have

deposited at the top surface of the membrane and not in the pore of the membrane.

35

 

+ Catalyst
+ No Catalyst

 

 

 

100;
80:
60~
40-

20-

Nickel Thickness (nm)

 

 

I j I ' I ' I ' l

0 5 1O 1 5 20
Time (minutes)

Figure 3.3: Rate of nickel deposition determined from Quartz crystal microbalance.
We estimated the thickness of nickel ﬁlm growing on polyelectrolytes by using
QCM. This was done by, adsorbing a —COOH terminated SAM onto gold coated QCM
crystals. Next polyelectrolytes are deposited on to the SAM followed by the deposition
of nickel. The weight of nickel deposited causes a shift in the resonance frequency of the
quartz crystal. Hence the rate of nickel deposition was calculated by measuring the
change in frequency of the quartz crystal. The thickness of nickel is plotted as a ﬁmction
of time and is shown in Figure 3.3. We have also created homogeneous nickel ﬁlms on
planar substrates. Measurement of these thin metal ﬁlms shows that they are conductive.
However, we have not independently measured the conductivity of the nickel nanowires.
Samples with no catalyst showed no controlled nickel deposition. Samples with the
palladium catalyst showed initial non-linear growth as the nickel agglomerated on the
surface and as time passed coalesced into a uniform coating. Once the surface is uniform

the deposition became linear at a rate of 5.9 nm per minute for plating times between ﬁve

36

minutes to ﬁfteen minutes. At about 20 minutes the plating rate slowed and became non-
linear before the nickel started delaminate and peel off due to the buildup of internal
stresses.

Based on the QCM data shown in Figure 3.3 and our previous work on the PEMs
[78], the PEM-nickel nanowire has a thickness of around 47 nm, 7 nm from the PEMs
and 40 nm ﬁom the nickel. Each thickness can be adjusted by changing the number of
PEM bilayers and metal deposition time. In addition, the width of nanowires can be
changed by using membranes with different sized pores.

Elemental analysis of the wires was done using energy dispersive spectroscopy
(EDS). The EDS spectra, in Figure 3.4, showed a strong nickel peak, along with other
elements from background. There was also a small carbon peak showing the presence of
the polyelectrolytes. For comparison a background spectrum was also taken where both
the nickel and copper peaks were absent. Also the control experiments gave indirect
evidence for the presence of carbon, in the form of polyelectrolytes that support the
nickel nanowires. Since there were no nanowires observed without polyelectrolytes

bilayer deposition step.

37

Spectrum 2

 

 

1pm Electron Image 1

Figure 3.4: SEM image showing the two regions where EDS spectra were obtained,
on the sample and on the background. The sample spectrum as shown in next page
has nickel and carbon peaks, showing presence of nickel from electroless deposition
and carbon as polyelectrolytes, which were absent in background spectrum
(continued).

38

2500
,3? 2000
1500 f

1000

 

Counts(a.u

500

 

 

 

 

 

 

 

2500 Background

Sr
2000 ' H

)

y—c
LII
O
O

 

1000 ' ()

Counts(a.u.

 

 

500 Na Al J

.cu

 

c:

‘l
4
out
'0']
t
l

1 L5 2

KeV

Figure 3.4 continued: Spectrum of nickel nanowires and background. Ni peak are
visible in sample but are absent in background spectrum.

39

3.4 CONCLUSIONS AND FUTURE WORK

Here we have demonstrated the step-edge like fabrication of nickel nanowires.
This is an easy technique to produce nanowires that does not require an elaborate set-up
and with a large amount of equipment. It can readily be extended to different metals
which can be electrolessly deposited. These nanowires are supported by polyelectrolyte
bilayers as base which makes these wires ﬂexible as they have polyelectrolytes
multilayers as support. These nanowires can possibly be printed on polymeric substrates
to achieve a printed nanowire array structure. Also it imparts functional anisotropy to the
composition of wires where one side is metal and other side polymer.

This has the added advantage of the ability to have two different molecules
adsorbed on single nanowires by functionalizing different sides of the nanowires.
Additionally bimetallic nanowires can easily be created by depositing a second metal on

top of the ﬁrst.

40

4 HIGH SENSITIVITY TYROSINASE BIOSENSOR USING LBL
DEPOSITION

4.1 INTRODUCTION

Tyrosinase is a multi-copper oxygenase and is derived ﬁom Streptomyces
glausescens, the ﬁmgi Neurospora crassa and Agaricus bioprus. Tyrosinase contains a
coupled binuclear copper active site and it catalyzes both the ortho-hydroxylation of
monophenols and the two-electron oxidation of o-diphenols to o-quinones. The fungal
tyrosinase is not a membrane protein unlike the human tyrosinase that is a membrane
protein. Tyrosinase has adapted itself to various physiological conditions, in fungi and
humans it catalyzes the ﬁrst step for formation of melanine from tyrosine. Whereas in
plants it causes oxidation of various phenolic derivatives [79, 80].

Tyrosinase in its native form occurs in inactive met-form where the binuclear
c0pper site is in the wrong oxidation state [Cu(II)] to bind dioxygen. Two-electron
reduction by a catechol converts met-tyrosinase to deoxy-tyrosinase, which then binds
with dioxygen to give wry-tyrosinase. Oxidation of a catechol leads to met-tyrosinase,
which cannot bind oxygen to regenerate wry-tyrosinase. Only in the presence of a second
catechol molecule is the met-tyrosinase reduced to deoxy-tyrosinase which then
regenerates the oxy form [81].The catecholase activity of Tyrosinase can thus be

described as

41

4+ 2- 4+

ony(cu2 " 02 ) + S 9 Etnet(cu2 ) + P ...... (4.1)
4+ 2+

Emet(CU2 )+ S 9 Edeoxy(CU2 ) + P .............. (4.2)

2+ 4+ 2-
Edeoxy(Cu2 )+ 02 —> ony(Cu2 - 02 ) ............ (4.3)

Where S is catechol molecule and P is quinone molecule.

Tyrosinase from A. bioprus is a heterotetramer, with heavy and light chains with
molecular weight of around 120 kDa. Tyrosinase enzyme has three domains, of which the
central domain contains two Cu binding sites, called Cut and C113. After comparing
sequences from different sources, the only conserved domain seems to be the central
copper-binding domain, which also shares sequence homology with hemocyanins (Hes).
Six conserved histidine residues which bind with a pair of copper ions in the active site of
the enzyme tyrosinase. This active side interacts with both molecular oxygen and its
phenolic substrate. The stability of protein is also determined by the location of cysteine
(cys). The number of Cys residues varies from one organism to another, as along the N-
terminal and central part of the protein, human and mouse tyrosinases have 17 Cys

residues and plants have 11, whereas the C-terminal domain contains 1 Cys residue [82].

Immobilization is the most important step in application of biocatalyses and
biosensors. There exists several paper discussing methodology to immobilize tyrosinase
in active conformation and then measure its activity. Further such methods are mainly
based on chemical and physical mechanisms [83]. One of the most commonly used
methods is by modifying electrode surface to impart amine groups and then using
glutaraldehyde to immobilize‘tyrosinase on it. This is an efﬁcient method to immobilize

enzymes without making it loose much of its activity [84-86]. Other method involves

42

using sol-gel method [87, 88] and also by forming pellets of graphite-teﬂon and

Tyrosinase [89].

In this chapter we utilize layer-by-layer deposition technique [27], commonly
abbreviated as LBL deposition technique, to deposit multilayers of tyrosinase. LBL
deposition technique, developed by Decher in early nineties, used electrostatic interaction
between oppositely charged surface and the polyelectrolytes in solution for layer by layer
adsorption of polyelectrolytes on the surface. It is done on a surface having certain
charge, positive or negative, and it is followed by dipping in solution containing
oppositely charged species. This oppositely charged species adsorbs itself onto the
surface and also leads to the charge reversal. Loosely bound molecules are removed by
washing in water and then it is followed by dipping the surface with one adsorbed species
into a solution containing oppositely charged species. This process is repeated to form
multilayer of these polyelectrolytes. We exploit net negative charge on the enzymes for
its adsorption on positively charged surface. As LBL deposition does not have any
covalent attachments so it should lead to more active immobilization onto the surface.
Also due to the versatility of LBL deposition technique we were able to fabricate various

architectures which in turn led to very high sensitivity for catechol sensor.

43

4.2 EXPERIMENTAL SECTION

4.2.1 Materials

Thioctic acid, poly-L-lysine (PLL) (molecular weight ~ 15,000), tyrosinase (Tyr),
sodium phosphate (monobasic and dibasic), cystamine, gold chloride trihydrate,
hydroxylamine, sodium citrate dihydrate, sulfonated polystyrene (Mw~70,000),
Poly(dimethyldiallyl amonium chloride (PDAC, Mw~100,000) were purchased from
Sigma. Polyallyl amine hydrochloride (PAH, Mw~60000) were purchased from
Polysciences, Inc. Silver membranes and copper grid for Transmission Electron
Microscopoy (TEM) were purchased from SPI supplies. Multiwalled carbon nanotubes
(8-15 nm diameter, 0.5-2um in length) from Cheaptubes, Inc. Ultrapure water (18.2MO)
was supplied by a Nanopure-UV four-stage puriﬁer (Bamstead International, Dubuque,
IA); the puriﬁer was equipped with a UV source and a ﬁnal 0.2 pm ﬁlter.

4.2.2 Preparation of Au nanoparticles

Gold nanoparticles were prepared by the standard procedure of reduction of gold
salt by sodium citrate. 500 mL of 1 mM HAqu was brought to a rolling boil with
vigorous stirring. Rapid addition of 50 mL of 38.8 mM sodium citrate to the vortex of
the solution resulted in a color change from pale yellow to burgundy. Boiling was
continued for 10 minutes; the heating mantle was then removed, and stirring was
continued for an additional 15 minutes [56].

4.2.3 Modification of multiwalled carbon nanotubes

Multiwalled carbon nanotubes (MWCNTs) are hydrophobic in nature.

Consequently to disperse them in aqueous solution, 1 mg/ml of MWCNTs was tip-

44

sonicated in 1 mg/ml of sulfonated polystyrene (SPS) for 30 minutes. The suspension was
ﬁltered through 220 nm membranes and ﬁltrate was collected and redispersed in de-
ionized water. This process of washing was repeated twice to get rid of free and loosely
bound polyelectrolyte. Then t0.5 mg/ml of poly-lysine was added and again sonicated for
30 minutes followed by three washings. The polyelectrolyte coating were characterized
by Transmission Electron Microscopy and by measuring the zeta potential of coated
nanotubes. PDAC coated MWCNTs were prepared by dispersing SPS coated MWCNTs
in 10 mM solution of PDAC sonicated for 30 minutes and followed by 3 washings.

4.2.4 Enzyme loading onto gold slides and silver membranes

Commercially available evaporated gold coated silicon wafers were cleaned in
Piranha (70% Sulfuric Acid and 30% Hydrogen Peroxide) solution for 20 seconds. This
was followed by thorough washing of gold with copious amount of de—ionized (DI) water
and ethanol. After cleaning the gold slides were dipped in 5mM amine terminated
alkanethiol (cystamine) in ethanol for 30 mins. Monolayer of cystamine molecules forms
on the gold surface. These amine groups impart positive charge over which negatively
charged tyrosinase (Tyr) was adsorbed by dipping modiﬁed gold slides into a solution of
tyrosinase in deionized water (0.25mg/ml). In other case before dipping cystamine (Cys)
modiﬁed gold electrode in the tyrosinase solution; the electrode was dipped in gold
nanoparticle (AuNPs) solution for 30 mins, washed and then dipped in tyrosinase
solution.

For other architectures, Piranha cleaned gold slides were dipped in 10 mM Lipoic
acid in ethanol for 30 mins. This was followed by layer-by-layer (LBL) deposition of 0.2

mg/ml of poly-L-lysine (PLL) in phosphate buffer (pH=8.4) and tyrosinase (Tyr) in

45

deionized water. LBL was done by alternative dipping of thioctic acid modiﬁed gold
electrode in as shown in Figure 4.1a. Instead of using polyelectrolytes, PLL coated
MWCNTs (ﬂVIWCNTs) were used for layer-by-layer self assembly using enzyme and
EMWCNTs as shown in Figure 4.1b. In another immobilization scheme polymer
nanospears, as fabricated in chapter 2, were coated with polyallyl amine hydrochloride
(PAH) to impart positive charge. A self assembled monolayer of cystamine was formed
on the gold slide. PAH coated nanospears were dropped on the cystamine modiﬁed gold
electrode and crosslinked onto the surface using glutaraldehye. Over this a monolayer of
Tyr was adsorbed by electrostatic interaction by dipping nanospears modiﬁed gold
electrode in a solution of Tyr. The whole procedure is illustrated in Figure 4.1c

In ﬁnal architecture instead of gold slides, commercially available silver
membrane with pore size of 5 pm was used. Silver membrane behaves similar to gold
electrode with respect to self-assembled monolayers formed by thiol terminated
compounds. Silver membranes were cleaned by plasma cleaner for 10 mins. They were
dipped in 10 mM lipoic acid in ethanol. The membranes was washed with ethanol and
dried with nitrogen. This was followed by LBL deposition of PLL and Tyr. A Scanning
electron micrograph of the silver membrane is shown in Figure 4.1d.

4.2.5 Amperometric i-t curves

Amperometric i-t studies were done using CHI-instruments. The enzyme loaded
gold electrodes were used as working electrode and it was maintained at -100 mV against
an Ag/AgCl reference electrode. A three electrode system was used and all the electrodes

were dipped in 125 m1 of lOOmM phosphate buffer at pH=7. Then catechol was used as a

46

analyte and a step current was observed for each addition of 8 uM of catechol in bulk. The
current response was studied for various architectures.

4.2.6 Cyclic Voltammetry

Silver electrodes used were porous structure with larger surface area per unit
projected area. So to determine the effective surface area cyclic voltammetry was carried
out in nitrogen purged 0.5M sodium sulfate solution in DI water. Scans were obtained at
scan rate of 0.1 V/s and range of scan was -0.35V to +0.15 V.

4.2.7 UV-Vis absorbance spectroscopy

UV-vis absorbance spectroscopy was used to determine the enzyme loading onto
the electrode. Different enzyme concentration was added to 5 mM catechol solution in
100 mM sodium phosphate buffer at pH=6.5 and reaction was allowed to continue for 10
minutes and then the absorbance was measured at 380 nm. This was used to make
calibration plot. Amount of enzymes onto electrode was determined by immersing
electrodes in 5 mM catechol for 10 minutes and measuring absorbance at 380 nm. SP-890
UV-Vis Turner Spectrophotometer was used for the experiment.

4.2.8 Microscopy

Scanning Electron Microscopy (SEM) was done on LBL deposited modiﬁed
carbon nanotubes on gold electrode and as received silver membrane. No sputter coating
with gold or osmium was needed and SEM used for high resolution imaging was JEOL
6300F with ﬁeld emission. TEM was done by coating copper grid with holey carbon ﬁlm
and drying small amount of suspended pure or functionalized carbon nanotubes.
Microscope used was JEOL 2200FS 200 kV ﬁeld emission TEM. The above mentioned

microscopes were located at Centre of Advanced Microscopy, MSU.

47

— "afforyuﬂm .

(Tyr) NH3 , g Tyrosinase
bilayers

     

    

 

 

 

Poly-lysine -—--'> J
(PLL)
Thioctic acid _,
S—S
l T
Gold
electrode (a)

PDAC-SPS or PLL-
. :,ﬂ,%ws.rss SPS functionalized
l MWCNTS.

     

m ++++ hit-“m “H

C00- C00- ”C00- ‘ ‘

$53»

1 Addition of Tyrosinase

Magﬁmqyrmr

C00- C00- NCOO- ‘ ‘

ésésés

(b)

Figure 4.1: Different architectures for catechol sensor: (a) Thioctic acid modified
gold electrode and layer-by-layer (LBL) deposition of tyrosinase and poly-lysine, (b)
same as (a) but functionalized multiwalled carbon nanotubes instead of poly-lysine
(continued).

48

Q ' NH;

 

((0
Figure 4.1: (c) Polymer nanospears drop coated and cross-linked onto cystamine
modiﬁed electrode and then a layer of tyrosinase adsorbed over it, and (d) Scanning
electron micrograph of silver membranes used for tyrosinase and poly-lysine
deposition using LBL deposition.

49

4.3 RESULTS AND DISCUSSIONS

4.3.1 Layer-by-Layer of Poly-L-Lysine and Tyrosinase

The current response for the biosensor showed very less reaction time with almost

a step response on the addition of catechol as shown in Figure 4.2.

 

 

 

 

300

A 250 —
NE

0 200 ~

21

r 150 -

C ..

a) 100 - Addition of8 [M

E catechol

0 50 - \

0 I I I I I I
0 1O 20 3O 40 50 60 70

Time(sec)

Figure 4.2: Tyrosinase biosensor for catechol as substrate, the electrode was
maintained at -0.1V vs Ag/AgCl as reference electrode. Each step is aliquot of
catechol added to achieve final concentration of 8 ”M in bulk.

On the architectures based on gold slides, the best response was observed for PLL- Tyr
bilayer system. As shown in Figure 4.3 the sensitivity increased as the number of bilayer
increased but after 6 bilayers it reached a plateau. This is due to the process now
becoming diffusion limited as additional number of layer of enzymes does not enhance
the sensitivity. This implies that the process in now limited by ﬂux of quinone to the
electrode through the enzyme layer, rather than the kinetics of the system. The build up

of bilayers was also characterized by Fourier transform infrared (FTIR) spectroscopy.

50

The addition of each layer increased the peaks of strong absorption at 1665 cm‘1 (C=O

stretching of amide group), 1540 cm'1 for N-H bending mode and 3300 cm'1 for N-H

stretching mode as shown in Figure 4.4.

 

 

 

 

8

7. l .
“E“ i l i l t
is] i
214‘ I
:3‘ i
3
02_

Q

1_

0 . . . I

0 2 4 6 8 10

Number of Bilayer

Figure 4.3: Bilayer versus Current sensitivity plot for PLL-Tyr biosensor. The

sensitivity with different number of bilayers increased till 6 bilayers and then
dropped and stabilized.

51

0.10

 

1665 cm'1

- 0.08

r 0.06

- 0.04

Absorbance

- 0.02

 

 

 

- 0.00

3600 3200 2800 2400 2000 1600 1200
Wavenumbers (cm'1)

 

Figure 4.4: FTIR spectra after each layer: 1665 cm'1 (C=O stretching of amide
group), 1540 cm'1 for N—H bending mode and 3300 cm‘1 for N-H stretching mode.

4.3.2 Incorporation of Multiwalled Carbon Nanotubes

As the process became diffusion limited at higher number of bilayers, a scheme
was developed were MWCNTs were functionalized with polyelectrolytes. We postulated
that if we can incorporate a conducting path by putting MW CNT 5 through the enzyme
layer it might increase the sensitivity or shift the saturation of biosensor to higher number
of bilayers. Functionalization was necessary as pure nanotubes are inherently
hydrophobic. So it is required to modify carbon nanotubes to achieve two properties.
Firstly, to make the carbon nanotubes hydrophilic as enzymes and hence, biosensors
works in aqueous media. Secondly, we needed a way to deposit nanotubes on the surface

of the electrode using LBL deposition technique. Hence, Sulfonated polystyrene (SPS)

52

was used as ﬁrst layer as SPS adsorbs readily on the carbon nanotubes and also imparts
sﬁong negative charge on the surface.

Therefore, MWCNTs were coated with SPS and its zeta potential shifted to a
negative value. Then these SPS-MWCNTs were coated with PLL to get positively
charged ﬂVIWCNTs for layer by layer self assembly. The shift in zeta potentials is given

in Table 4.1.

Table 4.1 Zeta potential of polyelectrolyte coated multiwalled carbon nanotubes

 

 

 

 

MWCNTs Zeta Potential
(mV)
SPS coated -49.00 i.89
PLL-SPS-coated 46.36 i 1.68
PDAC-SPS-Coated 53.85 i 1.31

 

 

 

 

TEM was also done to qualitatively show the coating of MWCNTs. Figure 4.5a is
a TEM micrograph of pure MWCNTs and Figure 4.5b shows the micrograph of coated
fMWCNTs. As it can be seen the surface of fMWCNTs is much rougher than pure
MWCNTs. The comparative sensitivities for initial system and new system at high
number of bilayers are shown in Figure 4.6. As seen in ﬁgure there is not signiﬁcant
difference in the sensitivity. It was also studied for the response at lower number of
bilayers and response was similar (data not shown).As it can be seen the sensitivity does
not increase with addition of carbon nanotubes with PLL-Tyr system. Hence addition of
CNTs is not advantageous in this system. One of the possible reasons for this is that the
diffusional resistance for initial PLL-Tyr system is already reduced due to the porous

bilayer structure of PLL-Tyr system. This was observed by Katz et al, where they showed

53

PLL forms porous bilayers and hence offers less resistance for the redox species difﬁrsing

through the bilayers [90].

 

Figure 4.5 a) TEM of pure multiwalled carbon nanotube and b) polyelectrolyte
coated MWCNTs.

54

Current (uAlpMcm’)
N U h 0' a: N

d
I

 

 

O

 

 

8 PL-SPS-CNTslTy 8 PLlTyl'o

Figure 4.6: Comparative plot of sensitivity for PLL coated MWCNTs and pure PLL
tyrosinase LBL system.

Additional experiment were also carried out, where a strong polyelectrolyte,
Poly(diallyl dirnethyl ammonium chloride) (PDAC) was used instead of PLL. PDAC is a
strong polyelectrolyte and should form a uniform layer on the surface and should offer
more diffusional resistance to diffusion of quinone to the electrode. In comparison a
similar system with PDAC-SPS coated MWCNTs were prepared and their zeta potentials
are tabulated in Table 3.1. Architectures of both, PDAC-Tyr bilayers and PDAC-
MWCNTs-Tyr were studied for their sensitivities and response as shown in Figure 4.73.

Figure 4.7b is a SEM image of PDAC—MWCNTs deposited by LBL technique.

55

30.00

 

25.00 -

A 20.00 -

'8

  
  
 

3 Bilayers

Current (M

10.00 -

5-00 1 Control

 

 

0:00 I I I I I
0.00 50.00 100.00 150.00 200.00 250.00 300.00
Tlme (Sec)

 

(a)

 

Figure 4.7: (a) Comparative response curve for 3 bilayers of PDAC coated
MWCNTs/Tyrosinase and pure PDAC/'1‘ yrosinase (Control) system, the difference
in sensitivity was significant, (b) SEM image of LBL fMWCNTs on electrode.

56

In this case, PDAC-MWCNTs containing biosensor yielded higher sensitivity as
compared to PDAC-Tyr system, though the overall sensitivity was much lower when
compared to other systems that were studied. The architecture with immobilized polymer
nanospears also showed lesser sensitivity as compared to PLL-Tyr system as there was
not sufﬁcient enzyme loading and polymer nanoparticles do not enhance conductivity of
the bilayers as well.

4.3.3 Silver membrane as electrode

Another architecture used a commercially available porous silver membrane as
electrode. As gold and silver has similar behavior towards the self-assembled thiol-
terminated monolayers. The same scheme was used as the one used for gold electrode.
The silver electrode are stable at the potential and pH range we test the electrode for
biosensor. As the silver electrode has porous structure, the amount of electroactive area
for a unit projected area is greater than the planar electrode. The effective electroactive
area was estimated by obtaining a cyclic voltammogram of porous silver electrode and a
planar silver electrode in 0.5M sodium sulfate at a scan rate 0.1V/s. The voltammogram
was obtained for blank silver porous electrode and planar electrode is shown in Figure
4.83. As the charging current is directly proportional to the product of the area of the
electrode and the scan rate, it can be used to determine the surface area of porous
electrode. The ratio of average charging current for a porous and planar electrode gives
the ratio of area of porous and planar electrode. It was approximately 3. So there is 3
times more active surface area in a porous electrode as compared to planar electrode.

Enzyme loading for each bilayer was determined using UV-vis absorbance.

Different amounts or units of tyrosinase were added to a ﬁxed volume (2 ml) of 5 mM

57

catechol solution. The reaction was allowed to continue for 10 minutes, where catechol
was oxidized to quinone and the absorbance was measured at 380 nm. The calibration
plot was made by measuring absorbance for quinone with known quantity of enzymes
added to catechol solution. This was followed by measuring absorbance for quinone for
solution with various electrodes immersed in the catechol solution for 10 minutes. This
gives the amount of equivalent active enzyme loading on the various electrodes. The plot
of enzyme loading versus number of bilayers and on silver membrane is given by Figure
4.8b.

Figure 4.8b shows increase in enzyme loading as number of bilayers increased
though the amount of enzyme loading increased beyond six bilayers but the current
saturates, as was shown in Figure 4.3, after certain number of bilayers because of
diffusion limitation of quinone that was produced in the enzymatic layer. It implies the
catechol that is oxidized to quinone has to diffuse through the enzyme layer before it gets
converted to catechol by the electrode. Though the amount of enzyme is increased at the

interface the current saturates because of diffusion limitation of quinone to electrode.

58

 

 

 

 

 

 

 

 

400.00 . . . r .
~0.4 -0.3 -0.2 -0.1 0 0.1 0.2

PotentialWolts)
(a)

 

600

 

§

.5
O
O

 

 

 

 

 

Active Enzyme Loading(nglcm2)
8
O

 

 

 

 

 

 

200
100
0
1
Bilayers
(b)

Figure 4.8: (a) Cyclic voltammogram of a silver planar as a control electrode and
porous silver membrane as electrode in 0.5M Na2S04 purged with nitrogen at scan
rate of 100 mV/s from -0.35V to +0.15V. (b) Amount of equivalent active enzyme
loading on planar electrodes per unit area until 8 bilayers and on porous silver
membrane. The amount of enzyme increased as number of bilayers increased.

59

Silver membrane electrode showed much larger enzyme loading than a planar
electrode because of its porous nature, consequently showing maximum sensitivity as
shown in Figure 4.10. This is due to higher enzyme loading as well as shorter diffusion
distance for quinone to diffuse through to the electrode. The comparative current versus
time response curve for most sensitive biosensor on planar electrode that corresponds to
six bilayers of PLL-Tyr and sensor on silver membrane is given in Figure 4.9. The
relative sensitivity plot for all the systems on planar electrode and silver membrane is
given by Figure 4.10. The sensitivity for porous electrode was much higher than for six
bilayers on planar electrode even though the tyrosinase loading was comparable. This is
because 6 bilayers increases the distance between the site, where catechol is oxidized to
quinone within the bilayers, and the electrode surface where actual potential is applied.
Hence quinone has to diffuse larger distance before it could reach the electrode whereas
on a porous electrode the diffusion distance is much lesser. This is illustrated in the
scheme shown in Figure 4.11. Figure 4.11(a) shows the case where the enzyme loading is
increased by increasing the number of bilayers and quinone (P) formed by catalysis of
catechol (S) has to diffuse through the enzyme layer before it feels the potential applied
on the electrode. Whereas in a higher surface area as shown in Figure 4.11(b) this

diffusion distance is greatly reduced.

60

120.00 -

100.00 -

Silver Memba\ne

40.00 * Addition of I

Catechol . ‘6 Bilayers PLL-Tyr

0.00 \ r I l I I

0.00 20.00 40.00 60.00 80.00 100.00
Time(Sec)

  
    
 

Current(|.rAlcm 2)
m a
C O
8 8

 

 

Figure 4.9: Comparative response curve for 6 Bilayer PLL-Tyr and catechol
biosensor on porous silver membrane.

20
18
16

Sensitivity(pA/(|1Mcm 2))
3

 

Figure 4.10: Comparative sensitivity plot for different architectures.

61

   

Applied
potentia

 

 

Applied ‘ ..
pote tial ]

 

 

(b)

Figure 4.11: (a) Diffusion of quinone (P) through the enzyme layer on a planar
electrode. The distance for quinone to diffuse to the electrode surface is much
larger. (b) As compared to the other electrode, which has higher enzyme loading
due the higher surface area while keeping the diffusion distance small (b).

62

It was also veriﬁed if the difference in sensitivity could be attributed to silver. It
was done by making same biosensor by LBL deposition process on a planar silver
electrode. Silver planar electrode was modiﬁed in the same manner as porous electrode
and bilayers of PLL-Tyr were built. It was tested for its activity against catechol and
sensitivity was found same as a biosensor built on planar gold electrode. Hence, there
was no difference in sensitivity for a catechol sensor made on a planar silver or gold

electrode.

Figure 4.12a gives the calibration plot for catechol sensor for 6 bilayers PLL-Tyr
sensor and Figure 4.12b gives calibration plot for catechol sensor on silver electrode. The
calibration plot for 6 bilayer sensor had longer linear range ﬁom 8 M to 40 uM and for
sensor of silver electrode had linear range from 2 M to 8 M. This could be because the
current density was very high at smaller concentration in silver membrane which could
lead to depletion of oxygen in enzymatic layer. The number of bilayers on silver
membrane was also increased but it did not increase the sensitivity (data not. shown)
possibly because of oxygen depletion in the enzymatic layer. As at such a high current,
the rate the consumption of oxygen within the enzymatic layer is very high.

Purging the buffer with oxygen increased the sensitivity of biosensor on porous
silver electrode by 25% since increasing the oxygen in buffer increased the oxygen ﬂux
to the electrode. Purging buffer with nitrogen decreased the sensitivity to negligible as
tyrosinase could not be regenerated to oxy-tyrosinase state from the deoxy-tyrosinase
state. This is because of absence of oxygen, retarding the regeneration of oxy-tyrosinase.
Whereas on planar electrodes purging buffer with oxygen did not show any appreciable

change in the sensitivity of the biosensor as sensitivity is governed more by diffusion of

63

quinone through the bilayers to the electrode. Oxygen is still present in sufﬁciently high

concentration to not to limit the sensitivity.

6 bilayers

 

or
8

§§§§

1004

Current (pAIcm 2 )

 

 

OS

 

I I I l I

20 30 40 50 60
Catechol Conc (uM)

O
a.
O

(a)

Silver Membrane

 

160
140 -
120 -
100 -
80 -
60 .
40 -
20 -
0 u l I I I

0 2 4 6 8 10 12

Catechol Conc (pM)

Current (pAlcmz)

 

 

 

(b)

Figure 4.12: Sensitivity plots for a) 6 bilayers of PLL-Tyr on planar gold and b) 1
layer of PLL-Tyr on porous silver electrode.

4.4 CONCLUSIONS AND FUTURE WORK

In this chapter we studied various architectures to fabricate catechol biosensor. A
layer by layer approach was used which can easily be extended to various kinds of
nanostructures. Poly-lysine-Tyr LBL self assembly architecture imparted highest
sensitivity for biosensor response for planar electrodes and additional nanostructures did
not impart better sensitivity. Though using high surface area supports like porous silver
membrane can enhance higher sensitivity without making the whole response process
diffusion limited. Also this process is versatile that can be extended to other form of
electrodes like carbon based electrodes where this sensor was fabricated on a 96 well

plate.

65

S NEUROPATHY TARGET ESTERASE BIOSENSOR

5.1 INTRODUCTION

Neuropathy Target Esterase (NTE), is a membrane-bound proteins found in
neurons of vertebrates [91-96], has been shown to be necessary for embryonic
development in mice, and is believed to be involved in cell-signaling pathways and lipid
trafﬁcking. [91] NTE has serine esterase activity and can hydrolyze ester, peptide, and
amide bonds. The nucleophilic serine residue (active site) of NTE attacks the carbonyl
carbon atom of the substrate, forming a covalent acyl-enzyme intermediate, which is
subsequently hydrolyzed. A consequence of this reaction mechanism is that the esterase
activity of NTE is susceptible to covalent inhibition by Organophosphorus Esters (OPS)
with which it forms an analogous phosphyl-enzyme intermediate. Irreversible binding of
some OP compounds to the active serine site results in a debilitating neural disease
known as (OP) Iinduced Delayed Neuropathy (OPIDN) [91]. Symptoms of OIPDN
include ﬂaccid paralysis of the lower limbs, which becomes evident two to three weeks
after exposure to neuropathic OPs. Recovery from this disease is usually poor, and there
is no speciﬁc treatment. Because NTE is difficult to produce for research purposes,
research to study its esterase activity is typically done using a fragment of the NTE
protein that contains the esterase activity and can be more easily produced One such
ﬁ'agment, known as NEST,[93, 94, 97] reacts with esters and inhibitors in a manner very
similar to NTE.

Because NTE plays a central role in both chemically induced and spontaneously

occurring neurological diseases, approaches that can help measure its esterase activity

66

and inhibition are of tremendous scientiﬁc and commercial importance. Conventionally,
the esterase activity of NTE (or NEST) is measured using two distinct steps. In the ﬁrst
step, a solution containing phenyl valerate is brought into contact with NEST or NTE
protein solution, whose esterase activity reacts with a portion of the artiﬁcial substrate
phenyl valerate to form phenol. In the second step, the concentration of phenol in the
solution is determined either colorimetrically, in the presence of 4-amino antipyrine [98],
or electrochemically, in the presence of tyrosinase enzyme [99, 100]. Tyrosinase
converts phenol ﬁrst to catechol and then to o-quinone, which can be measured
electrochemically at an electrode [95]. The current generated by the electrode increases
with the amount of o-quinone present, thus giving an indirect measurement of the amount
of NTE esterase activity present during the ﬁrst step. To test for esterase inhibition, this
procedure is repeated both in the absence and presence of a putative inhibitor (e.g., an OP
compound). A reduced signal indicates inhibition of the esterase activity. This method
has the disadvantages of being slow, and requiring two steps, making it unsuitable for
some important applications, such as high-throughput screening of compounds for NTE
inhibition and continuous, on-line, enviromnental monitoring to detect chemical warfare
agents that target NTE.

This work presents the ﬁrst continuous, electrochemical biosensor for real-time,
rapid measurement of NEST (or NTE) esterase activity. The biosensor was fabricated by
co-immobilizing NEST protein and tyrosinase enzyme on an electrode using layer by
layer assembly approach by Decher [27]. To our knowledge, this is the ﬁrst time NEST
has been immobilized in an active conformation on an electrode. Potential applications of

this sensor include detecting the presence of chemical weapons that target NTE,

67

screening industrial and agricultural OP compounds for NTE inhibition, studying the
fundamental reaction kinetics of NTE, and investigating the effect of NTE mutations
found in Amyotrophic Lateral Sclerosis or Lou Gehrig’s disease (ALS) patients on

NTE’s enzymatic pr0perties.

5.2 EXPERIMENTAL SECTION

5.2.1 Materials

Thioctic acid, poly-L—lysine (PLL) (molecular weight ~ 15,000), tyrosinase (Tyr),
sodium phosphate (monobasic and dibasic), ethylenediaminetetraacetic acid (EDTA),
sodium chloride, 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate
(CHAPS) and isopropyl tlriogalactoside (IPTG). Ultrapure water (18.2MQ) was supplied
by a Nanopure-UV four-stage puriﬁer (Bamstead International, Dubuque, IA); the
puriﬁer was equipped with a UV source and a ﬁnal 0.2 pm ﬁlter.

5.2.2 NEST expression and purification

NEST was expressed and puriﬁed according to published procedures [93].
Brieﬂy, DNA fragment encoding NEST was cloned into pET-21b vector, and the
resulting expression vector was transformed into E. coli BL21 (DE3). An overnight
culture of transformed Ecoli was inoculated with M9 media containing ampicillin and
grown in a ferrnentor. IPTG was added to the resulting cell culture after a day to induce
the expression of NEST. The resulting cells were collected 4 h after induction by
centrifugation and subjected to protein expression techniques. Brieﬂy, 5 g of cell paste
was suspended in 30 m1 of PEN buffer (SOmM sodium phosphate/0.3 M NaCl/0.5 mM

EDTA, pH 7.8) containing 2% CHAPS and tip sonicated four times. The cell lysate was

68

centrifuged at 12000 rpm for 30 min at 4°C, the supernatant was collected, and about 7
mL of supernatant was added to a mini column (volume 10 mL) containing 3 mL of Ni—
nitrilotriacetic acid (NTA) resin. The mini column was rotated at room temperature for 20
min, centrifuged at 2000 rpm for 20 sec and then the top solution was drawn off. The
histidine tagged NEST was eluted from the Ni-NTA resin using 10 mL of PEN buffer
containing 0.3% CHAPS and 0.3 M irnidazole. The protein purity was determined using
SDS PAGE and protein concentration was determined using BioRad Dc protein assay kit.

For long term storage, 25% glycerol was added to the protein solution, which was then

stored at -20°C.
5.2.3 Preparation of gold electrode for NEST biosensor

Tyrosinase is a copper-containing oxidase [101, 102], which possesses two

different activities, as illustrated in reaction 1.

phenol —— catechol
: o-quinone + H20 ....(5.1)

 

The ﬁrst step is referred to as the enzyme’s hydroxylase activity (also known as
cresolase activity) where phenol is hydroxylated by the aid of molecular oxygen to
produce catechol. In the second step, known as the catecholase activity, the enzyme
oxidizes catechol to o—quinone and is simultaneously oxidized by oxygen to its original
form, with the production of water. The reaction product, o-quinone, is electrochemically
active and can be reduced back to the catechol form at low applied potentials, as

illustrated in reaction 2.

o-quinone + 2H+ + 29- -) catechol ............................ (5.2)

69

These characteristics of tyrosinase were exploited by us to fabricate a NEST
biosensor, capable of measuring the NEST’s esterase activity and its inhibition, by co-
immobilizing NEST and tyrosinase on a gold electrode using layer by layer assembly
approach.

The molecular architecture of the biosensor interface is shown schematically in
Figure 5.1. Gold electrodes cleaned in Piranha solution, were dipped in 5 mM solution of
thioctic acid in ethanol for 30 min. The electrodes were washed with ethanol, dried under
nitrogen and dipped in PLL solution for 45 min. The PLL solution was prepared by
adding 12 mg of poly-L—lysine in 50 mL of 20 mM phosphate buffer (pH 8.5). The
electrodes were then rinsed with water and dipped in an aqueous solution of Tyrosinase
(Tyr) (0.2 mg/ml) for 1 h. The last two steps were repeated varying number times to
create PLL-Tyr bilayers with PLL being the topmost layer. The electrodes were washed
with water and dipped in a solution of NEST protein (0.1 mg/ml) in 100 mM phosphate
buffer, pH (7.0) for l h. The electrodes were then washed with water, dried under

nitrogen and dipped in phosphate buffer (0.1 M, pH 7.0) for testing.

70

     

Poly-lysine
and
tyrosinase
bilayers

NH3+ }

Gold
electrode

Figure 5.1: Molecular architecture of NEST biosensor

5.2.4 Preparation of phenyl valerate solution

To prepare phenyl valerate solution, 15 mg of phenyl valerate was dissolved in 1
mL of dimethylformamide (DMF), and 15 mL of water containing 0.03% Triton was
added slowly under vigorous stirring. For potential step voltammetry experiments, small
aliquots of the resulting phenyl valerate micellar solution (5.286 mM) were added to the
phosphate buffer to obtain the desired concentrations.

5.2.5 Ellipsometry

Ellipsometric measurements were obtained with rotating analyzer ellipsometer
(model M-44; J.A. Woollan Co. Inc., Lincoln, NE) using WVASE32 software. The
thickness values for dried ﬁlms were determined using 44 wavelengths between 414.0
and 736.1 nm. The angle of incidence was 75° for all experiments. Refractive indices of

ﬁlms containing PLL and proteins was assumed to be n=1.5, k=0. These optical constants

71

compare well with those determined for 4 bilayer ﬁlms consisting of poly-L—lysine and
tyrosinase using ellipsometry.

5.2.6 Potential step voltammetry and other measurements

The electrodes (sensors) were maintained at a potential of -100 mV (vs Ag/AgCl
reference electrode) using a BAS CV-50W electrochemical analyzer. The esterase
activity of NEST biosensor was monitored by measuring the output current for a variety
of phenyl valerate concentrations, under stirred conditions. The NEST protein converts
phenyl valerate to phenol, which gets converted to o-quinone by tyrosinase. The 0-
quinone gets reduced at the electrode’s surface, resulting in the generation of current. The
electroreduction of o-quinone produces catechol which again gets converted to o-quinone
by tyrosinase, thus amplifying the signal.

To measure inhibition of the esterase activity, a known quantity of phenyl valerate
was added to the phosphate buffer (pH 7.0), under stirred conditions. After the
stabilization of current, a known amount of NEST inhibitor was added, and the resulting

drop in current was measured.

5.3 RESULTS AND DISCUSSION

5.3.1 Ellipsometry

Ellipsometry were used to conﬁrm the deposition of different layers that make up
the NEST biosensor. As shown in Figure 5.2, the thickness increase following the
addition of ﬁrst PLL and Tyr bilayer was approximately 9.3 i 0.4 nm. The thickness

increase for the next two PLL-Tyr bilayers was the same and equal to approximately 7.2:l:

72

0.3 nm. The thickness increase following the addition of ﬁnal PLL-NEST bilayer was

approximately 6.6 i 0.3 nm.

 

 

 

 

35
30 - 5 1"
E 25 ‘ i (d)
w 20~
8 s
_ (c)
g 15
.9
ES 10 “ i (b)
5 -4
..(3)
0 ” . . . .
0 1 2 3 4 5

Number of bilayers

Figure 5.2: Ellipsometric thicknesses after the successive addition of following
layers: thioctic acid (point a), PLL-Tyr first bilayer (point b), PLL-Tyr second
bilayer (point c), PLL-Tyr third bilayer (point (i), and PLL and NEST final bilayer
(point e).

5.3.2 Dependence of current response on working potential and pH

The various experimental parameters (such as pH and applied potential), which
can affect the amperometric determination of phenyl valerate, were optimized. The effect
of applied potential on the amperometric response of the sensor was tested in the range

between 0.05 and -0.20 V. Figure 5.3a illustrates the signal and background for the whole

73

 

Current (pA/cmz)

m

A
-

.0 .0 .O .O t" T‘ .—‘
O N b O) 03 -‘ N h C)
r r r r 1 r r

 

 

l

I T I I

-025 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1
Potential (V)

-L
.h

 

Current (uA/cmz)
.o .o .o .4
-h 0) on A N

.0
N

 

O

 

 

01
O)
V _
on
(0

pH

Figure 5.3: (a) Effect of working potential on the response current of the enzyme
electrode in 0.1 M phosphate buffer (pH 7.0) with (i) and without (ii) 12 pM phenyl
valerate solution, in 0.1 M phosphate buffer at an applied potential of -0.1 V (vs
Ag/AgCl). (b) Effect of pH on the response current of the electrode, in the presence

of 12 pM phenyl valerate solution, in 0.1 M phosphate buffer at an applied potential
of -0.1V (vs Ag/AgCl).

74

range. The highest signal-to background ratio, was obtained at -0.1 V. At working
potential more negative than -0.1 V, higher signals were obtained, but the background
current also increased distinctly. Therefore, a working potential of -0.1 V was used for
further studies. The effect of pH was also studied in the pH range 5.5 to 8.0 in 0.1 M
phosphate buffer at working potential of -0.1 V. As shown in Figure 5.3b, the response
current attained a maximum value at pH 7.0. This pH was used for further studies.

5.3.3 Measurement of esterase activity using NEST biosensor

Figure 5.4a displays a typical current-time response under the optimal
experimental conditions after the successive addition of aliquots of 4 uM phenyl valerate
to the phosphate buffer. A well deﬁned reduction current, proportional to the amount of
phenyl valerate, was observed. The response time of the electrode was less then 20 5, due
to the nano-scale thickness of the interface. The response to phenyl valerate was linear
(r=0.991) in the range 0.5 uM to 12 uM, and it reached saturation at approx. 30 pM
(Figure 5.4b). The limit of detection was 0.5 uM at a signal-to-noise ratio of 3. The
reproducibility of the sensor was investigated at a phenyl valerate concentration of 4 uM;
the mean current was approximately 348 nAcm'Z, with a relative standard deviation of
9.9%. Figure 5.5 shows a control experiment which was done on an electrode containing
only poly-L-lysine and tyrosinase bilayers. As expected, a relatively very small rise in
steady state current was observed on the addition of phenyl valerate. The small rise can
be attributed to the presence of small amount of phenol produced due to auto hydrolysis

of phenyl valerate solution.

75

 

1.50

Addition of 4uM
Phenyl valerate

\

0.00 " . l .
0 50 100 150 200

Time (see)

(a)

Current (uA/cmz)

.0 .0 .0 7"

or a: «a N

o o o o
I l l I

 

 

 

 

 

 

 

 

0 r I I I I I
0 5 10 1 5 20 25 30 35

Concentration (uM)

(b)

Figure 5.4: (a) Current time response of the NEST biosensor to the addition of
aliquots of 4 pM phenyl valerate, in 0.1 M phosphate buffer, pH 7.0, at an applied
potential of -0.1V (vs Ag/AgCl). (b) Calibration plot.

76

p
8

 

 

 

 

NA

E 0.30 4

é’

3 0 20 , Addition ofauM

g ' Phenyl valerate

t:

3
0 0.10 4W

O.m I I I I I I I I I

0102030405060708090100
Time(sec)

Figure 5.5: Control experiment: Current time response on an electrode containing

only tyrosinase. The electrode was assembled in exactly the same way as NEST
biosensor, except that the ﬁnal NEST layer was not deposited.

77

5.3.4 Inhibition of esterase activity

To measure inhibition of the esterase activity, an aliquot of phenyl valerate was
added to the phosphate buffer. After a steady biosensor signal was obtained, a known
quantity of phenylmethylsulfonyl ﬂuoride (PMSF), a non-neuropathic compound
previously shown to inhibit NEST (or NTE) esterase activity, was added to the phosphate
buffer solution, and the resulting drop in current was measured. At very low
concentration there was not appreciable drop in signal as shown in Figure 5.6. As shown
in Figures 5.7, a 20% (i 3%) decrease in response on the addition of 100 pM PMSF and
a 70% (i 4%) decrease on the addition of 1000 uM PMSF was observed as shown in
Figure 5.8. PMSF inhibition of NEST esterase activity reduces the amount of phenol and
subsequently o-quinone produced. Therefore, less of o-quinone gets reduced at the
electrode surface, leading to a drop in current. These results suggest that the NEST

biosensor can be used for dose dependent detection of NEST inhibitors.

78

1.00

 

    

 

 

 

 

0.90 - Addition of 10‘5M
GTE-a 0.80 - / PMSF
0 0.70 -
Z 0.60 -
£5 0.50 -
C
5 0.30 -
0 020 . Addition of 8pM
0.10 _ / Phenyl valerate
0.00 . . .
0 50 100 150
Time (sec)

Figure 5.6: Current time response of NEST biosensor to the addition of phenyl
valerate in phosphate buffer (0.1 M, pH 7 .0) to obtain a final phenyl valerate
concentration of 8 pM followed by the addition of NEST inhibitor PMSF to obtain a
final PMSF concentration of 10 pM.

79

 

  
   
 

 

 

 

 

1.00
0'90 ' Addition of 104M
0"; 0'80 “ PMSF
0 0'70 ' 20 % decrease
2 0.60 -
:1
v 050 -
d—l
5 0.40 .
g- 0.30 - Addition of 8pm
0 0.20 _ / Phenyl valerate
0.10 «“4
0.00 I l l I l
0 20 40 60 80 100
Time (see)

Figure 5.7 : Current time response of NEST biosensor to the addition of phenyl
valerate in phosphate buffer (0.1 M, pH 7.0) to obtain a final phenyl valerate
concentration of 8 pM followed by the addition of NEST inhibitor PMSF to obtain a
ﬁnal PMSF concentration of 100 pM.

80

 

Addition of 10*3 M
PMSF

 

  

 

 
 

Addition of mm
Phenyl Valerate

 

 

 

0.00 N . . .
0 50 100 150

Time (see)

Figure 5.8: Current time response of NEST biosensor to the addition of phenyl
valerate in phosphate buffer (0.1 M , pH 7.0) to obtain a ﬁnal phenyl valerate
concentration of 8 pM followed by the addition of NEST inhibitor PMSF to obtain a
final PMSF concentration of 1000 pM.

81

5.3.5 Higher sensitivity NEST biosensor

NEST biosensor sensitivity could be increased by increasing the number of
bilayers of PLL-Tyr to six. As discussed by Kohli et. al [103] in the mathematical model
developed for the NEST biosensor, that the sensitivity can be improved more efficiently
by increasing the amount of tyrosinase immobilized on the surface rather than increasing
the amount of NEST. Hence, we developed a sensor by increasing the bilayers to six as
the sensitivity of catechol sensor was maximized with 6 bilayers as discussed in previous
chapter.

The same procedure was followed to fabricate biosensor. The gold electrode was
ﬁrst modiﬁed with thioctic acid followed by the deposition of PLL-Tyr bilayers. After
addition of 6 bilayers a layer of NEST was added. The sensor was tested for NEST
activity by phenyl valerate assay. The sensitivity went upto 847 :t 158 nA cm'zuM'1 for
the addition of phenyl valerate. The plot for phenyl valerate is shown in Figure 5.9. The
NEST inhibition was also done by addition of 100 uM and lmM of PMSF. The decrease
in current was same as observed with 3 bilayers of NEST. The plots for inhibition of
NEST on 6 bilayers of PLL-Tyr are shown in Figure 5.10 and 5.11 for 100 uM and 1 mM
respectively. There was no remarkable decrease in current with 10 pM addition of PMSF
(data not shown). Hence higher sensitivity NEST biosensor can be obtained by increasing
the amount of Tyrosinase on the interface. It also implies that better sensors can be
prepared with marginal increment to cost as tyrosinase is commercially available and can

be easily deposited on to the electrode by using LBL deposition.

82

8.00

 

 

 

 

 

 

 

 

 

7.00 -
—Phen lValerate

,7- 6-00 ‘ Addition of _c tyl
E 4 M of Phen I °" m
u 5.00 - I" y
i Valerate
3': 4.00 - \
E
g 3.00 -
3
0 2.00 -

1.00 _ \ Control

0.00 . T TEL .

0.00 50.00 100.00 150.00 200.00
Time(Sec)

Figure 5.9: Current time response of NEST biosensor with 6 bilayers of PLL-Tyr
underneath the NEST layer. The sensor was tested in phosphate buffer (0.1M) with
electrode maintained -0.1V vs Ag/AgCl reference electrode. In control the electrode
was prepared in exactly the same manner but NEST layer was not added.

83

12.00

 

  
 

  

 

 

 

 

Addition of

10.00 - / 0-1 mM
“'5 8.00 -
i Decreasei
... 6.00 - current
‘5
2 . .
.5 4,00 , Addition f
0 8pm Phe I

2,00 . Valera:

0.00 I I I I

0.00 50.00 100.00 150.00 200.00 250.00
Time(Sec)

Figure 5.10: Current response of NEST biosensor with 6 layers of PLL-Tyr and
followed by inhibition of NEST by addition of 0.1 mM PMSF. First phenyl valerate
was added to get the ﬁnal concentration of 8 uM in bulk solution. The current

response was allowed to achieve steady state before addition of an aliquot of PMSF
to get final concentration of 0.1 mM.

84

14.00
Addition of
12.00 — MW 1 mM
“ PMSF

8.00 - 70%
reduction in
6.00 l

current km“
4-°° ‘iAddition of 8 [M of
2 00 _Pheny| \Rlerrte

0.00 . i
0.00 50.00 100.00 150.00 200.00
Time(Sec)

Figure 5.11: Current response of NEST biosensor with 6 layers of PLL-Tyr and
followed by inhibition of NEST by addition of 1 mM PMSF. First phenyl valerate
was added to get the ﬁnal concentration of 8 pM in bulk solution. The current

response was allowed to achieve steady state before addition of aliquot of PMSF to
get final concentration of 1 mM.

 

‘
.°
O
O
r

 

Current(pA/cm2)

 

 

 

 

 

I

85

5.3.6 Signiﬁcance of NEST biosensor

This new biosensor approach to measuring NEST esterase activity and its
inhibition can in principle easily be extended to full length NTE. The approach offers
several advantages over the old two step method. First, it requires only a single step to
measure NEST (or NTE) esterase activity. Because the NEST esterase activity is co-
irnmobilized with tyrosinase on the sensor interface, the presence of phenyl valerate
triggers sequential reactions that result in an electrical signal. Second, the nanometer-
scale thickness of layers in the sensing interface provides a very short diffusion path
giving a rapid response time (less than 10 seconds). Third, the biosensor is suitable for
continuous, real-time measurements of esterase activity. Fourth, the biosensor is
designed to achieve signal ampliﬁcation via recycle of o-quinone to catechol, thus
increasing the sensitivity of the sensor. Fifth, the biosensor interface is generated by
ﬂexible, layer-by-layer (LBL), molecular self-assembly methods that would allow it to be
assembled on electrodes inside microﬂuidic channels, thus enabling the production of
high-density biosensor arrays consisting of various esterases for high-throughput
applications.

This combination of desirable properties makes this interface well suited for
important applications, including studying the kinetic properties of esterases such as
NEST protein, high-throughput screening of compounds for NEST (or NTE) inhibition
and continuous, on-line, environmental monitoring to detect chemical warfare agents that

target NEST (or NTE) and other esterases.

86

5.4 CONCLUSIONS AND FUTURE WORK

A biosensor has been developed that allows the activity of NEST to be measured
continuously. The biosensor was fabricated by layer-by—layer assembly approach to co-
immobilize NEST and tyrosinase on a gold electrode. Ellipsometry provided evidence
for the sequential assembly of the multiple layers that make up the interface. Constant
potential voltammetry allowed NEST enzyme activity to be measured with a rapid
response time (< 10 s). The biosensor gave dose-dependent response to known non-
neuropathic (PMSF) NEST inhibitors. As further studies, the NEST sensor can be

fabricated on higher sensitivity sensors as discussed in previous chapter.

87

6 FABRICATION OF POLY-LACTIC ACID PARTICLES USING A
HIGH SHEAR RATE MIXER

6.1 INTRODUCTION

Functionalized nanoparticles are used in a wide variety of biological application
such as biosensors, biocatalysis, and drug delivery. Nanoparticles in drug delivery are
gaining widespread interest due to their size, as the larger drug delivery vehicles can get
opsonized in blood stream or taken up by the reticuloendothelial system [104, 105].
These nanoscale drug delivery vehicles can potentially circumvent this problem and can
be more efﬁcient for drug delivery. Nanoparticles can be loaded with drugs during
formation of particles and/or the prefabricated particles can be functionalized using
various surface treatment for anchoring site-speciﬁc molecules for target [106].
Fabrication of polymeric nanoparticles can be broadly classiﬁed in two schemes; one
involves polymerization of monomers, whereas second involves starting from preformed
polymers. Methods to form nanoparticles from preformed polymers include emulsion
diffusion method [107, 108] and nanoprecipitation [3, 109].

In emulsion diffusion, ﬁrst polymer is dissolved in a solvent and the solution is
added into a non-solvent, like water, containing some stabilizer (or surfactant) and then
they are homogenized to form emulsion having tiny droplets of polymer solution
dispersed in water. Then the solvent is allowed to diffuse into the non-solvent by adding
excess amount of non-solvent phase and mixing for certain duration of time, thus leading
to the formation of nanoparticles [110, 111]. In nanoprecipitation, solvent like acetone or
ethanol is used to dissolve polymer and then the polymer solution is added to nonsolvent

such as water. Nanoprecipitation differs from emulsion diffusion as there is no formal

88

emulsion formation. As polymer solution is added, acetone starts to diffuse out into
nonsolvent with some polymer chain associated with it. As it diffuses further, the

polymer chains aggregate to form nanoparticles [112].

This work involved fabrication of polymer nanoparticles using the thin-ﬁlrn spin
system, “T.K. FILMICS® Model 80-50”, from PRIMIX Japan. A scheme of mixing
chamber of the high shear rate mixer is shown in Figure 6.1. It consists of concentric
cylinders (inner dia. 5.2 cm and outer dia. 5.8 cm) where inner cylinder can rotate at high
speeds imparting high shear forces on the materials being mixed. This mixer can impart
high shear force on the particles. Also mixing occurs in an open system as the mixer has
an opening that is also used as injection port. The turbine can achieve high
circumferential speed of 50m/s. Reynolds number equivalent to the circumferential speed
with water as ﬂuid is plotted in Figure 6.2. The non-linear trend can be explained by the
heat generated inside the chamber which raises the temperature of the water. Higher

temperature decreases the viscosity of water thereby changing the Reynolds number.

89

 

 

 

 

 

 

 

 

 

 

 

 

 

    
 

Cooling
Jacket

0))

Figure 6.1: (a) Schematic of mixing chamber of the nanomixer. It has cylindrical
turbine which rotates at peripheral speed of 10m/s to 50m/s. (b) A cross-sectional
cartoon, it can have one or two injection port with continuous processing.

90

2500

 

 

 

 

A O
"o 2000 -
E
g 1500 - ,
E
.3.
2 e
O
E.
a, 500 — .
in
O
0 i l i i i
0 10 20 30 40 50 60

Speed(mls)

Figure 6.2: Turbine peripheral speed and equivalent Reynolds number for Couette-
Taylor geometry ﬂow.

6.2 THEORETICAL BACKGROUND

6.2.1 Nanoprecipitation

Nanoprecipitation is a process of spontaneous precipitation of polymer into a non-solvent
phase giving rise to polymer nanoparticles. Polymer is dissolved in a solvent and the
solution is added to a non solvent where solvent-nonsolvent are miscible and hence
solvent diffuses into non-solvent and polymer chains associated with it precipitate out to

form nanoparticles.

According to literature [110], the size of nanoparticles during nanoprecipitation is
governed by solvent-nonsolvent solubility. A solubility parameter (5) of solvent or

nonsolvent is deﬁned as

91

(AH -RT)
Vm

a: ........................................... (6.1)

where AH is heat of vaporization (J mol'l) , R is gas constant (J K'1 mol'l), T is
temperature (K) and Vm is molar volume (m3 mol'l). Difference in solubility parameters
(A6 solvent-nonsolvent) of solvent and non-solvent is an important parameter. Lower the

A8, the smaller will be the nanoparticles.

At slow speeds smaller particles were prepared as the process is probably
governed by nanoprecipitation mechanism. But at high shear rates we get particle size
increase to microns scale. This could be because, as the smaller particles form during

initial stages, they agglomerate or coalesce because of high shear rate. As the temperature
in the mixer is beyond the glass transition temperature Tg, the sticky polymer particles

can gel together to form larger particles.

6.2.2 Emulsion Diffusion

In this work we investigate the inﬂuence of mixing rates on the formation of
polymeric particles and their possible agglomeration during mixing process. Some of the
parameters that can inﬂuence particle size are viscosity, density of solvent and non-
solvent, concentration of polymer in solvent phase and shear rate. Shear rate can cause
dispersion of particles or enhance emulsion process but it can also cause agglomeration
or coalescence of particles and emulsion droplets.

Role of shear rate can be more easily studied by using emulsion diffusion process

for nanoparticles formation. As illustrated in Figure 6.3 in emulsion diffusion process,

92

  
  
 

Water

with

3:32?” Oil in Water
Emulsion

  
  
   
  

 

Emulsiﬁcation '— .: +144. 0.;
L. I". 0.. — _’
'_ re _
591%‘3 .°

 

 

 

Initial
Emulsion
droplet
Solvent
diffusion
Stable
particles
nuclei

Stable
nanoparticle

 

Figure 6.3: Emulsiﬁcation is carried out in the nanonrixer by adding water with
surfactant and polymer in oil (solvent). This was followed by diffusion step in which
emulsion was added to excess amount of water for solvent phase to diffuse out.

93

emulsion can be formed in the mixer and then the particles can be formed in second
stage by diffusing out the solvent by addition of excess amount of non-solvent [107].
Hence we can possibly determine whether formed droplets agglomerate or coalesce due
to high shear rate.

To analyze this problem and determine parameter that should affect particle size,
we made a very simpliﬁed estimate for the turbulent ﬂow at high speeds by assuming the
geometry of mixer to have Couette-Taylor ﬂow, that is ﬂow between two concentric
cylinders with only inner cylinder rotating [113]. As discussed in literature, drops placed
in turbulent conditions could break upon the action of viscous or inertial stress [114,
115]. Either of the stress could be dominant depending on the size of the smallest
turbulent eddies in the ﬂow [116-118] .

80 called “Kolmogorov Scale” is used to approximate the size of smallest eddies

[115], A, given by equation 6.2

_1 3 _3‘
A z e Ancﬂ pc / ................................ (6.2)

where nc is viscosity (Pa 8") and pc is density of the ﬂuid (kg m'3)or continuous phase
and 8 is the rate of energy dissipation (mzs'3). Hence faster the rate of agitation, which
correlates to rate of energy dissipation, smaller should be the size of the turbulent eddies.
Estimation of the diameter of droplets can be done, with emulsiﬁcation in inertial
turbulent regime, for drops with viscosity similar to that of water. Kolmogrov-Hinze
theory gave an estimate of maximum stable drop [117], dKH in inertial turbulent regime as

given by equation 6.3.

94

dKH=A1 e '2’56 3’5 pc'3/5 ............................................ (6.3)

where A. is a constant of proportionality of the order of unity and O is interfacial tension
(N m").

In case of viscous turbulent regime, the drop breakup occurs under the action of
viscous stress inside the turbulent eddies. The maximum stable drop diameter [117] de,

was estimated as given by equation 6.4

1 1 1/2

dKV=A2 e ' znc - 2pc. 0 ..................................... (6.4)

where A2 ~ 4, is a numerical constant

Hence, these equations give an estimate about how the diameter of emulsion
droplets should vary by varying different parameters like viscosity, density and interfacial
tension. Though this theory does not account for viscosity of the dispersed phase but
these equations are valid for dispersed phase viscosity less than the continuous phase’s
viscosity. There are modiﬁcation of aforementioned equations to account for higher
viscosity dispersed phase [117]. Hence, for the known value of energy dissipation, all the
three values A, de, dKH can be estimated. If equations predict, A < dim then the
emulsiﬁcation is in inertial turbulent regime. Whereas if de < A then the emulsiﬁcation
occurs in the viscous regime. If all three values are approximately equal then
emulsiﬁcation occurs in transitional regime. If above conditions are not met, modiﬁed

equations which accounts for viscosity of dispersed phase could be used.

95

Experimentally the regime can be determined by changing the no, as changing nc_
strongly affects A and de, but not dim. Hence if diameter changes by changing viscosity
it is in viscous regime.

Rate of dissipation of energy per unit mass characterizes the hydrodynamic
condition during emulsiﬁcation. For very high Reynolds number and Couette-Taylor kind
of ﬂow, the rate of energy dissipation can be a calculated by a simple approximation
[119] as given by equation 6.5

e =(AU)3/Ar ................................................... (6.5)
where AU is velocity difference across distance Ar. Ar is difference between inner and
outer radii of cylinders. Approximate rate of energy dissipation as a function of
peripheral speed is given by Figure 6.4a.

Substitution of values in above equations by assuming ethyl acetate as solvent and
water as continuous phase we could estimate all the parameters. We got the drops to form
in turbulent inertial regime as A < dim for all mixing speeds. Figure 6% gives the plot of
as A and dig; for each mixing speed. This simpliﬁed approximation of mixing in turbulent
range, gives us the trend that the droplet sizes should follow by changing the mixing
conditions. Smaller emulsion droplet sizes can be achieved by transiting to viscous
turbulent regime by increasing viscosity of continuous phase or by bringing down the
interfacial tension can be inferred from equation 6.4. Hence it provides us the parameters

that can be varied to inﬂuence the mixing conditions and possibly the particle size.

96

1000 -

 

 

 

 

 

 

 

 

,3" O
2 O
,5 100 — .
‘5
a.
'ﬁ 0
.9
3 10 -
o
0
iii 0
n:
1 I I I I I
0 10 20 30 4o 50
Peripheral Speed(mls)
(a)
6.00 -
5 00 _ o Eddy Diameter
' " - th-Inertial
E 4.00 —
3
8 3.00 -
o
E
5 2.00 - '
8 I
1.00 —
o I
o
o
0.00 . . r . I
0 10 20 30 40 50
Peripheral Speed(mls)

Figure 6.4: (a) Rate of energy dissipation as a function of peripheral speed and (b)
Plot of eddy diameter, A and droplet diameter for inertial turbulent regime dim, for
each mixing speed assuming ethyl acetate and water system.

97

6.3 EXPERIMENTAL SECTION

6.3.1 Materials

Poly(DL)lactic Acid ﬁom LakeShore Biomaterials(California, USA), Poly(L)Lactic Acid
from Sigma Aldrich, KCl (analytical Grade) and Acetone from F luka. Pluronic F68,
Cetyl Trimethyl Ammonium Bromide (CTAB), gold chloride trihydrate, hydroxylamine
and sodium citrate dehydrate were purchased from Sigma and de-ionized (DI) water was
used at 18.2 M!) purity.

6.3.2 Experimental Details

For nanoprecipitation lSmg/ml of PLA was dissolved in acetone. 15mg/ml of
Pluronic F 68 and lOmM CTAB was dissolved in water. 60 ml of this surfactant solution
was added in mixing chamber of FLIMICS ﬁom Primix Japan. Mixer was closed and set
to run for certain time duration at different peripheral speeds varying from 10 m/s to
50m/s. Water in cooling jacket was maintained at 40C. Polymer solution was injected
after mixer achieved its maximum speed.

For emulsion diffusion, ethyl acetate and water were mutually saturated to avoid
any inter-diffusion during emulsion formation. 15mg/ml PLA solution was prepared
using saturated ethyl acetate. 15 mg/ml solution of Pluronic F68 was prepared in
saturated water. Emulsion was prepared by adding 20 ml PLA solution in 60 ml of F68
solution. It was emulsiﬁed at different speeds for speciﬁc preset time duration. The
emulsion was collected and added to 300 ml of pure DI water, stirred at 600 rpm in a
beaker for diffusion step to take place. Once the particles have formed the particle

suspension was kept in partial vacuum for 2 hour to allow ethyl acetate to evaporate.

98

Gold nanoparticles (AuNPs) of mean diameter 18 nm were prepared using a
standard procedure [120]. AuNPs suspension was mixed in nanomixer to study the effect
on agglomeration of nanoparticles. AuNPs were mixed at different speeds inside the
mixer and agglomeration was measured using UV-vis absorbance at 540 nm [121].

6.3.3 Dynamic Light Scattering (DLS)

DLS was used to estimate the mean size of the particles. DLS was done in lOmM
KCL solution using DLS Analyzer (90Plus) from BrookHaven instruments. Average of
mean diameter was obtained by 3 runs for each sample. The uniform sphere option was
selected for measuring the particle size.

6.3.4 Microscopy

Electron microscopes were located at the Centre of Advanced Microscopy at
MSU. Scanning Electron Microscope (SEM) was used to estimate mean size as well as
coefﬁcient of variation for particle size. SEM used for high resolution imaging was JEOL
6300F with ﬁeld emission. PLA particles were coated with gold ﬁlm to view under
electron microscope. Transmission electron microscopy (TEM) was done on PLA
nanoparticles using JEOL 2200FS 200 kV ﬁeld emission TEM. Copper grids coated with
carbon were used to support PLA nanoparticles. Optical Microscope was used to image
ﬂuorescent polystyrene particles which were subjected to high shear in the nanomixer
along with PLA emulsion. A Nikon Eclipse E400 microscope was used to capture
ﬂuorescent images.

6.3.5 UV-vis absorbance spectroscopy

UV-vis absorbance spectroscopy was used to determine the AuNPs

agglomeration. The spectrophotometer used was SP-890 UV-Vis Turner

99

Spectrophotometer. AuNPs solution mixed for 10 minutes at different peripheral speeds
was collected and its absorbance was measured at 540 nm.

6.3.6 Zeta Potential

Zeta potential was measured using 90Plus Brookhaven Instrument. PLA particles
were added to KC] solution of different ionic strengths. Zeta potential measured is an
average of 3 runs for each sample.

6.3.7 Differential Scanning Calorimetry

Differential scanning calorimetry was done using TA Instruments Q100. Small
quantity of polymer was weighed and placed inside the chamber with continuous ﬂow of
nitrogen. The temperature range was chosen from 25°C to 200°C with heating rate of
SOC/minute.

6.3.8 Thermogravimetric Analysis

Therrnogravirnetic analysis was done using 2950 TGA HR V5.4A from TA
instruments. Small quantity was weighed a placed inside the chamber with the nitrogen
ﬂow rate of 60ml/minute. The samples were heated from 25°C to 200°C at the heating

rate of SOC/minute.

6.4 RESULTS AND DISCUSSION

6.4.1 Nanoprecipitation

Nanomixer had negligible inﬂuence on the particle size during the precipitation
process at low mixing speeds of 10 m/s and 20m/s as shown in Figure 6.8. The particle

size was similar to precipitation done in a beaker with moderate stirring. It complies with

100

the theoretical background where the particle size during precipitation is governed more
by thermodynamic factors like solvent parameters which determine the miscibility
between two solvents. Hence theoretically mixing rate should not have inﬂuence on
particle size.

The application of high shear rate led to the formation of particles of larger mean
diameter as shown in Figure 6.8. This observation was counter intuitive to what was
expected as shear rate should bring down the particle size. In nanomixer with small
mixing volume, high shear rate can lead to the increase in the temperature of solution.
With the water/acetone mixture, the temperature inside the nanomixer can reach the
temperature of 65 :l: 2 0C at the mixing speed of 50m/s. This is beyond the glass transition
temperature of PLA which is around 55°C. Hence these particles are heated beyond their

glass transition temperature and the nanomixer causes these sticky particles to collide. As
these particles are beyond their Tg, they coalesce to form larger particles as shown in

Figure 6.5.

 

Figure 6.5: SEM image of PLA particles prepared at 50m/s mixing speed.

101

In the ﬁgure we observed very large particles with more than few microns in
diameter whereas at lOm/s the mean diameter was around 151 um i 6 nm. We also
observed the particles which were coalescing during the process of mixing at high speeds
within the mixer as shown in Figure 6.6. As can be seen in the image the larger particles

which are encircled appear to be coalescing and forming larger particles.

 

Sticky
/par\'ticles
' i

 

 

 

Collide and
coalesce

 

 

 

Figure 6.6: As can be seen in the SEM image the larger particles are heated above
glass transition temperature and colliding and coalescing into larger particles.

102

The zeta potential for the particles formed by using Pluronic F68 as surfactant
was measured in the presence of varying molar concentration of KCl. The zeta potential
is a function of the ionic strength of the surrounding medium. According to Guoy-
Chapman theory [122, 123] , as the charge on a surface of particle is counter balanced by
the ions in the surrounding medium, it forms a compact static layer of oppositely charged
ions called Stern Layer and then by loosely bound ions called diffuse layer. The zeta
potential is considered as the potential at the end of stem layer. When the ionic strength is

varied the zeta potential varies and is given by the following equation 6.6.

( 415T) tanh(—:k T)CXP(-KA) ....................... (6. 6)

Where

e is the electronic charge (Coulombs); k: Boltzmann Constant (J K-l);

T: Temperature (K); ‘I’o: Surface Potential (millivolts); K: Reciprocal thickness of diffuse

layer (m4); 2;: Zeta Potential (millivolts); A: Distance to the plane of zeta-potential from

the surface (m).

Log of zeta potential function in equation 6.6 was plotted against the inverse of
Debye length (data not shown). The intercept of that curve can be used to estimate
surface potential on the surface of particle. Estimated surface potential was used to

determine the surface charge density (00) by using the following equation 6.7

l
ze‘I’o
06 =l(8an€o £02 sinh( 2“.) ......................... (6.7)

 

where so and a, are perrnittivities (Farads/m); 11 number concentration of each ion in

. -3
solutron (m ).

103

PLA particles were also fabricated by precipitation in presence of 10 mM CTAB.
It is positively charged surfactant with amine groups providing the PLA particles a
positive charge. The particles were formed by adding 15 mg/ml solution of PLA in
acetone to lOmM solution of CTAB in DI water. PLA solution in acetone was injected in
the nanomixer running at a set speed and solution was allowed to mix for 10 minutes.

Zeta potential for the particles fabricated using both the surfactants is plotted in
Figure 6.7. As the ionic strength in the surrounding medium increases, the zeta potential
decreases because there is more efﬁcient screening of surface charge on the particles. The
particles prepared with Pluronic F68 as surfactant had lower zeta potential in magnitude
than the particle with CTAB as surfactant. CTAB has quaternary amine groups as polar
head group which imparts strong charge to the particles. Log of zeta potential was also
plotted as a function of ionic strength of solution for both the particles and intercept was
used to determine surface potential and hence surface charge density. The surface charge

density and surface potential calculated are tabulated in the Table 6.1.

Table 6.1 Effect of surfactant type on the surface charge and surface potential on

 

 

 

nanoparticles formed
Surfactant Surface Potential (mV) Surface Charge
(11 C/cmz)
Pluronic F 68 -34 -O.45
CTAB 48 1.57

 

 

 

 

 

104

CTAB as surfactant

Zeta Potential (mV)
Adventures-a0:
UIOUIOUIOUIO O

 

O

0 100 200 300 400 500 600
Gone KCl(mM)

(a)
Pluronic F68

Zeta Potential (mV)

 

0 10 20 30
Cone KCI (mM)

0))

Figure 6.7: Zeta potential of PLA particles (a) CTAB as surfactant; (b) Pluronic F68
as surfactant; as a function of varying ionic strength in DI water. Increasing the
ionic strength decreases the zeta potential of the particles.

105

From the zeta potential measurement, an estimate of surface charge on particles
can be made. The particle size for each surfactant is also plotted as ﬁmction of mixing
speed as shown in Figure 6.8. The particle size in both the cases went up as the mixing
speed was increased. The temperature inside the vessel is also shown in the Figure 6.8.

As the particles are mixed at higher speeds, the temperature inside the vessel rises beyond

the glass transition temperature of PLA. Above Tg the softened particles impinge and

coalesce to form larger particles. This agglomeration was also observed by mixing gold
nanoparticles in the nanomixer. Gold nanoparticles were prepared by standard gold salt
reduction using sodium citrate as catalyst and also stabilizer in solution under boiling

conditions [1 20].

. F68
2500 - . c1' AB -— so
A TEMPERATURE -— 70
E 2000 - l __ 60 A
5 o
8 i __ 50 3..
a 1500 — l g
3
: -~ 4° 5
0
g 1000 - —— 30 a-
o. a E
c i __ ‘”
1'0 6 20 I-
§ 500 -
l I "T 10
. - .
o I l I I l o

 

 

 

0 10 20 30 40 50 60
Peripheral Speed(mls)

Figure 6.8: Mean particle size of PLA particles with Pluronic F68 and CTAB as
surfactant. It also shows the temperature in the vessel at each mixing speed.

106

P
Q
l

AuNPs

9
0|
1

30mls 10 mins
‘ 40mls 10 mins

P
.5
1

\ SOmIs 2 mins

\.
\ 50mls 10 mins
\

.°
N
1

Absorbance (A. U.)
c
U

 

 

0'1 A W
0 I I I l
o 200 400 600 800
Wavelength (nm)

Figure 6.9: Decrease in the peak intensity at 540 nm after mixing gold nanoparticles
inside the nanomixer.

0.8
0.7 -
0.6 -

 

AuNPs

A.U.)
9
UI

AuNPs
Aggregates

Absorbance (
P P P
N u A

P
d
1

 

 

O
'l

 

I I I

200 400 600 800 1000
Wavelength (nm)

0

Figure 6.10: It shows the signature shift in the peak intensity from 540nm to 700 nm
after agglomeration of gold nanoparticles.

107

The gold particles obtained before agglomeration had a mean diameter of 185:2
nm. Free dispersed gold nanoparticles showed their characteristic absorption peak at 540
nm in UV-vis absorbance spectroscopy [56] as shown in Figure 6.9. As the particles were
mixed, AuNPs agglomerated and collapsed out of the solution and the peak intensity
went down with the mixing speed. There was no shift in the peak or a separate peak after
AuNPs were mixed showing that there were no stable gold agglomerated in the solution
and all the agglomerates precipitated out from the solution. The Figure 6.10 shows a
typical shift in absorption peak to around 700 nm caused by agglomerated gold
nanoparticles that are still suspended in solution. However when the AuNPs are
subjected to high shear in the nanomixer they collide and form larger aggregates that
eventually precipitate out of the solution.

Hence this gives us additional evidence that nanoparticles under high shear rate in
nanomixer are subjected to two opposing phenomena. One is high shear force that would
want to shear them apart. Whereas the same shear force also causes the dispersed
particles to collide into each other and can lead to agglomeration and coalescence. In case
of PLA particles, it formed larger particles as was observed directly when the speed was
increased.

6.4.2 Emulsion Diffusion

Emulsion was prepared by ﬁrst saturating ethyl acetate with water and vice versa.
Polylactic acid was dissolved in saturated ethyl acetate and Pluronic F68 was dissolved in
water saturated with ethyl acetate. The saturation of solvents is done to avoid any
precipitation of polymer during emulsiﬁcation. The emulsion was added to excess DI

water for ethyl acetate to diffuse out. Ethyl acetate has solubility of around 10% in D1

108

water. So, 300 ml of DI water was taken for diffusion step as emulsion is added to pure
DI water. During this step most of the ethyl acetate can diffuse out of the particles into
the water phase. The particles were characterized by dynamic light scattering and
scanning electron microscopy.

As the temperature rises up close to glass transition temperature at 40m/s mixing
speed and further beyond the glass transition temperature at the mixing speed of 50m/s.
The mixing times were varied for different speeds. For initial results the mixing time was
10 minutes for lOm/s to 30 m/s and 2 minutes for 40m/s and 1 minute for 50m/s. The
mean particle diameter aﬁer diffusion step and the size of emulsion droplets was
measured by dynamic light scattering and is plotted as a function of speed in Figure 6.11.
As expected the particle size went down by increasing the mixing speed. High shear rate
resulted in smaller droplet size in emulsion as shown in Figure 6.11. It can also be
followed from the equations as discussed in section 6.2 where high shear rate leads to
higher rate of energy dissipation. The droplet size that is stable under that turbulent
condition gets smaller. Hence increasing mixing rate leads to decrease in droplet size that
eventually leads to smaller particle size.

The emulsion dr0plet size, hence the particle size went down with the speed.
Figure 6.12 shows a simple digital photograph with particles from lOm/s mixing speed up
to SOm/s mixing speed. The suspension turned clearer from whitish appearance due to the
decrease in the size of particles. Larger particles appear milky as they interfere with light
passing through the solution whereas small particles size <100 nm appears clear because
their size is much smaller than the wavelength of visible light. It gives us qualitative

indication of particle size gets smaller with increasing mixing speed.

109

 

 

 

 

o Emulsion Droplets
700 i { I Particles
600 a
A 500 T
C
‘5' 400 - i
.5
U) 300 _ 5
O
200 — i
i f .
100 - I I
o I I I I I
0 1o 20 30 40 50 60
Speed (mls)

Figure 6.11: Mean particle size as obtained by dynamic light scattering for particles
prepared at different mixing speeds in Nanomixer.

 

10 20 30 40 50

Figure 6.12: Emulsions were made at different speeds and then added to excess
amount of water for particle formation.

110

Scanning electron micrographs of particles were obtained by drying particle
suspension on a glass slide and coating it with a 7 nm layer of gold. Gold coating was
required as PLA is not conducting. The scanning electron micrographs were obtained at
working distance of 8 mm and accelerating voltage of 3 KV as higher accelerating
voltage led to the damage of particles as discussed later in the chapter. Figure 6.13 show
scanning electron micrograph of the particles obtained when emulsiﬁcation was carried
out at lOm/s (EDlO) and 20m/s (ED20) mixing speed with the mixing time of 10
minutes. It is clearly seen that the particle size decreased as the mixing speed was
increased. Higher magniﬁcation images in Figure 6.14 show particles are not that
uniform at both the mixing speeds of lOm/s and 20 m/s. The coefﬁcients of variation are

18% and 11% at the speeds of 10 m/s and 20m/s respectively.

111

 

Figure 6.13: Scanning electron micrographs at 7500X of PLA nanoparticles
Obtained by emulsifying a) 10 m/s and b) 20m/s for 10 minutes.

112

r‘

r...
’3
(N

u A
. . pr"
4‘ P 1‘ Ain‘t ~ \.
m .., . ,.. _,_

- i

’X

 

Figure 6.14: Higher magniﬁcation (15000X)scanning electron micrographs of PLA
nanoparticles obtained by emulsifying a) 10 m/s and b) 20m/s for 10 minutes.
Illustrates PLA particles at l0m/s are more polydisperse than particles at 20m/s.

113

In another set of experiments when the mixing speed was increased to 30m/s
(ED30), the particle size reduced further. At 40m/s (ED40) and 50 m/s (EDSO) mixing
speed the time of mixing had to be reduced to 2 minutes and 1 minute respectively as the
particle size grew larger at longer mixing time due to coalescence of PLA particles. The
temperature in vessel also reached around or beyond the glass transition temperature of
PLA causing particles to coalesce. Scanning electron micrographs shown for particles in
Figure 6.15 are at same magniﬁcation as Figure 6.14 to illustrate particle size is much
smaller than at slower emulsiﬁcation speed. The following Figure 6.16 illustrates the
particles at ED30 and ED40 at higher magniﬁcation. The particles appear polydisperse at
higher magniﬁcation images especially in the case of ED30. The coefﬁcient of variation
for ED30 was 15% and ED40 was 10%.

At SOm/s (EDSO) the particle size was slightly smaller than ED40 but with
slightly higher coefﬁcient of variation of 11%. This trend of small decrease in particle
size from 40 to 50 m/s was similar to what we observed in theoretical treatment of droplet
in turbulence. At higher turbulence conditions, the rate of decrease of particle size gets
smaller. Therefore, we observe a faster reduction in particle size at slower speeds as in
the case of EDIO to ED20 as compared to ED40 to EDSO. The TEM and SEM images of
particles obtained at SOm/s are shown in Figure 6.17. The mean diameter for each method
was also approximated by actual measurement of particle diameters from the images. The
mean diameter from SEM images was in good agreement with that obtained from

dynamic light scattering experiment as shown in Figure 6.18.

114

 

Figure 6.15: Scanning electron micrographs (15000X) of PLA nanoparticles
obtained by emulsifying at a) 30 m/s for 10 minutes and b) 40m/s for 2 minutes.

115

 

Figure 6.16: Scanning electron micrographs (65000X) of PLA nanoparticles
obtained by emulsifying a) 30 m/s for 10 minutes and b) 40m/s for 2 minutes.

116

 

ﬁgure 6.17: TEM (a) and SEM image (b) of PLA nanoparticles obtained by
emulsifying at 50m/s for 1 minute.

117

700 -

 

 

 

 

 

 

coo -
E 500 —
'5
GE) 400 d o DLS
(0
S
m 200 -
2 i i

100 - 3 :

0 I T I I I T

O

1 0 20 30 40 50 60
Mixing Speed(mls)

Figure 6.18: Mean diameter of the particles as obtained from DLS and as calculated
from SEM images.

As discussed in previous section the particles obtained from emulsion diffusion
had large coefﬁcient of variation and after observing the SEM images it can be inferred
that particle size distribution is bimodal in nature. Particles can be divided into set of two
particle size distribution with different means.

The particle sizes that were measured from images were divided into two set of
particle size distributions. The particles were categorized by simply putting particles with
diameter greater than an arbitrary choice of border diameter in larger particle size
category and the rest in smaller size category. The mean and standard deviation for each

set was calculated and are plotted in Figure 6.19.

118

 

 

 

 

 

900
800 -
P<<0.05

700 - . -
E 600 -
73' 500 _ a 10mls
5 IZOmIs
% 40° ' I30mls
5 300 -
n.

200-

22:: ‘J

0 _ .
Large Small Combined

.‘
:33
m

 

 

 

Figure 6.19: The particle sizes can be divided into two categories as large and small.
The mean diameter of each categories is plotted at mixing speeds of lOm/s to 30mls.

It was difﬁcult to divide particles prepared at 40m/s and SOm/s as two set of
particle distribution as the particle did not appear to be distinctively separable. But in
lOm/s, 20m/s and 30 m/s it was more evident and particles were easily separable.

The emulsiﬁcation was carried for varying time duration followed by diffusion
step. Mean particle size was estimated by dynamic light scattering experiment. It was
found that the particle size decreased with increasing mixing time at mixing speeds of 10
m/s to 30 m/s as shown in Figure 6.20. At higher mixing speeds of 40m/s and SOm/s,
particle size increased sharply as emulsiﬁcation time was increased. This phenomenon is
similar to as observed in nanoprecipitation where higher mixing speeds led to the
impingement and coalescence of PLA particles due to the increase in temperature beyond

glass transition temperature of the PLA.

119

As illustrated in Figure 6.20 the particle size went down as the mixing time was
increased (P<0.05). It was most clear in the case of mixing speed of lOm/s. The emulsion
was made by mixing ethyl acetate and water mix in nanomixer for different time period
varying from 1 minute to 60 minutes. The mean particle size dropped from around 800
nm to 250 nm. Also the mean diameter of large particle size distribution and small
particle size distribution from SEM image data match well with the mean diameter at 1
minute and 60 minutes (30 minutes for 30m/s) respectively.

Hence it shows that the mixing in nanomixer have different mixing zones with
different ﬂow conditions as illustrated in Figure 6.21. These different zones can have
different turbulent conditions. As discussed in Section 6.2.2 the difference in turbulence
conditions can lead to difference in the rate of energy dissipation in that zone. This
difference in rate of energy dissipation can then affect the droplet size that is stable in that
zone. Therefore, as the time is increased the percentage of droplets that passes through
the zone with smaller droplet as stable diameter increases. Hence the particle size is
reduced with increasing mixing time which as clearly evident at the mixing speed of

10m/s.

120

1000

 

900 O 10mls
A 20mls
A 800 -¥ . 3W8
,5: 700 - <> 10mls Large and Small Mean
:5 600 - x ZOWs Large and Small Mean
2 500 - O 30mls Large and Smal Mean
to
E 400 A I
g 300 - t l
i
200 - A
100 F ° ° é ‘5
o I I I I I I

 

 

 

0 10 20 30 40 50 60 70
Time(mins)

Figure 6.20: The mean diameter of particles at the different mixing times. Hollow
legends are used to mark the large particle diameter and small particle diameter as
observed in SEM images.

 

 

 

4 ‘ \4
® 0 : . G)
$
0 00000 0
00 000000 00
0 00000 0

 

 

Figure 6.21: Illustrates the fluid motion inside the mixer and how emulsion droplet

can encounter different turbulent conditions giving rise to difference in stable
droplet size.

121

At the mixing speeds of 20m/s and 30m/s, the particle size decreased with
increase in the mixing time from 1 minute to 60 minutes. But the decrease in particle size
was not as remarkable as in the case of lOm/s. At 30m/s there was also increase in the
particles size as they start to collide and coalesce together to form larger particles. With
40 m/s and 50m/s the particle size went up dramatically due to higher collision rate and
coalescence of PLA particles (data not shown).

The coalescence of emulsion droplets at higher mixing speed was also
qualitatively studied by addition of commercially available ﬂuorescently tagged
polystyrene particles of 1 micron mean diameter. The emulsion at 50 m/s second was
prepared for 1 minute. Then the ﬂuorescently tagged polystyrene particles were added to
the emulsion. It was further mixed for 2 minutes at 50m/s and the diffusion step was
carried out as described in experimental section. As a control pure ﬂuorescently tagged
polystyrene particles were mixed in nanomixer for 2 minutes at SOm/s. Both particles
were taken and observed under optical microscope for signature ﬂuorescence of
polystyrene particles.

PLA emulsion with ﬂuorescent particles and pure particles were imaged and are
shown in Fig 6.22. As expected the pure polystyrene particles looked uniform in size
under microscope whereas the particles mixed with emulsion appeared much larger in
size. As there was random collision of particles inside the nanomixer, the emulsion

droplets collided with ﬂuorescent polystyrene particles and coalesced to give rise to

larger ﬂuorescent particles. Whereas in case of pure polystyrene particles as Tg of

polystyrene is around 105°C [59] they collide but did not coalesce to form larger

particles as the process was well below the Tg of polystyrene.

122

 

(b)

Figure 6.22: a) Optical image of fluorescent particles: PLA emulsion prepared at
50m/s for 1 minute followed by addition of 1 micron ﬂuorescently tagged pure
polystyrene particles and further mixing for 2 nrinutes at 50m/s. b) Pure 1 micron
fluorescently tagged pure polystyrene particles mixed at 50m/s for 2 minutes. The
length of scale bar in both the images is 20 pm.

123

6.4.3 Effect of addition of glycerol

Effect of addition of glycerol at the mixing speed of 50 m/s was also studied in
the nanomixer. As discussed in section 6.2.2 changing viscosity has affect on the various
parameters. Inertial diameter (dm) is independent of viscosity whereas eddy diameter (1»)
and viscous diameter (de) are a function of viscosity. Hence we can move ﬁom inertial
regime (l. < dm) to viscous regime (de < l.) by changing the viscosity of the continuous
phase.

Different volume fraction of glycerol was added along with water. The particles
were prepared by adding PLA solution in ethyl acetate to various volume fraction of
water/glycerol mixture with 15mg/ml of Pluronic F 68. The emulsiﬁcation step was
carried out at 50m/s for 1 minute and then the emulsion was added to pure water for
diffusion step. The particle size was estimated by dynamic light scattering and is plotted

in Figure 6.23.

Effect of Glycerol

 

i

If 0

 

Mean Particle Siz

O

 

 

 

0 o o 0 9
l l I I I I I I I I

0 510152025303540455055
% Glycerol(vlv)

Figure 6.23 :The mean particle size as a function of glycerol volume fraction at
mixing speed of 50 m/s.

124

The particle size remained constant until 40% volume fraction of glycerol and
went up suddenly beyond that point. In the following Figure 6.24, all the three
parameters: eddy diameter (7t) inertial diameter (dim) and viscous diameter (dxv) are
plotted along with mean diameter of particles as a function of glycerol volume fraction
mixed at the mixing speed of 50m/s for 1 minute. The interfacial tension was calculated
by using the theory of interfacial tension for partial miscible liquids where one liquid is
mixture of water/glycerol and second liquid is ethyl acetate. For the case of partial
miscible liquid, the formulation developed by Girifalco and Good [124] was used and is

given in equation 6.28.
‘/2
y= YA + YB -2(yA/ YB) .................................... (6.28)

Where 7 is the interfacial tension (N/m), YA is the surface tension of liquid A (N/m),, 7};

is the surface tension of liquid B (N/m),.

For ethyl acetate, the literature value of surface tension at room temperature was
used [124]. Water/glycerol system the surface tension was used from the plot as
published by Connor et al [125]. Density and viscosity data was used from the physical
properties of glycerol-water solution available in literature [126]. The values obtained
were used to calculate the eddy diameter, inertial stable diameter (dim) and viscous stable

diameter (de).

125

3000

 

~I- Eddy Diameter
+ th-lnertial

+ Dkv-Wscous
+ Mean Diameter

2500 —

 

 

 

 

 

’g 2000 —

5

E 1500 _ Inertial 3 L VISCOUS
g Regime Wu F Regime
(U

.5

 

 

 

 

 

 

Glycero|(v/v%)

Figure 6.24: The above figure illustrates how the eddy diameter increases and is
greater than both dxn and dxv at glycerol volume percentage greater than 35%. The
mean diameter of particles for emulsion prepared at 50m/s for 1 minute also
increased beyond 40%.

As shown in the Figure 6.24, eddy diameter is less than the dKH for solution with
glycerol volume percentage less than 30%. Hence the mixing is in the inertial turbulent
regime. As discussed in section 6.2.2, the emulsion droplet size is governed by the
equation which is independent of the viscosity. Therefore we do not observed a large
change in particle size with increasing viscosity. But beyond 40% volume ﬁaction of
glycerol there was a sudden increase in the particle size with large standard deviation in
mean. Also at 50% viscosity at SOm/s the temperature inside the vessel reaches 78 :l: 2°C
which is close to the boiling point of ethyl acetate. Hence it becomes an extremely

complex system where the droplets are caught in eddies along with ethyl acetate

126

evaporating from the droplets. To determine whether mixing at high viscosity alone was
the main reason for the particle size to increase, the emulsion step at 50m/s with 50%
glycerol was done for 20 seconds. The particle size went down and is plotted in Figure
6.25. Figure 6.25 illustrates that the mixing time plays a critical role in controlling the
particle size. As increasing the mixing time led to heating and it inﬂuenced the size of the

particles as observed at 50% glycerol at 50 m/s.

 

 

 

 

 

 

 

 

Mixing Speed 50mls
1400
3 + Mean Particle Size
1200 r -I— Eddy
... 1000 q + th-lnertial
E —><— Dkv-Viscous
z 800 - /
3
a.»
E 600
.2
o 400
200 F 20 g 3
0 I I 3 I Secondsl
10 20 30 40 50
Glycerol(vlv%)

Figure 6.25: Mean particle size as a function of glycerol percentage. The mixing
time at 50% glycerol was brought down to 20 seconds to avoid heating of the
emulsion to 78 C.

Particle shape was also affected by the viscosity which in turn affects the heating
inside the nanomixer. At 50% viscosity, the particles stayed spherical with 20 seconds of
mixing time but when the mixing time was increased to 1 minute the particle shape

changed from spherical to large hollow shells as shown Figure 6.26.

127

 

From 20 sec
to 1 min

 

Figure 6.26: Effect of time on the particle size and shape at 50% glycerol at 50m/s
mixing speed. Particles changed from solid nanospheres to micron sized hollow
hemispherical shells.

The effect of glycerol leads to two simultaneous changes in condition: ﬁrstly it
increases the viscosity of the continuous phase and secondly, it also increases the
temperature inside the vessel. The temperature inside the vessel increased to 78i2°C

which is close to boiling temperature of ethyl acetate. Hence to distinguish between two

128

cases a series of experiments were done. The mixer has a cooling jacket, where the water
is circulated to cool the vessel. The temperature in the outside jacket was maintained at
4°C for all the previous experiments. In ﬁrst case the coolant temperature in the jacket
around the mixing vessel was increased to 170C for the 40% glycerol as continuous
phase, so the temperature inside the vessel increases to the same temperature as 78 :l:2°C.
If it is only the heating that is causing particles to agglomerate into the shell structure we
should observe similar particles as obtained with 50% glycerol. The particles obtained at
40% glycerol remained spherical and did not change shape to open shells. The image for
particles obtained at 40% glycerol is shown in Figure 6.27a.

The other experiment was done where the emulsion was prepared at 20m/s for 10
minutes and was added to the heated mixture of 50% water and 50% glycerol by volume
at 78°C, while it is being stirred in a beaker. The particles obtained remained spherical in
shape as shown in Figure 6.27b illustrating heating alone itself does not cause particles to
take shell shape. This also conﬁrms that the nanomixer imparting high shear rate in
viscous turbulent regime plays a critical role in particles taking the shape of shells.
Transmission electron microscopy of particles, as shown in Figure 6.28a, before forming
the shell shaped particles revealed that particles are not hollow and are solid particles.

Hence the change shape occurs because of combined inﬂuence of high
temperature and high shear rate in viscous turbulent regime. It causes the particles to
take peculiar shape as they are mixed in viscous turbulent regime. The viscous turbulent

regime is illustrated in the Figure 6.28b, where the droplets are caught inside eddies.

129

 

(b)

Figure 6.27 : a) SEM image of particles formed at 40% glycerol when the internal
temperature was raised to 78°C. b) Particles formed by adding emulsion grepared
at 20m/s for 10 minutes to water/glycerol (50% v/v) mixed in a beaker at 78 C.

130

 

Inertial Regime Viscous Regime

(b)

Figure 6.28: a. TEM image of PLA nanoparticles showing that the particles are not
hollow. b) Illustrating the mixing condition in inertial and viscous turbulent
regimes, in inertial turbulent regime droplets are outside eddies and in viscous
turbulent regime they are trapped inside the eddies.

131

In inertial regime as discussed in Section 6.2.2 the eddy diameter is less than the
stable droplet diameter and the droplets are located outside the eddies whereas in the
viscous turbulent regime the droplets are located inside the eddies. As the viscosity is
increased there is shift in the mixing regime from inertial to viscous turbulent regime and
the droplets are caught inside eddies. Also involved is the heating of the system to
temperatures around the boiling point of ethyl acetate that causes these droplets to lose
ethyl acetate and this polymer containing droplets coalesce and grow larger as has been
observed in other cases as well. But as this coalescing droplets are caught inside the
eddies the polymer is shaped into open shell due to combined effect of evaporating ethyl
acetate and sustained shape due to rotational motion of the surrounding eddy. Figure 6.29
illustrates the possible process where droplets are caught in the eddy and coalesce to form
bowl shaped particle. Hence the ﬁnal shape of open bowl is obtained. Similar shapes
have also been obtained just by evaporation of solvent by Xia et al [127]. In their set-up
they freeze dried the emulsion to form hollow spherical shell. The emulsion of poly-
methyl metha acrylate was made and ﬁ'oze it in liquid nitrogen. Toluene was evaporated
under vacuum at temperature below 0°C. Temperature below zero was needed to prevent

collapse of hollow particles from pressure exerted by liquid water [127].

132

Mixing continues
Increasrng T

 

  
   

 

Removal
(evaporation)
of solvent
(D)
Bowl shaped
retained after
mixing is
stopped

 

Figure 6.29: Scheme of the fabrication of bowl-shaped polymer particles in a
turbulent eddy. At a short mixing time emulsion droplets caught in an eddy. Flow
induced self-assembly of nanoparticles in an eddy. (B) At a long mixing time,
emulsion droplets start coalescing in an eddy. (C) Solid form after loss of solvent.
(D) Bowl shaped polymer particle.

In our case the particles retained the shape because droplets were caught in eddies
and rotation caused particles to maintain its shape while the ethyl acetate was evaporating
away from the emulsion. This was not true in the case where the emulsion was added to
the glycerol water mixture in a beaker and spherical particles were observed. So the
nanomixer unique ability to have turbulent conditions inside the vessel caused the

emulsion droplets to coalesce and give rise to these unique shapes of polymer shells. We

133

also looked at the 60% glycerol system and mixing speed of 40m/s. We observed, as
shown in Figure 6.30, similar trend where particles under viscous turbulent conditions

combined to form open shell structure as was observed in with 50% glycerol.

 

Figure 6.30: Scanning electron micrograph of poly-lactic acid open shells obtained
by mixing at 40m/s at 60% glycerol by volume for 4 minutes.

The effect of mixing speed at 50% glycerol was also studied. As the mixing is in
viscous turbulent regime at the speeds ﬁom lOm/s to 50m/s and if we substitute the
viscosity at the maximum temperature that is measured inside the vessel in the equations
discussed in section 6.2.2 we observe that the droplet size is unchanged beyond the
mixing speed of 30mls. The particle size should be constant from 30 m/s to 50 m/s. But
at SOm/s, even with smaller mixing times, the particle size was slightly greater that of the
particle size obtained at 30m/s. This could be because at 50m/s at 50% glycerol the

temperature reaches 78°C; this remarkably brings down the viscosity of the whole

134

continuous phase and also the temperature is slightly above the boiling point of ethyl
acetate.

The particle size, the eddy diameter and the stable viscous diameter-de are
shown in Figure 6.31 and it can be seen that the de is almost steady from mixing speed
of 30mls to 50 m/s. The measured particle size using DLS is also shown in the plot and
they seemed to follow similar trend as expected. Hence we obtained smaller particles
without running the mixer at maximum speed as the particle size is affected by a
combination of various parameters like viscosity and rate of energy dissipation.

Therefore, smallest size can be obtained by not operating at maximum mixing speed.

 

 

 

 

 

 

 

 

 

 

50% (vlv) Glycerol
12 1
+Eddy

1° i +Dkv 3- 0.8
E 8 _ +Particle Size
é —- 0.6
3 6 -
0
E -- 0.4
.2 4 -
a

2 _ -- 0.2

0 0

0 10 20 30 40 50 60

Mixing Speed(mls)

Figure 6.31: Effect of mixing speed on particle size at 50% glycerol volume percent.
At all the speeds it is in viscous turbulent regime and the droplet size achieves a
stable size at 30mls. The mean particle size also reached minima at 30mls and
slightly increased at 50m/s.

135

Transmission electron microscopy of PLA particles obtained at 30mls with 50%
glycerol is shown in Figure 6.32. Signiﬁcant portion of particles looked smaller than 87
nm :l: 4 nm mean diameter as measured by dynamic light scattering experiment in
solution. The mean diameter of these smaller particles as measured from TEM images
was around 32 nm 1: 6 nm. As the whole mixing in nanomixer is a dynamic process with
continually changing temperature and viscosity initially. When the mixer is started it
takes 10 to 30 seconds for the solution to achieve a steady state in terms of temperature.
As viscosity is a strong function of temperature viscosity keeps on changing initially.
Also at 50% glycerol, the mixing is in viscous turbulent regime and the droplet diameter
is a function of viscosity. Therefore, during that initial time period, droplets might feel
higher viscous drag due to higher viscosity. Hence we get a portion of particles with
small diameter. It was not as evident at lower glycerol percentages as the mixing is in
inertial turbulent regime and the diameter of droplets is independent of the viscosity and
by increasing mixing time the particle size can be made smaller.

We calculated theoretically, if the values to viscosity are chosen at the initial
temperature of 4°C, the stable droplet diameter is 60% of what it would be if the droplet
diameter was calculated using the maximum temperature reached inside the mixer. Hence
in viscous turbulent regime there is signiﬁcant portion of particle size that stays much
smaller than the larger particles. Also the coefﬁcient of variation at 30mls, 50% glycerol
is much larger at 46% whereas the coefﬁcient of variation at 30m/s, no glycerol present,
is 15%. The coefficient of variation for just the smaller particles as seen in Figure 6.32 B

is 22%.

136

 

(B)

Figure 6.32: TEM images of PLA nanoparticles prepared at 30 m/s with 50%
glycerol. A) Shows the particles images with relatively larger particles. B) PLA
nanoparticles with mean diameter around 30 nm. The scale bar in both the images
is 100 nm.

137

6.5 EFFECT OF ELECTRON BEAM ON PLA PARTICLES

6.5.1 Poly-Lactic Acid and Beam Damage

Poly-lactic acid (PLA) is extensively used as biomaterial for its biocompatibility
and biodegradability. Poly-lactic acid, though insoluble in water, is hydrolyzed by water
and can be degraded by ester hydrolysis [128]. The rate of hydrolysis is conventionally
governed by glass transition temperature, crystallinity and molecular weights. Recently,
studies have been done to understand the behavior of biopolymers under the inﬂuence of
radiations. Poly-lactic acid and other biopolymers like Poly(lactide-co-glycolide) (PLGA)
can have their properties altered by exposing the polymer to high energy electron beam
[129]. Boey et.al. showed that PLA and PLGA can have their properties altered by
exposing them to the electron beam.

The main mechanism of polymer degradation as given by radiation chemistry is
as follows. The polymer materials get excited under the exposure of radiation to form
active species, such as cations, anions or radicals [130]. These active species are able to
react among the polymeric chains or can react with one another giving rise to changes in
the material properties. The combination of two radicals leads to cross-linking, while the
chain transfer and subsequent splitting lowers the molecular weight [129, 130]. The
electron beam energy can directly disrupt the attractive forces the atoms, thus disrupting
the molecules [129, 130]. This radiation energy breaks the chemical bonds causing chain
scission. As a result, a radiation source of shorter wavelength and higher frequency or
energy, often packs sufﬁcient energy to cause chain scission through the breaking of

these chemical bonds.

138

6.5.2 Deformation of PLA Particles by Electron Beam

The electron beam has dramatic effect on the poly-lactic acid particles. As
mentioned in the previous section the electron beam can cause polymer chains to break
down and they can undergo transformation giving rise to different thermal and
morphological properties. As shown in Figure 6.33a, the poly-lactic acid particle is
shown before beam damage and Figure 6.33b shows particles after damage. The particles
were imaged under a ﬁeld emission scanning electron microscope with lOKV of
accelerating voltage at a working distance of 15 mm.

As PLA particles can not be viewed directly under electron microscope, the
particles were also coated with 7 nm of gold for imaging. The particles were imaged at a
low magniﬁcation so large area was scanned and the exposure of particle to beam was
limited. The particle was imaged at low magniﬁcation at a scan rate of 20 sec/frame and
then the particle was exposed to electron beam for 30 seconds by increasing the
magniﬁcation 3-4 times. The PLA particle was damaged and deformed due to the high
energy electron beam. As discussed in previous section it has been observed that poly-
lactic acid can get damaged by electron beam because of chain scission by high energy
electrons. As the energy of electron is sufﬁciently high it can simply break the atom—
atom bonds. It can cause molecular weight of polymer to go down. The morphology of
polymer can change as amorphous region in polymer can become more crystalline due to
rearrangement of these shorter polymer chains as was observed by Boey et. al. Boey at al
also observed that breaking of poly-lactic acid can also lead to release of carbon dioxide

from the polymer.

139

 

 

(b)

Figure 6.33. Polylactic acid (a) before and (b) after exposure to lOKeV electron
beam.

140

A control experiment was conducted by dissolving polylactic acid in acetone and
drop coating a ﬁlm on a glass slide. It was done to conﬁrm if the deformation was
enhanced due to the processing in high shear rate nanomixer. The drop coated polymer
ﬁlm was again coated with a 7 nm ﬁhn of gold and was investigated using a FESEM at
an accelerating voltage of lOKV. The drop coated polymer ﬁlm was also damaged due to
the electron beam confrrrning that the deformation is because of beam damage to polymer
and processing with nanomixer did not induce any change to polymer physical properties.
We also carried out differential scanning calorimetry (DSC) and thermal gravimetric
analysis (TGA) for PLA particles and did not ﬁnd any difference in polymer before and
after processing in nanomixer. DSC did not show any change in the glass transition
temperature of PLA or any change in physical properties of PLA. Whether there is any
residual solvent that might be getting released during electron beam damage was tested
by TGA. TGA also was exactly the same as for pure PLA and had no evidence of any
trapped mass within the polymer that could cause the deformation in particles.

Figure 6.34 shows the image of polymer ﬁlm formed by drying PLA solution in
acetone before and after the electron beam damage. It is visible that the PLA ﬁlm
deforms on the exposure of electron beam. As it is evident from comparison of images (a)
before damage and (c) after damage that the ﬁlm is damaged and has sagged where it was
exposed to the beam. It also provides indirect evidence that nanomixer processing does
not have any inﬂuence on the deformation behavior and is induced by the high energy

carried by the electrons as evident clearly from images (b) and (d).

141

 

(C) 00

Figure 6.34: The effect electron beam damage on PLA ﬁlm dried on a glass slide
coated with 7 nm gold film. (a) and (b) is an image of PLA film before damage and (c)
and (d) are the images of same area after electron beam damage. The length of scale
bar in each image is 5 pm.

142

6.5.3 Effect of Gold Coating on the Poly-Lactic Acid Deformation

As discussed in previous section the electron beam is causing the poly-lactic acid
particles to deform. We studied the inﬂuence of gold ﬁlm that is used to view the poly-
lactic acid in the SEM. Electron beam has a certain penetration depth depending on the
material property and beam energy. For a bulk sample the beam penetration depth (D)
can be estimated by an approximation developed by Kanaya Okayama [131] and is given
by following equation.

D (um) = 0.0276AE01'67Z‘0'89p" ......................... (6.9)
Where: A is the atomic weight (g/mole), Z is the atomic number, p is the density (g/cm3),
E0 is electron beam energy (KeV) .The depth of penetration for gold and PLA is

tabulated in the following table for different accelerating voltages.

Table 6.2: Beam penetration depth for PLA and gold as a ﬁmction of accelerating
voltage

Accelerating Voltage (KV) Beam penetration Depth(nm)

 

 

 

 

Gold PLA
10 270 2500
3 36 330

 

 

 

 

 

If the thickness of the materials is less than the beam penetration depth then the
electron beam interaction with that material can be divided into three parts, fraction
absorbed (fA), fraction transmitted (ﬁr) and fraction back scattered (f3). Incase of
polylactic acid coated with a thin layer of gold, the fraction of transmitted beam would
cause damage to the underlying polymer ﬁlm. Hence we studied the effect of varying the

gold ﬁlm thickness by coating 7 nm, 35 nm and 70 nm gold ﬁlm on polylactic acid.

143

 

As the thickness of gold is increased it should reduce the effect of beam damage
on the polylactic acid. This was observed on poly-lactic acid during SEM. Figures 6.35,
6.36 and 6.37 illustrates the effect of beam damage of poly-lactic acid that has been
coated with 7nm, 35 nm and 70 nm of gold ﬁlms.

As can be seen, the damaging effect of electron beam with lOKeV of energy is
reduced as the thickness of the ﬁlm in increased. This is because the fraction of electrons
that can pass through the gold ﬁlm reduces as the thickness is increased, as discussed by

Dapor et. al. Dapor developed a model to quantify the fraction of beam that can pass

through any materials by doing an iteration scheme that estimates fA, fT and ﬁg [132]. It

assumes a certain thickness of material (5) and adds a small thickness (Ag) to existing
thickness and estimate the change in the three ﬁactions.

The beam damage is reduced because more electrons are either back scattered or
absorbed by the gold ﬁlm as the thickness is increased as illustrated in Figure 6.38.The
beam has exaggerated tapering though typical angle is ~ 0.30. The fraction of beam
transmitted at different thicknesses at accelerating voltage of lOKV is given in Table 6.3.
As seen from the table, the fraction of electrons transmitted is very low at 70nm that there

is not sufﬁcient damage to observe drastic change in the particle shape and size.

Table 6.3 Fraction of beam (10 K V) transmitted at diﬁ'erent gold ﬁlm thicknesses [132]
Gold Fihn Thickness(nm) Fraction Transmitted (%)

 

 

 

 

7 98%
35 62%
70 20%

 

 

 

 

144

 

 

(b)

Figure 6.35: SEM image of PLA particles coated with 7 nm of gold film (a) before
damage and (b) after damage by lOKeV electron beam.

145

 

(b)

Figure 6.36: SEM image of PLA particles coated with 35 nm of gold ﬁlm (a) before
damage and (b) after damage by lOKeV electron beam.

146

 

(b)

Figure 6.37: SEM image of PLA particles coated with 70 nm of gold ﬁlm (a) before
damage and (b) after damage by lOKeV electron beam.

147

    
    
   
  

Beam

Absorbed
Electron

Backscattered
Electron

  
  
   

<— Gold Film

(8)

Continuous
Beam
Exposure

’

(b)

Figure 6.38: a) Illustrates how beam damage is reduced with increasing thickness of
gold ﬁlm over polylactic acid particle. b) Illustrates the shrinking of particles during
electron beam damage.

148

6.5.4 Effect of Structure of Poly-Lactic Acid on Deformation

Effect of PLA structure on damage by electron beam was also studied. Poly-L-
lactic acid (PLLA) has more crystallinity than the poly-DL-lactic acid that has been used
for previous studies. Differential scanning calorimetry revealed that the Poly-L-lactic
acid has a recrystallization peak and melting point peak present at the 100°C and 180°C
respectively which was absent in the case of poly-L-lactic acid shown in Figure 6.39.
Poly-L-lactic acid particle were prepared by emulsion evaporation technique. First PLLA
was dissolved in chloroform at the concentration of 15 mg/ml and then emulsion was
formed by adding 2 ml of PLLA solution in 6 ml of DI water with 15mg/ml of surfactant,
Pluronic F68. Emulsiﬁcation was done in bath sonicator for 1 hour. This was followed by
evaporation step under partial vacuum for 2 hours.

The particles were again drop coated on a glass slide and coated with 7 nm of
gold ﬁlm. The particles were imaged under the accelerating voltage of lOKV. The
particle before and after beam damage are shown in Figure 6.40. The beam damage
appears similar to what was observed for amorphous poly-lactic acid hence crystallinity
does not reﬂect in surface feature that change with beam damage. Figure 6.41 shows the
TGA of pure crystalline and amorphous PLA and there was not difference in the TGAs
obtained for the particles formed. Hence the change in shape observed for poly-lactic acid
particles can be attributed to the beam damage done by high energy electrons in SEM.
This damage can be reduced by coating PLA particles with thicker layer of gold or by

imaging the particles at low accelerating voltage in SEM.

149

PLLA-Cyrstalline

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.2
0 1 Recrystallization
' Peak
.3 0 - . . T
§ .0 14p \80 120 160 W
‘3" Glass
5 "0'2 ‘ Transition
.5 .0,3 - Temperature
0
I -0.4 -
4,35 - Melting
Point
-0.6
Temperature(°C)
(a)
PLA-Amorphous
0.1
0.05 — /
a 0 . . . W
E
‘3' £0540 so 120 160
2
El: -0.1 -
ﬂ -0 15 _ Glass Transition
1 ' Temperature
-0.2 -
-0.25
Temperature(°C)
(b)

Figure 6.39: DSC curves for poly-lactic acid: a) Crystalline and b) Amorphous.

150

 

 

(b)

Figure 6.40: SEM images of PLLA particles coated with 7 nm of gold ﬁlm a) before
and b) after damage.

151

110

 

 

 

 

 

 

 

Crystalline
.. 100
,. ‘7
f» Amorphous
o
3 90
80 I ﬁ T
0 50 100 150 200

Temperature(°C)

Figure 6.41: Thermogravimetric Analysis of crystalline and amorphous poly-lactic
acid revealed that there was not significant mass loss in the PLA particles fabricated

in the nanomixer and the shape change is due to the beam damage caused by the
electron beam.

152

 

6.6 CONCLUSIONS AND FUTURE WORK

In this chapter we have developed a fast and efficient way to fabricate poly-lactic
acid nanoparticles using the high shear rate nanomixer. The inﬂuence of shear rate is
evident in both nanoprecipitation as well as emulsion diffusion. High shear rate can form
smaller particles in case of emulsion diffusion process but can also lead to agglomeration
and coalescence of particles due to high temperatures inside the mixer. In emulsion
diffusion the particle size went down with increasing mixing speeds but is controlled by
mixing time. Longer mixing time led to the coalescence of droplets to form larger
particles and is true for different kinds of particles as shown with gold nanoparticles and
polystyrene ﬂuorescent particles.

Varying the viscosity also can inﬂuence the shape and size of the particles by
shifting from inertial to viscous turbulent regime. It was also evident that the coalescence
of droplets can be completely different at high viscosities. The droplets at high viscosity
can lead to shell shaped structures that are formed by droplets getting caught inside the
eddy and coalescing at the same time. The PLA particles are also inﬂuenced by the
electron beam under SEM. This happens because the electrons in the beam can have
sufﬁcient energy to damage the PLA chains and deform the particle shape. It can be
reduced by increasing the gold coating that needs to be done on PLA particles before
imaging them under SEM. The increasing gold thickness dramatically reduces the
transmitted beam fraction, hence reducing the extent of the damage done to the PLA
particles.

There is a lot of scope for future work while studying the inﬂuence of nanomixer

in nanoprecipitation and emulsion diffusion. As the particles have tendency to

153

agglomerate or coalesce in the mixer, two different kinds of phases or materials can be
mixed to form biphasic or composite particles. Also in the emulsion diffusion method,
the inﬂuence of deuterated water on the emulsion formation can be studied. Further
experiment to study the inﬂuence of particles shape in viscous turbulent regime can be

carried out by controlling the viscosity and mixing conditions inside the vessel.

154

10.

11.

12.

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