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FORMATION, CHARACTERIZATION AND APPLICATIONS
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GOLD

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JANELLE DAWN SECL NEWMAN

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FORMATION, CHARACTERIZATION AND APPLICATIONS OF GOLD
NANOPARTICLES AND SURFACE-MODIFIED GOLD

By

Janelle Dawn Secl Newman

A Dissertation
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Chemistry

2007

ABSTRACT
FORMATION, CHARACTERIZATION AND APPLICATIONS OF GOLD
NANOPARTICLES AND SURFACE-MODIFIED GOLD

By
Janelle Dawn Secl Newman

Gold nanoparticles (AuNPs) have gained interest due to their unique properties.
This dissertation will encompass three areas of current nanoparticle research. The first
sections will discuss the formation of AuNPs using amines as both the reducing and
stabilizing agent. Simple electrochemical measurements showed that the presence of the
oxidation peak for an amine between the oxidation and reduction peaks of a solution of
HAuC14 was a marker for thermodynamic feasibility of AuNP formation. In addition to
thermodynamic considerations, kinetics of formation are also important and were
explored using time-resolved ultraviolet-visible spectroscopy to measure PR evolution. It
was seen that in amine reducing agents with a single amine per reducing agent molecule,
that increasing oxidation potential resulted in a decrease in the rate of PR evolution. This
is consistent with the AuNP formation occurring in the Marcus inverted region.
Poly(ally1amine hydrochloride) (PAH) was explored as a simple polymeric amine
reducing agent. The thermodynamics and kinetics of the PAH system were explored as
for the monomeric amines. The equilibrium behavior of the PAH-AuNP composite was
also investigated and found to be readily controlled.

The characterization of quartz crystal microbalances with alkanethiol self-

assembled monolayers (SAMs) adsorbed on the Au electrodes was explored using

impedance spectroscopy. The time-evolved impedance spectra for the SAMs were fitted
to an equivalent circuit model and information was extracted about the assembly and
viscoelastic behaviors of C6-C13 SAMs. The initial deposition of the alkanethiols was
found to occur within the first minute of QCM immersion. The organization rates for the
C6-C16 SAMs were found to be similar, indicating the aliphatic chain length had little
impact on the rate of SAM assembly. The viscoelastic properties of the C5 monolayer
differed from those of the C9-C16 SAMs and indicated that the shorter aliphatic chain
resulted in a different solvent-monolayer interface than was seen for the longer aliphatic
chain lengths. Additionally C13 was found to exhibit different behavior from the other
SAMs. This was attributed to solubility limitations of the ClgSH at the experimental
deposition temperature.

Finally the application of AuNPs in sensors for organophosphate/phosphonate
(OPP) compounds was explored. The sensors were created fiom the covalent attachment
of AuNPs to silica gel or planar quartz. The AuNPs were then modified with zirconium-
phosphorous (ZP) chemistry to create a sensor which was class-selective for OPP
compounds. Both the silica gel and planar quartz substrates exhibited a spectral blue-
shifi when exposed to OPP compounds which could be attributed to changes in the local
dielectric environment associated with OPP binding. The limit of detection was seen to
be in the range of 5 x 10'7 to 5 x 10'5 M for the silica gel and planar quartz platforms,
respectively. Additionally, the equilibrium of the binding was explored using the silica

gel-AuNP-ZP sensor and estimated to have a K ~ 2 x 106 M".

To my parents, John M. and Gloria Secl

iv

ACKNOWLEDGEMENTS

First, and foremost, I would like to acknowledge my advisor, Dr. Gary Blanchard.
I came up with more than a few crazy ideas in my graduate research career and his
willingness to let me explore taught me a lot about not only research but also about
myself as a scientist. I would also like to thank my committee, Dr. Greg Swain, Dr.
Merlin Bruening, and Dr. David Weliky for being supportive throughout my graduate
career. They are all great educators and I have learned more about chemistry from them
than I can begin to count. The Blanchard research group members, past and present, have
made the past five years fly by. Thanks to them for insightful and revitalizing
discussions. It has never been boring.

I would also like to take the opportunity to thank several individuals who were
very helpful in this journey. First, I am grateful to Pavel Krysinski, for his helpful insight
into the electrochemical studies. Also thanks to Ewa Danielewicz and Xudong Fan for
their assistance in microscopic imaging studies. I would also like to thank Lisa
Dillingham for all her assistance throughout my entire graduate career. She has gone
above and beyond for me on many occasions and for that I am very grateful. I have had
the opportunity to work with several undergraduate students during graduate school and I
would especially like to thank one talented undergraduate, John Roberts. He is especially
talented and his work was vital to the success of the sensor project. Best of luck to him in

his future endeavors.

Special thanks is owed to Dr. William MacCrehan. He took a chance on the
research ability of a first year masters student and without that opportunity I would most
certainly have not have gotten this far. His suggestion to return to graduate school has
most certainly changed the course of my life. I would also like to take this chance to
thank Dr. William Wulff and his research group for all their delightful and insightful
discussions. More than one scientific inspiration came over a bottle or two of wine on a
Friday evening. Thanks for the honorary group membership.

I am most grateful for the support and encouragement l received from my family.
Thanks to my mother-in-law and father-in-law, Diane and Tim Newman, for always
keeping me grounded and providing an escape from the daily grind. Having someplace
to go made being here much more enjoyable. It truly is great to have your in-laws be two
of your greatest champions and true friends. Thanks to my brother, Matthew Secl, for
always being there. I find your creativity inspirational and hope that when I grow up I
can be as successful in my life as you are in yours. Thanks to my parents, Gloria and
John Secl, for their endless encouragement and support. You have never set limits to me
and have always provided me with every opportunity to explore whatever I wanted to.
You helped me to understand the value of education and I really could not have done this
without you.

Finally, thanks to my husband, Cory Newman. Perhaps only you can truly
appreciate this journey. There is no one else I can imagine having been on it with. I
never dreamed when I started graduate school that I would come out of it richer in life
than I was in knowledge, but in you I found a friend and partner. I feel blessed. Thank

you for everything you have done and have yet to do.

vi

TABLE OF CONTENTS

LIST OF TABLES .................................................................................... ix

LIST OF FIGURES .................................................................................. x

KEY TO SYMBOLS OR ABBREVIATIONS ................................................. xiv

CHAPTER 1

INTRODUCTION .................................................................................... 1
Historical Perspective ....................................................................... 1
Theory and General Spectroscopic Behavior ............................................ 2
Synthetic Advances ......................................................................... 5
AuNP Characterization .................................................................... 10
Applications of AuNPs .................................................................... 12
Conclusion .................................................................................. 19
References ................................................................................... 21

CHAPTER 2

FORMATION OF GOLD NANOPARTICLES USING AMIN E

REDUCING AGENTS ............................................................................. 28
Introduction .................................................................................. 28
Materials and Methods ...................................................................... 31
Results and Discussion .................................................................... 32
Conclusion .................................................................................. 48
References ................................................................................... 51

CHAPTER 3

POLYMERIC AMINE REDUCING AGENT .................................................. 54
Introduction .................................................................................. 54
Materials and Methods .................................................................... 56
Results and Discussion .................................................................... 57
Conclusion .................................................................................. 68
References ................................................................................... 69

CHAPTER 4

INVESTIGATIONS OF ALKANETHIOL SELF -ASSEMBLED
MONOLAYER ORGANIZATION AND VISCOELASTIC PROPERTIES
USING IMPEDANCE SPECTROSCOPY ...................................................... 73

Introduction ................................................................................. 73

vii

Materials and Methods .................................................................... 79

Results and Discussion .................................................................... 80
Conclusion ................................................................................... 93
References .................................................................................... 95

CHAPTER 5

OPTICAL ORGANOPHOSPHATE SENSOR BASED ON GOLD

NANOPARTICLE F UNCTIONALIZED SILICA GEL ....................................... 98
Introduction .................................................................................. 98
Materials and Methods ................................................................... 101
Results and Discussion .................................................................. 104
Conclusion ................................................................................. 1 19
References .................................................................................. 121

CHAPTER 6

OPTICAL ORGANOPHOSPHATE SENSOR BASED UPON GOLD

NANOPARTICLE FUNCTIONALIZED QUARTZ ......................................... 127
Introduction ................................................................................ 127
Materials and Methods ................................................................... 129
Results and Discussion .................................................................. 132
Conclusion ................................................................................. 143
References .................................................................................. 145

CHAPTER 7

CONCLUSIONS AND FUTURE WORK ..................................................... 150

viii

Table 2.1.

Table 2.2.

Table 5.1.

Table 5.2.

Table 5.3.

Table 6.1.

Table 6.2.

Table 6.3.

LIST OF TABLES

Oxidation potentials of amines in a 0.5 M LiClO4 electrolyte
solution .............................................................................. 37

Summary of kinetic data from UV-visible

spectroscopy ........................................................................ 39
Response of sensor to OPP compounds compared to the ethanol

control sample .................................................................... 114
Sensitivity of sensor to DECP .................................................. 115

Influence of solvent refractive index on S/N ratio and background
absorbance ......................................................................... 118

Plasmon resonance band shifts for APDMES sensor exposed to

analytes ............................................................................ 137
Plasmon resonance band shifts for MPTMS sensor exposed to

analytes ............................................................................ 139
Sensitivity to DECP ............................................................. 141

ix

Figure 2.1.

Figure 2.2.

Figure 2.3.

Figure 2.4.

Figure 2.5. .

Figure 2.6.

Figure 2.7.

Figure 2.8.

Figure 3.1

Figure 3.2.

Figure 3.3.

LIST OF FIGURES

a) Comparison between the CV of HAuCl4 (solid line) and

tryptophan (dashed line) in aqueous 0.5 M LiClO4 electrolyte

b) CV of HAuCl4 0.5 M LiClO4 electrolyte (MeCN) and

c) CV of pyridine in 0.5 M LiCIO4

(MeCN) .............................................................................. 34

Structures of reducing agents examined in this work ......................... 35

Relationship between oxidation potential and initial growth rate
when one nitrogen is present per mole reducing agent ....................... 40

CV of aniline in 0.5 M LiClO4
(MeCN) ............................................................................. 43

1H NMR in CDCl; for a) aniline after completion of electrochemistry
and b) prior to electrochemical oxidation ...................................... 44

Time resolved US-visible spectrum of 100:1 0.005 M aniline-0.005 M
HAuCl4 conversion to poly(aniline) ............................................. 45

a) CV and b) UV-vis of 3-aminophenol ....................................... 46

UV-visible spectrum of a) 100:1 0.005 M 4-aminophenoI-0.005 M
HAuCl4 and b) excess Au0 and 0.005 M 4-aminophenol conversion
to poly(4-aminophenol) .......................................................... 48

AuNP imbedded in PAH matrix ................................................ 58

First order plot of the dependence of the plasmon resonance band

growth rate on the concentration of HAuCl4 for nominally constant

PAH concentration. The line is a calculated best-fit, with the slope

being 0.86 log(a.u.)/log(M HAuCl4) (1.39 a.u./M HAuCl4) ................. 59

Concentration effect on plasmon resonance band growth rate at
100:1 PAHzHAuCl4. The dashed line is a fit of the data to a third
order growth curve ................................................................. 60

Figure 3.4.

Figure 3.5.

Figure 3.6.

Figure 3.7.

Figure 3.8.

Figure 3.9.

Figure 3.10.

Figure 4.1.

Figure 4.2.

Figure 4.3.

Plasmon resonance band spectral growth for a 100:1 PAHzHAuCl4
mixture at room temperature. Solution concentrations were
0.005 M for both PAH and HAuCl4 ............................................. 61

Polymer-AuNP composites with mass ratio increasing from left to

right. A) Sample 1.3:]; B) Sample 45:]; C) Sample 92:];

D) Sample 13.1 :1; E) Sample 204:] (This image is presented in

color ................................................................................. 62

UV-visible spectra of the five polymer-AuNP composites and the

aqueous PAH. Mass ratios for the spectra are (a) 1.3:] PAH :HAuCl4,
(b)4.5:1,(c)9.2:1,(d)13.1:1, and (e) 20.4:1.

Baseline offset for highest polymer concentrations is due to light
scattering ............................................................................ 63

Linear relationship between polyrner-to-gold mass ratio and plasmon
resonance maximum. The line through the data is a best-fit line .......... 64

1H NMR spectra of a) poly(allylarnine hydrochloride) in D20 and
b) AuNPs in a poly(allylarnine hydrochloride) (from a 1:1 molar ratio)
matrix in D2O ...................................................................... 65

TEM images of a) a sample made from 4.5:1 polymer-to-gold ratio
solution, with particle sizes ranging from 5-15nm in diameter
(measurement bar indicates 100 nm) and b) a sample made from 20.4:1
polymer-to-gold ratio solution, with particle sizes ranging from 35—50nm
in diameter (measurement bar indicates 50 nm) .............................. 66

Particle size distributions recovered from TEM micrographs for AuNPs
synthesized and imbedded in PAH. For each distribution, the
PAHzHAuCl4 ratio is indicated in the legend .................................. 67

(a) BVD equivalent circuit where the left arm of the circuit is the static
capacitance (Co) and the right arm is the motional arm where Lm is the
motional inductance, Cm is the motional capacitance, and Rm is the
motional resistance. (b) Equivalent circuit describing the unperturbed
(L1, C1, R1), liquid loaded (R2, L2) and mass loaded (L3) portions of the
QCM crystal ........................................................................ 77

Comparison of experimental data (-) and model fit (—) for a blank
QCM crystal ........................................................................ 81

Comparison of experimental data (-) and model fit (—) for a C16-SH
monolayer on QCM crystal ....................................................... 82

xi

Figure 4.4.

Figure 4.5.

Figure 4.6.

Figure 4.7.

Figure 4.8.

Figure 4.9.

Figure 4.10.

Figure 5.1.

Figure 5.2.

Figure 5.3.

Figure 5.4.

Figure 5.5.

Figure 5.6.

Figure 5.7.

Comparison of grth behavior of L2 (I) and R2 (0) ........................ 84

Time-dependent impedance responses of a) Cg-SH and b) Clg—SH
SAMs on Au-coated QCM. Spectra were acquired starting at time
zero, at 1 hour intervals for a period of 12 hours .............................. 85

C9 monolayer growth from ethanolic solution ................................. 86

Initial growth rate of L2 for alkanethiol SAMs of varying carbon chain
length ................................................................................ 87

Inductance of alkanethiol SAMs of varying carbon chain length. . . . . . .88

Viscoelastic response (p11) for alkanethiol SAMs of varying carbon
chain length ......................................................................... 90

QCM crystal with a) Cut-SH monolayer and b) ng-SH monolayer.
Both SAMs were deposited from ethanolic solution ......................... 92

OPP analytes a) methylphosphonic acid and
b) diethylchlorophosphate ...................................................... 101

SEM image of AuNPs prepared by the citrate reduction method.
Average particle size is 20 nm ................................................. 102

SEM image of a) unfimctionalized silica gel beads with average particle
size of approximately 63.6 um and b) AuNP-functionalized silica gel
beads with average particle size of approximately 10 um .................. 105

SEM image of the surface of a) unfunctionalized silica

gel beads (as seen in Figure 5.3a) and b) AuNP-functionalized silica

gel beads (as seen in Figure 5.3b). The measurement bar represents

200 nm in each 1mage106

UV-visible spectrum of AuNP-functionalized silica gel beads with the
spectrum of unfunctionalized silica gel beads subtracted in ethanol
(black line) and citrate stabilized AuNPs (gray line) ........................ 108

UV-visible spectra of silica gel sensor exposed to varying
concentrations of DECP. Absorbance has been normalized to

450 nm absorbance .............................................................. 109

Response of MPTMS functionalized silica gel sensor to 24 hour
exposure to a) ethanol, b) 2 mM MPA, and c) 2 mM DECP ............... 114

xii

Figure 5.8.

Figure 5.9.

Figure 6.1.

Figure 6.2.

Figure 6.3.

Figure 6.4.

Figure 6.5.

Figure 6.6.

Comparison of observed PR bands to 1:1 free-complexed AuNP model
(black trace), 2:1 free-complexed AuNP model (dark gay trace) and

3:1 model (light gray trace) ..................................................... 1 l7
S/N of sensor in a) DMSO (S/N = 42), b) DMF (S/N = 23.1), 0) ethanol

(S/N= 5.8), and d) chloroform (S/N = 36.2) .................................. 119
Representation of the AuNP-functionalized quartz sensor ................. 131

SEM images of AuNPs on ITO substrates at 10,000 times magnification
A) APTES linker, B) APDMES linker C) MPTMS ........................ 133

SEM images of AuNPs on ITO substrates at 100,000 times magnification
A) APTES linker, B) APDMES linker C) MPTMS ........................ 135

APTES linker PR before (dashed line) and after (solid line) exposure to
2 mM MPA. PR blue shifted 4 nm upon exposure ......................... 136

a) MPTMS PR before (dashed line) and after (solid line) exposure to

2 mM MPA. PR blue shifted 13 nm upon exposure.

b) MPTMS PR before (dashed line) and after (solid line) exposure to

2 mM DECP. PR blue shifted 10 nm upon exposure.

c) MPTMS PR before (dashed line) and after (solid line) exposure to
ethanol. No blue shift observed ............................................... 138

Observed PR response of the sensor to variations in DECP
concentration ..................................................................... 142

xiii

APDMES
APTES
AuNPs
BVD
CWC
MPTMS
OPP
PAH

PR
QCM
SAM
SEM.
SERS
SHE
TEM

ZP

KEY TO SYMBOLS OR ABBREVIATIONS
3-aminopropyldimethyethoxysilane
3-aminopropyltriethoxysilane
Gold nanoparticles
Butterworth-Van Kyke
Chemical Warfare Convention
3-mercaptopropyltrimethoxysilane
Organophosphate/phosphonate
Poly(allylamine hydrochloride)
Plasmon resonance
Quartz crystal microbalance
Self-assembled monolayer
Scanning electron microscopy
Surface enhanced Raman scattering
Standard hydrogen electrode
Transmission electron microscopy

Zirconium phosphate

xiv

Chapter 1

Introduction

Historical Perspective

Gold is one of the oldest metals known and has been used since the time of
ancient civilizations.M It is known as a noble metal, due to its inertness, and occurs in
nature in its native form."3 Gold is present in the Earth’s crust at a concentration of
approximately 0.004 parts per million3 and it is presumed that due to this relative rarity it
was and has continued to be used both in coinage and as the basis for many monetary
systems worldwide?” 5 Additionally, gold is a soft metal whose malleability makes it
usefirl as a material in jewelry manufacturing, dentistry, and in electronics." 2 In its bulk
form, gold is a yellow metal and it is this color which is responsible for its chemical
symbol, Au, which is derived from the Latin word for yellow, aurum.2 In addition to
gold being useful in its bulk form, it has also been used in its colloidal form for centuries.
This thesis will address preparation, modification and characterization of colloidal gold,
also known as gold nanoparticles (AuNPs), as well as the development of analytical
techniques useful in applying AuNP chemistry to broader chemical problems.

AuNPs have been utilized by artisans for centuries. One of the earliest
preparations of colloidal gold was in “purple of Cassius”, which has was used as a

colorant in art glasses and ceramic paints. 3’5 This colorant, as its name suggests, is

purple and is believed to have been formed by mixing tin(II) chloride with a solution
containing the chloroaurate ion ([AuCl4]').3’ 4 Solutions of gold ions have also been
reduced to colloidal suspensions by reducing agents including tannin and phosphorous.3'5
Throughout history, the preparations of these solutions were largely phenomenological.
In the late 1850’s, Michael Faraday determined that the color of ruby glass actually
resulted from the incorporation of small particles of gold.“3 Faraday reduced yellow
solutions of chloroaurate ions with phosphorous and created AuNP solutions. 4‘ Faraday
suggested that “if a piece of this substance (phosphorous) be placed under the surface of a
moderately strong solution of chloride of gold, the reduced metal adheres to the
phosphorus as a granular crystalline crust”.6 He also discovered, in a broad sense, that by
altering the concentration of the gold chloride he could get colloids ranging from ruby
colored solutions to darker solutions which formed a gold precipitate.6 Faraday was
instrumental in introducing the study of these colloids using transmitted light. He noted
the variety of colors that were present in the solutions prepared under various conditions.6
It was his understanding of the colloidal color coming from the finely divided gold

particles which paved the way for the harnessing of these gold clusters in the 20th century

and beyond.

Theory and General Spectroscopic Behavior

The color of colloidal gold solutions is one piece of evidence for the behavior of
these solutions of finely divided gold having properties which fall between that of atomic
gold and bulk gold. These properties are both optical and electronic in nature, but it is

the optical properties which have drawn the greatest amount of interest in the study of

AuNPs. In the early part of the 20th century, Gustav Mie solved Maxwell’s equations
with special conditions for small spherical metal particles.7 This series of equations, now
known as Mic Theory, elucidated the relationship between the size of the metal particles,
the medium in which the particles were suspended, the absorption wavelength, and the
absorbance intensity. In a general sense Mie determined that the optical response of
AuNPs was the result of the summation of the electronic and magnetic oscillations and
could be described as the extinction cross-section, cm. This value could be further
described as ow = cabs + 030,, where cabs is the absorbance cross-section and osea is the

10

scattering cross-section.7' Further, it was determined that for small particles the

scattering cross-section was minimized so the majority of the optical response was due to

the absorbance cross-section of the AuNPs (equation l)8’ “’ '2

8'

2:24 2R3 % 1
00,, fl 8'" (r:'+2.c,,,)2+(.s")2 ()

 

The absorbance cross-section and the wavelength of absorbance (7») can be shown to be
related to the size of the particles which make up the analyte (R), the dielectric constant
of the medium surrounding the particles (em), and the real and imaginary components of
the dielectric constant of the metal which makes up the particles (8’ and a”, respectively).
While surface modifications have the potential to affect e’ and a”, this equation is most
useful in its demonstration that the wavelength at which these colloidal solutions absorb
energy can be predicted by the particle size and medium dielectric constant. The utility
of this relationship will be discussed in greater detail later in this chapter.

While specific to spherical particles, Mie’s equations allowed for the modification

of Beer’s Law in order to understand the absorbance behavior of AuNPs (equation 2).'3

3 la
Aabsusca = i = c abs 500 (2)
IO 4Rp

Here the value c represents the concentration of AuNPs in the analyzed solution, I is the
pathlength, and crabs»,ca is the absorbance or scattering cross-section of the colloidal
solution, R is the nanoparticle radius, and p is the density of Au. However, it is clear that
Mie’s elucidation of these relationships and their subsequent application to Beer’s Law
can be used to explain the variations in the colors and intensities of AuNP solutions used
in both the arts and sciences. Most importantly variations in the particle sizes14 and the
medium surrounding the particles can be directly correlated to the wavelength of light
which the particles absorb. With these AuNPs, the absorbance wavelength is in the
visible region, leading to the red, blue and purple colors exhibited by these solutions.

The characteristic absorption exhibited by solutions of AuNPs is the result of the
resonant excitation of the surface plasmons of nanoparticles which are much smaller than
the wavelength of incident light and is most pronounced for AuNPs smaller than 25 nm”‘
15 This absorption, called the plamson resonance (PR), is the result of the collective and
concerted oscillation of the conduction electrons on the surface of the AuNP.l4 This
oscillation occurs, in the presence of an electric field, due to the immobile nature of the
positively charged ions and the mobile nature of the free conduction band electrons on
the particle surface. Equation 1 predicts that if a” is small the resonance fiequency can be
predicted to fall at the wavelength where e’ = -280, the resonance condition.‘6 The

frequency of this oscillation can also be described by equation 3'7

Ne2
wp=V£m (3)
0 e

 

Where (up is the frequency of the plasmon oscillation, and N is density of the surface
electrons. N is effectively related to the surface to volume ratio, or size and shape, of the
nanoparticle. In practice, this predicted size dependence has been widely described and

the position of the PR of AuNP colloids is widely variable with particle size.5’ 10’ I" ”"5:

”'21 As discussed previously, for small particles there is a dependence on both the
particle size as well as the local dielectric environment surrounding the particles.
However, for larger particles (> ca. 20 nm) the PR response is dependent only on the
AuNP size”, up to the limit where the particle aggregates to form bulk metal. Bogatyrev
and co-workers have demonstrated that in the AuNP size range of approximately 18-30
nm a linear increase was seen in the PR repsponsezz. Additionally, they found that
increasing the particle size also resulted in an increase in the peak half-width and
magnitude of absorbance, as would be predicted by equation 2.22 Similar results have

been reported by numerous others as well and are the basis for the utility of AuNPs in a

variety of applications to be discussed later in this chapter.

Synthetic Advances

With the mathematical insight provided by Mie, AuNPs became a more
interesting target of research due to the ability to theoretically predict and understand the
spectroscopic behavior of the solutions. In the 1950’s AuNPs became even more usefirl
as chemical targets when a more clear understanding of the reduction of [AuCl4]‘ into
colloidal gold was elucidated through a series of experiments by John Turkevich, et al.23
Turkevich studied the reduction of [AuCl4]' by the citrate ion in detail, including efforts

to determine the behavior of the reaction at both the nucleation and grth stages.”26

AuNP synthesis using the [AuCl4]' and citrate ions was simple and involved combining
boiling dilute aqueous solutions of [AuCl4]' and citrate, from either citric acid or sodium
citrate, and stirring vigorously.23 Turkevich examined the various stages of the AuNP
preparation using transmission electron microscopy (TEM) to understand and attempt to
delineate the nucleation and grth phases.23 He understood nucleation to be the key

23 The actual nucleation event, however, was less well

step in the formation of AuNPs.
defined. Turkevich proposed the nucleation of reduced gold atoms resulted from the
complexation of gold atoms inside of a matrix of the reducing agent. When enough of
these gold atoms came into close spatial proximity, a spontaneous reorganization would
occur which resulted in the formation of a nucleus. Following the nucleation event, the
AuNPs would grow through aggregation of remaining gold ions and atoms into the final
AuNP.23 This growth was described by equation 4 and which predicts that the final
particle size was dependent on the size of the nucleus created as well as the mass of the

remaining Au in the solution”‘ 26

M.+M
D =Ds—‘——i 4
f 0" Mn. ()

Subsequent investigations into this type of nucleation and growth, sometimes referred to
as seed-mediated nucleation, have supported this mechanismz7 The AuNP solutions
resulting from this method are stabilized by the citrate ions which surround the reduced
AuNP core. The negative charges of the citrate ions on the particle surface create an
electronic hindrance to the further aggregation of Au atoms. Thus the final particle size
is also dependent on the amount of reducing agent in this synthetic method as it also acts
as the stabilizing agent as well. Frens exploited this reduction and stabilization

mechanism and explored the effect of altering the ratio of the gold ion to the reducing

agent.28 Like Turkevich, he found that the reaction reached completion quickly with no
visual change in the colloid occurring after approximately five minutes of mixing.28
Frens also found that simple alterations in the ratio of AuC14' to citrate ion resulted in the
accessibility of a broad range of particle sizes and resulting spectral resonances and
resulted in relatively monodisperse solution of the AuNPs in the range of 1.2-15 nm.28
He suggested, as did Turkevich and co-workers, that the final particle size available from
any AuNP synthesis was able to be determined primarily by the number of nuclei created
and not by changes in the efficiency of the reduction event resulting from changes in the
ratio.28 Additionally, Jana reported that the rate at which the reducing agent is added also
plays a role in the final AuNP size as well as the degree of dispersity in the final
colloid.27 The understanding of both the mechanism of the citrate reduction and the
simple control over the final particles has resulted in this method being widely prevalent
in the AuNP literature even as other methods have been discovered.

The citrate reduction method has a number of significant advantages as a
synthetic method for creating AuNPs. First, it is a simple and time-efficient synthesis as
it requires only boiling and mixing of the aqueous Audi and citrate solutions. In less
than 15 minutes a stable colloidal solution results, requiring only stirring to complete the
reaction. Additionally, as discussed previously, there is a great deal of control over the
size of the AuNPs simply through alteration of the relative concentration of the Au” and
citrate components. Finally, the particles are reduced and stabilized by the same
compound, which minimizes the need for and additional stabilizing agent as well as
ensuring that the particles are surrounded by only the citrate ions as opposed to another

functional group which may be in the solution. Despite these advantages, there are

problems with the citrate reduction method. First, the AuNPs created in this synthesis are
generally larger in size. This creates difficulties if the particles are to be removed from
the aqueous solution and re-suspended in another aqueous or organic solution. This is a
disadvantage if a stabilization shell is desired which is not soluble in aqueous solution.
Much of the established gold surface chemistry is done in organic solvent, so this limits
the surface chemistry being applied to these particles in the solution phase. Finally, while
displacement of the citrate shell is desired with an aqueous-phase modifier is possible, it
is still difficult to verify the complete exchange of one stabilizer for another. This can be
a concern if a high concentration of the replacement stabilizer is required for further
AuNP chemistries.

In the mid-1990’s a new class of methods were developed which allowed for

29' 3° Brust and co-workers

creation and stabilization of AuNPs in organic solvents.
specifically introduced these methods as a technique for creating thiol stabilized AuNPs.
In the two-phase method, first proposed in 1994, an aqueous solution of HAuCl4 was
combined with a solution of tetraoctylarmnonium bromide (TOAB) in toluene. The two
solutions were agitated with the TOAB acting as a phase transfer agent, moving the Au3+
ions from the aqueous phase and into the toluene. Once the ion transfer is complete, a
solution of the desired thiol stabilizer in toluene was added and the mixture reduced with
aqueous sodium borohydride (N aBH4).30 A second method, reported in 1995, utilized a
single organic phase.29 Brust utilized p-mercaptophenol as the stabilizing thiol and did
the entire synthesis in toluene, eliminating the need for the TOAB phase transfer agent.

Again, the reduction was accomplished in this synthesis using NaBH4. In these cases, the

resulting AuNP colloids are sterically stabilized by the relatively bulky surface-bound

thiols. The AuNPs resulting from both these methods are in the 1-3 nm range and require
reaction times in the range of 3 hours and equilibration times in the range of 4 hours.” 30
Due to the smaller particle size, it is possible to readily remove these AuNPs from
solution for storage and to subsequently re—suspend them in a desired organic solvent.30
Despite the obvious advantages that arise from the use of this method, including the
much broader range of stabilizers that can be used, there are several disadvantages as
well. First, while the use of organic solvents broadens the scope of possible reducing
agents and allows the accessibility of much of the modification chemistry established for
planar gold surfaces, it is also less environmentally and biologically friendly than using
aqueous solvents. Secondly, the use of NaBH4 as the reducing agent is required for
AuNP formation. This not only adds an additional step to the synthetic procedure, but it
can also be hazardous due to the violent nature of the reaction which occurs when the
NaBH4 is added to the organic phase containing the Au3+ and thiol stabilizer.

The first part of this thesis addresses the formation of AuNPs using amines as
simultaneous reducing and stabilizing agents. These reactions are attractive as they have
many of the advantages of both the Turkevich and Brust methods while minimizing the
disadvantages associated with both these methods. Like the citrate reduction method,
formation of AuNPs using amines allows for simultaneous reduction and stabilization,
making the synthesis a single step. It also has the advantage of employing a wide variety
of solvents, both aqueous and organic, depending on the desired amine. Finally, the
ubiquitous nature of amines provides a wide variety of possible choices for
reducing/stabilizing agents with multiple functionalities possible. This thesis addresses

the study of monomeric and polymeric amines as reducing and stabilizing agents for the

formation of AuNPs via reduction of Au“. These studies will include investigations of
the thermodynamic potential of amines of various structures to act as reducing agents for
Au“. Following determination of the thermodynamic potential, kinetic investigations
will be discussed where ultraviolet-visible spectroscopic investigations will be used to
determine if amines with the appropriate electrochemistry actually demonstrate the
presence of a PR, indicative of the formation of AuNPs. Reactions using both

monomeric and polymeric amines will be discussed.

AuNP Characterization

Once synthesized, it is important to be able to characterize the AuNPs. While
there are a number of parameters of interest in the measurement of AuNPs the size,
polydispersity, degree of aggregation, and PR position are the characteristics of greatest
interest to nanoparticle scientists. Despite challenges inherent to the relative size
domains present challenges in AuNP colloids two primary methods have emerged as the
analytical techniques most commonly employed in AuNP metrology: imaging techniques
and UV-visible spectrometry.

Imaging. Imaging of AuNPs is, perhaps, the most powerful characterization tool
available. Visualization of AuNPs can be accomplished by either scanning electron
microscopy (SEM) or transmission electron microscopy (TEM), though the latter is more
valuable as it does not require a conductive substrate. The importance of these imaging
techniques comes from the ability to examine multiple characteristics simultaneously.

Through examination of images obtained from microscopic techniques information can

10

be obtained about the structure of the nanoparticles5 as well as the size and shape
distribution of the AuNPs in a colloidal solution.

U V-visible spectroscopy. As discussed previously, the awareness of the
existence of AuNPs came from the characteristic red to purple color. This leads to UV-
visible spectroscopy being a very useful characterization tool for AuNP colloids. The
presence of a strong absorption peak, the PR, in the UV-visible spectrum in the range of
500-600 nm is considered diagnostic of the presence of AuNPs in colloidal solutions.
Also discussed previously is the ability to use the size and shape of this PR peak to
determine the approximate size of the AuNPs in a given medium. 5’ '0‘ I" ”45’ ”'2' The
speed and simplicity of this kind of measurement makes it the most widely used
characterization method for AuNPs, both in solution and when attached to transparent
surfaces.

Impedance spectroscopy. Impedance spectroscopy relies on measurement of the
oscillation of quartz in the presence of an electric field. This oscillation can be monitored
through a range of frequencies by an impedance analyzer. The impedance measurements
are useful for a number of reasons. First, the Sauerbrey equation (equation 5) can be used
to determine the mass of an adsorbed film by measurement of the shift of the resonance

frequency.3 I

-2Am7y’02
Af=—-—- (5)
Aime)”

This relationship can be used in any case where the properties of the adsorbed monolayer
are similar to the quartz used in the QCM. However, when the magnitude of the
oscillation is measured across a range of frequencies near the resonance frequency, it is

possible to also obtain information about the line-shape and line-width of the peak

11

associated with the oscillation. Through modeling the QCM system to an equivalent

32'37, information can be obtained

circuit such as a modified Butterworth-Van Dyke circuit
about the films attached to the QCM crystal. This information can be obtained for
measurements obtained in both air and liquid and can be used for either equilibrium or
kinetic measurements.

The second part of this thesis describes initial studies using impedance
spectroscopy and corresponding equivalent circuit models to study the viscoelastic
properties of thin films on gold. In the these initial studies, self-assembled monolayers
(SAMs) of alkanethiols of various carbon chain lengths were chemisorbed, in situ,.onto
planar gold quartz crystal microbalance (QCM) substrates. A great deal is known about
the behavior of alkanethiol SAMs38, including information about the adsorption kinetics
and basic information about monolayer formation. Impedance measurements offer
another layer of information about the film-substrate interface. While these studies were
conducted on planar substrates, the same techniques could be applied when investigating

the properties of AuNPs bound to the QCM substrate or the properties of, for example, an

AuNP composite material such as that described in Chapter 3.

Applications of A uNPs

Biological applications. One major advantage of understanding the formation of
AuNPs by amines, as outlined in chapters 2 and 3 is the potential of these types of
composite materials to be biologically compatible. Gold has been used for centuries as a

remedy for a variety of health problems.4’ 5 Not surprisingly, as understanding about the

12

nature of gold nanoparticles has expanded so has the interest in using these nanoparticles
for medicinal purposes.

AuNPs have been explored for use in a variety of different applications. One of
the promising areas in which AuNPs have been investigated is in the area of drug and
gene delivery.39‘" In these applications, the nanoparticles are incorporated into a system
which is responsive to temperature. Therefore, when the AuNP composite material is
exposed to radiation at its resonant wavelength the matrix into which the AuNP has been
incorporated undergoes a transition which releases the drug or gene. Thomas and co-
workers have reported on the synthesis of spiropyran functionalized AuNPs which were
used to bind and release amino acids based on thermal and photochemical reactions of the

0 In this case the zwitterionic form of the spiropyran-AuNP composite

spiropyran.4
associates with amino acids and forms a relatively stable complex."'0 Upon irradiation of
the AuNP-spiropyran—amino acid complex at the PR wavelength of 520 nm, the complex
dissociates and releases the amino acid.40 The authors suggest that this mechanism could
be used, for example, to deliver L-DOPA, for treatment of Parkinson’s disease or
hypertension.40

One of the most promising areas for the medical use of AuNPs is in cancer
research. There are currently two major areas where AuNPs have seen use. The first is
imaging for cancer diagnostics and the second is cancer treatments. In the area of
diagnostics, AuNPs have shown promise for use in enhancement of imaging of early
stage tumors.” 4244 Here, the ability of radiation in the near infra-red (N IR) region to

harrnlessly penetrate tissue, and the tunability of the PR wavelength of AuNPs are both

exploited. The AuNPs, often synthesized with a dielectric core39’42’ 43‘45, are created as to

13

have their PR in the desired wavelength regime. They are then subjected to a variety of
imaging techniques which create a cross sectional image, illuminating the tumor with its
enhanced uptake of AuNPs39’ 42‘ 43. Tumor strains ranging from breast carcinoma cells
(SK-BR-3) to oral epithelial live cancer cells (HOC 313 close 8, HSC 3) have been
imaged using modifications of this kind of technique.” 4244 The same types of AuNP
composites which have been exploited for tumor imaging have also been explored for
their potential in the destruction of tumor cells. Hirsch and Halas at Rice University have
been instrumental in the field of NIR thermal therapies for tumor ablation. In these
studies, gold nanoshells are prepared via coating of surface firnctionalized silica particles
with 1-3 nm AuNPs.“ 46’ 47 The size of these particles makes it possible for the particles
to pass into the tumors where they accumulate.” 47 Following uptake, the area of the
tumor is irradiated with light in the NIR and localized heating occurs, thus destroying the
tumor cells through a thermal mechanism.” 47 These studies have shown that mice with
both SK-BR-3 human breast epithelial carcinoma and CT26.WT murine colon carcinoma
tumor cells have been successfully treated in animal studies using this technique.46’ 47
After Additionally, animals with tumors but treated only with NIR irradiation in the
absence of AuNPs showed experimental results similar to those of animals with tumors
which remained untreated.” 4’

All of these applications point to the need for biologically-compatible AuNP
composites. The enhanced understanding of amines as reducing and stabilizing agents in
AuNP formation will be potentially useful in the creation of such AuNP materials.

Surface Enhanced Raman Spectroscopy (SERS) Substrates. Another area where

AuNPs are quite useful is in the area of surface-enhanced Raman spectroscopy. As

14

discussed previously the PR exhibited by AuNPs is due to the collective oscillation of
surface electrons on the particles. This excitation is also associated with an increase in

16

the local electromagnetic field surrounding the particles. The Van Duyne and Schatz

groups have pioneered much of the work in the area of nanoparticles and SERSIO‘ '6’ “'52
and it has come to be focused primarily on the use of silver nanoparticles. However, the
same basic enhancement principles which apply to the use of silver in SERS apply to
gold as well and Raman intensity enhancements in the range of 105 (for AuNP solutions)
to 1010 (for AuNP arrays) have been reported.”‘ 54

As early as 1982, Mabuchi and co—workers reported on the SERS spectrum of
citrate ions adsorbed onto AuNPs after preparation using the Turkevich method.54 After
preparation, more sodium citrate was added to the colloidal solutions and they were aged
for several days to ensure that all reactions had reached the maximum degree of
aggregation.54 They found that they were able to detect the sodium citrate in the Raman
spectrum with and enhancement of about 105 times over samples with no AuNPs.
Mabuchi proposed that the enhancement occurred as a result of the excitation of the
surface plasmons of the AuNPs in the solution, in line with current understanding of the
SERS mechanism.54

In more current evaluations of AuNPs as enhancing agents, AuNPs are typically
bound to a substrate and the SERS activity of the substrate probed with Raman active
molecules, such as p-aminophenol.55 Zhu and co-workers found that surface-bound
AuNPs showed the greatest enhancement of Raman intensity when there was a greater
surface coverage and larger AuNP size, with the greatest enhancement coming from the

55

latter. They also found further confirmation of the need for a high surface density of

15

AuNPs in SERS substrates when the examined the effect of interparticle spacing on the
degree of Raman signal enhancement and found that increasing the distance between
AuNPs on the surface resulted in a significant decrease in the ability of the substrate to

1.55

enhance the Raman signa However, they also verified, that while weak, Raman

enhancement by single, uncoupled AuNPs was also possible to measure.55

AuNPs provide a useful tool in SERS thorough their ability to significantly
enhance the observed Raman signal. This is particularly important in increasing the
utility of Raman spectroscopy to evaluate very low concentration signals or to measure
otherwise weakly-Rarnan active molecules.

AuNP-based Sensors. In addition to their use as diagnostic and therapeutic
agents, AuNPs have also been used in sensory applications. These AuNP-based sensors
can be used for the detection of molecules and interactions of biological interest or for
more traditional chemical sensing applications.

When used in biosensing, there are two primary AuNP properties which are
exploited. One sensing mechanism is based on AuNP aggregation of induced by
interaction with the biomolecule of interest. This can be manifested in either the AuNP-
biomolecule complex having properties unique from that of either component or a shift in
the PR position.39 The other mechanism is utilization of the AuNP light scattering to
label biomolecules which are otherwise not optically active.3'9

As an example of the first mechanism, Cao and co-workers have functionalized
AuNPs with antibodies and Raman dye to create an assay for small molecule (antibody)-

protein interactions.56 These AuNPs were exposed to proteins which were spotted onto a

planar array.56 When an interaction occurred between the proteins on the surface and the

16

Raman labeled AuNPs, a gray spot became visible on the array which could be further
evaluated using a Raman spectrometer.56 Additionally, the authors note that this method
can be used in an analogous sense to test for protein-protein interactions as well.56 A
similar experiment was described by Frederix where thin films of AuNPs were
immobilized onto quartz substrates and fimctionalized with anti-human serum albumin
(anti-HSA).57 The UV-visible spectrum was obtained for the sensor prior to exposure to
human serum albumin (HSA). The presence of HSA was manifested as increases in both
the PR wavelength and observed absorbance.57 Similar mechanisms have also been
utilized to detect specific strands of DNA, in immuno-sensing applications, in sugar
sensing, and in the evaluation of biological interactions through conjugation with
biomacromolecules.4’ 58’ 59 Nath and Chilkoti utilized the changes observed in the
spectrum of surface bound AuNPs in the detection of streptavidin.60 They found that the
peak position shift and change in absorbance could be correlated to the degree of binding
and consequently the streptavidin concentration.60 In addition to the sensor being simple
to construct it was also useful because it required nothing more than a simple UV-visible
spectrometer for analysis, thus making the sensor widely applicable.60

In addition to being useful as biosensors, the ability to utilize both the PR position
in the visible region of the spectrum and the well established chemistry available on gold
surfaces makes AuNPs useful as the basis of sensors for a variety of other compounds as
well. There have been several recent examples of sensors created based upon the energy
absorption properties of AuNPs. In 2004, a colorimetric sensor was reported on by
Matsui and co-workers, which was useful in the detection of small molecules ranging

61

from adrenaline to 2-phenylethylamine. It was found that a molecularly imprinted

l7

polymer with AuNPs imbedded within the matrix could be used in conjunction with UV-
visible spectrometry to detect the target analytes in aqueous solutions.61 The detection
mechanism was based on a spectral blue-shift observed when the polymers bound to the
analytes of interest};1 This report is also interesting because the AuNPs themselves
played no role in the binding of the polymer and the analytes of interest. Therefore, it
demonstrated that the AuNP PR can be used as an indicator for other interactions taking
place in the vicinity of the particles. In 2005, He and co-workers reported on a
colorimetric and fluorometric Cu(ll) sensor based on the quenching of a pyridyl-
terminated perylene chromophore by AuNPs.62 In the absence of a Cu(II) ion, the AuNPs
will bind to the chromophore by interaction with the pyridyl N. However, because the
Au—N interaction is weaker than the Cu-N interaction, when Cu(II) is present, the latter
interaction is preferred and the quenching observed in the blank case is not seen.62 They
report a detection limit for Cu(lI) of 1.0 x 1045 M.62 AuNPs have have also been reported
as a sensor interface in a chemiresistor sensor device for detection of volatile organic
compounds (VOCs).63’ 64 Pang and co-workers described the incorporation of AuNPs,
either protected with monolayers or thiols, into a 3-D sensor matrix.63’ 64 The resistance
and capacitance of the films were measured in the presence of VOCs with varying
degrees of humidity. They showed that using AuNPs as the electrical interface in this
sensor they were able to achieve fast reponse and low noise VOC detection at the
hundreds of parts per million levels. These examples demonstrate the wide potential
AuNPs have in the area of chemical sensing.

The final part of this thesis discusses the modification of citrate—stabilized AuNPs,

through displacement of the citrate ion shell and subsequent functionalization with

18

zirconium phosphate (ZP) chemistry, in two optical sensors for detection of
organophosphate/phosphonate (OPP) compounds. There have been examples where
AuNPs were incorporated into OPP sensors65’ 66, however the previous examples rely on
the measurement of acetylcholinesterase (AChE) inhibition. In the work discussed in
Chapters 5 and 6, the detection scheme described is purely chemical in nature and does
not rely on the use of the AChE, making it much more robust as it does not use enzymes.
The AuNPs were bound to either silica gel or planar quartz using silanes. They were then

modified using ZP chemistry“76

to create a Zr4+ terminated surface. This terminus takes
advantage of the affinity of Zr4+ for phosphorous compounds and binds OPP compounds
upon exposure. These sensors exhibited a blue-shift in the visible spectrum upon
exposure to OPP compounds. This blue-shift, due to changes in the local dielectric
environment experienced by the AuNPs upon OPP binding, is characteristic of the
presence of OPPs. These sensors demonstrate the potential for using the characteristic

PR of AuNPs to create sensors which are simple to construct, robust, and require minimal

training to analyze.

Conclusion

The synthesis, characterization, and application of AuNPs have been the three
areas of primary interest to researchers in the field of AuNPs. Since the initial
understanding of the optical behavior of AuNP colloids by Michael Faraday and Gustav
Mie, there has been tremendous grth in the area of understanding how to control,
measure, and use AuNPs. This thesis will cover my contributions to this field. First I

will discuss how the electrochemical behavior of the reducing agent and gold source can

19

be used in the prediction of when AuNPs will be formed. I will also discuss the role of
the kinetics in the formation of the AuNPs in the optical behavior of the colloids. Then I
will discuss the characterization of SAMs on a QCM crystal with Au electrodes using
impedance spectroscopy. While used in this example on planar gold, the information
obtained about the measurement of the viscoelastic properties of the SAMs could be
applied to the measurement of the viscoelastic properties of composite materials which
incorporate AuNPs. Finally, I will discuss my contribution to the field of chemical
sensing using AuNPs. The design and development of optical sensors for OPP
compounds will be discussed. These sensors, created on silica gel and planar quartz

substrates, have shown promise for the simple optical detection of OPPs.

20

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27

Chapter 2

Formation of Gold Nanoparticles Using Amine Reducing Agents

Introduction

Amines have been reported to be usefirl as both reducing agents and stabilizers in
the formation of AuNPs“. A wide variety of amines have been used to reduce gold salts
to AuNPs including primary amines4‘ 6, multifunctional amines3, and amino acids" 2’ 7.
They are attractive choices for reducing agents for several reasons. First, they are
ubiquitous in nature and a wide variety of amine compounds are readily available
commercially. Additionally, their prevalence in biological systems indicates that
composite materials formed from the reduction of HAuCl4 by amines may have the
potential to be biologically compatible. This could be useful in potential applications of
the resulting colloidal materials.

One of the earliest reports of the use of amines in the spontaneous formation of
AuNPs was by Turkevich in 1951. In this instance, hydroxylamine hydrochloride was
used to facilitate the reduction of HAuCl4 into colloidal golds. Turkevich examined

8-

much more closely the reduction of HAuCl4 by citrate ions H and much of the research

on AuNPs which followed used this reduction method. In the mid-1990’s thiols began to
emerge as stabilizing agents after a series of reports by Brust and co-workers which

established organic solvent based methods, which were amenable to use with thiolsn’ '3

28

Amines were less examined than thiols as reducing and stabilizing agents because the
amine-gold bond (bond strength ~ 6 kcal/mol”) is weaker than the sulfur-gold bond seen
in thiol adsorption (44 kcal/mol's). However, in 1996, Leff reported that amine stabilized
AuNPs were similar to AuNP colloids stabilized with thiols4. In this instance, the
HAuCl4 was added in aqueous solution while an alkylamine was added in toluene. The
reduction was facilitated by sodium borohydride and the resulting AuNPs were found in
the organic phase at reaction completion4. This body of work demonstrates that amines
can be used to successfully stabilize AuNPs, but it was not until the early 2000’s that
amines began to be used as reducing agents as well as stabilizers.

In 2004, Selvakannan and co-workers reported on AuNPs formed by the
spontaneous reduction of HAuCl4 by tryptophan in aqueous solution at elevated
temperatures, resulting in AuNPs dispersible in waters. UV-visible spectroscopy showed
the appearance of spectral features in the region characteristic of AuNP PR. The
demonstrated peak grew in intensity with reaction time and also blue shifted as the
reaction neared completions. This is characteristic of the growth and aggregation of
AuNPs in solution. The authors in this case report that it was the indole group of the
tryptophan, a secondary amine, which was singly responsible for the reduction of
HAuCl4, to the exclusion of the primary amine segment, glycines.

Aromatic amines were discussed in 2005 as reducing agents capable of forming
AuNPs from HAuCl4. Subramaniam and co-workers reported on the use of 2-methyl
aniline, a primary amine, as a reducing and stabilizing agent in AuNP synthesis. This
synthesis, like Selvakannan’s, was carried out in aqueous solution at elevated

temperature. In addition to the UV-visible spectrum demonstrating a PR in the

29

characteristic range, Subramaniam also demonstrated that the position of the PR could be
blue-shifted by increasing the volume of reducing agent added to the synthetic solutions.
In 2006, Kuo and co-workers reported on the formation of gold threads from the

reduction of HAuCL. by triethylaminelb.

In this case the reduction was carried out by
combining triethylamine, a tertiary amine, and HAuCl4 (20-fold excess of triethylamine)
in aqueous solution at room temperature. Kuo reported that a characteristic PR was also
seen in the visible region of the spectrum. As expected, the PR band increased in
intensity with increasing reaction time. The PR band also blue-shifted with reaction time,
due to a spontaneous change in the form of the reduced gold from small spherical
particles to organized gold thread structures”.

Despite these and other reports about use of amines in the reduction and
stabilization of AuNPs, most of the information about which amines are appropriate for
use as reducing agents is phenomenological. Little is known about the properties which
make some amines function well as reducing agents and others do not successfully
reduce HAuCl4. A brief overview of the types of amines which have been used reveals
that primary, secondary, and tertiary amines as well as alkyl, aromatic, and aliphatic
amines have all been successfirl. Additionally, reduction has been accomplished in
organic and aqueous media. Understanding what the most important properties of a
potential reducing agent are is important in gaining control and predictability over
nanoparticle formation, growth, and stabilization. This study investigates the

thermodynamic and kinetic factors which can be used to gain predictability over whether

a given amine will function as a reducing agent for HAuCl4.

30

Materials and Methods

Chemicals. HAuCl4-3H20 (99.9+%), 3-aminophenol, 4-aminophenol, triethylamine,
acetone, indole, 1,4-phenlyenediamine, aniline, 4-bromoani1ine, 3-indolepropionic acid,
3-amino-1-propanol, l-methylindole, pyridine, citric acid, hydroxylamine hydrochloride,

sodium citrate, D,L—tryptophan, and lithium perchlorate were purchased from Aldrich
Chemical Co. and used as received. Glycine was purchased from Tyron (Okemos, MI)
and used as received.

Electrochemistry. HAuCla solutions were prepared at a concentration of 0.1 M in 0.5 M
LiClO4. Solutions of amines to be evaluated as reducing agents were prepared to a
concentration of 0.1 - 0.5MLiCIO4 in either acetonitrile (MeCN) or H20, depending on
solubility. Due to solubility limitations, D,L-tryptophan was prepared as a 0.005 M
solution in 0.5 M LiClO4 (aq). Cyclic voltammetry was conducted using an
electrochemical bench (CH Instruments model 604B, Austin, TX) in cyclic voltammetry
(CV) mode with a scan rate of 50 mV/s and a sensitivity of 10'5 A full scale. A standard
three electrode configuration was used with a glassy carbon working electrode, a
platinum wire counter electrode, and a reference electrode. The reference electrodes used
were Ag/AgCl with 3 M KCl (aq) for aqueous measurements and 1 mM AgNOs (MeCN)
for nonaqueous measurements. The potentials measured using the Ag/AgCl reference
electrodes are reported against a standard hydrogen electrode (SHE) to facilitate
comparison to reported tabular values for Au reduction potentials. Three forward and

three reverse scans were recorded for each sample.

31

Kinetics and Stability Studies. AuNPs were synthesized by combining a 0.005 M
reducing agent solution (5 mL) with a 0.005 M I-IAuCl4 (50 IL) solution in the same
solvent and shaking to agitate. The reducing agents used for the kinetics studies were

4-aminophenol, triethylamine, glycine, indole, 1,4-phenylenediamine, tryptophan, and
sodium citrate. The solutions were combined and analyzed at room temperature, and UV-
visible spectra were obtained at intervals for the resulting undiluted solution. The spectra
were obtained at either 10 or 15 min intervals for up to 100 scans or until a depletion of
the absorbance was noted. The duration of a given measurement was determined by the
rate of the process being monitored. The spectra were acquired from 350 to 800 nm using
a Cary model 300 UV-visible spectrometer (Varian) and acquisition time was ca. 1 min
per spectrum. Initial rate information was extracted from the temporal dependence of the

plasmon resonance absorbance band following the initial mixing of the reactants.

Results and Discussion

The purpose of these studies is threefold. The first goal is determining which
amine compounds are thermodynamically capable of reducing HAuCl4 to AuNPs. The
second goal is to verify the ability of the amines to successfully reduce HAuCl4 to form
gold nanoparticles and to subsequently investigate the formation rate. The third goal is to
understand any side reactions which may either compete with or facilitate the formation
of AuNPs by amine reducing agents.
Electrochemistry. The formation of AuNPs occurs through an electron transfer reaction

wherein electrons are transferred from the reducing agent to the Au3+ source. In the case

32

of amine reducing agents, the reduction of HAuCl4 to Auo can be described generally by
Scheme 1.
HAuCl4 + 3 NR3 —. Au" + 3 NR3+' + H’“ + 4 0' (Scheme 1)

The Au0 may then undergo association with other AuO atoms to begin the
nucleation and grth of AuNPs in the solution. The radical amine species is transient
and will be either quickly quenched or act as the initiation step to a resulting polymeric
species. NMR experiments suggest the presence of oligomeric species of the amine
reducing agent in some cases and will be discussed in greater detail later in this chapter.

The most important information to be acquired fi'om electrochemical
measurements is the determination of whether the reaction shown in Scheme I is
thermodynamically allowed. This can be derived through comparison of the oxidation
potential of a given amine with the reduction and oxidation potentials of the HAuCl4 and
Au0 in solution. Evaluation of HAuCl4 in both aqueous and MeCN solution shows
oxidation and reduction potentials present. In aqueous solution the reduction potential of
HAuCl4 to Au0 was found to be 0.853V (vs SHE) and the oxidation potential of Au0 to
Aul+ was found to be 1.425 V (vs SHE), in 0.5 M LiClO4 solution. In MeCN the
reduction potential of HAuCl4 to Au0 was found to be 0.888 V (vs SHE) and the
oxidation potential of Au0 to Au1+ was found to be 1.555 V (vs SHE), in 0.5 M LiClO4

solution (Figure 2.1, panels a and b, respectively).

33

Figure 2.1.
(dashed line) in aqueous 0.5 M LiClO4 electrolyte b) CV of HAuCl4 0.5 M LiClO4
electrolyte (MeCN) and c) CV of pyridine in 0.5 M LiClO4 (MeCN).

1.5x10‘3
1.0x10'3
mm“
0.0
-5.0x10“
-1.0x10'3
-1.5x10”
.2.0x10'3
.2.5x10'3

Current (A)

V

HAuCl

1.0x10‘5

5.0x10‘5

urrent (A)

"-5.0x10*5

.1.0x10'5

.1.5x10‘5

A

1.5x10'5

V
H

a .
g 1.0x10'5

U
50:00"

0.0

-5.0x10‘

0.0

2.0x10'5

 

-— HAuCI‘ .'
-— Tryptophan

7.0x10'5 -a
6.0x10"§,

 

' I

I

  
   
 

 

 

I V

S"
9
—n
O
'J!

 

I
M

(v) manna 012qu

v I 1

.4‘
S’
.—
O

3.01.:10'5
2.0x10'
1.0x10'
0.0

-1.0x10‘s

'I.jf1
M M

I
’ O
t ‘
.-
-----u.--o-’ __-----t
.--.--.._..--.----....-.---O-

 

 

 

Potential vs. SHE (V)

‘1

 

1.2 1.4

potential vs. SHE (V)

 

 

l

1.8

0.6 A 0.8 i 1.0 l 1.2 1.71.64
Potentialvs.SHE(V)

J

#20 2.2

a) Comparison between the CV of HAuCl4 (solid line) and tryptophan

These oxidation and

reduction potentials represent the upper and lower boundaries for the

oxidation potential of the amines examined in this work (Figure 2.2).

34

A. A /

 

N
H
4-am inophenol aminophenol triethylamine indole
/
N
l,4-phenylenediamine aniline 4-bnomoaniline l-methylindole
H
N
I o
M I 0.. “bx/1k
H0 NHZ \ 2 on
3-amino— l-propanol pyridine 3-indole propionic acid glycine
Na
0 0' O OH
HO ”O
-0 0 HO OH
+

Na 0 Na+ O

0 O

. . ' ' 'd
DL-tryptophan sodium curate cm": ac:
0
CI OH
NH, H/ —-< HO
/
O O O
hydroxylamine HCl acetone oxalic acid

Figure 2.2. Structures of reducing agents examined in this work

In these systems the reduction of HAuCl4 to AuO in the form of AuNPs is irreversible, as
is the conversion of the amine to its corresponding oxidized product. For this reason, a
comparison will be made between the reduction and oxidation potentials of the gold
solution and the oxidation potentials of the amine species. The first requirement for the

reduction reaction to proceed is for the oxidation potential of the amine to be greater than

35

the reduction potential of the HAuCl4 and less than the oxidation potential of Au0 in the
same solvent system (Figure 2.1a). When the oxidation potential of the amine is lower
than that observed for the HAuCl4, AuNPs are not expected because the reaction will not
proceed spontaneously. Conversely, if the oxidation potential of the amine is. greater than
that observed for the oxidation of Au0 to Au”, AuNPs would not be expected to form as
the Au0 would be oxidized before the amine. This is the case for pyridine (Figure 2.1,
panels b and c). For the non-amine reducing agents previously reported to spontaneously
form AuNPs without additional reducing agents (eg. acetone, citric acid, and sodium
citrate)8, the oxidation potential was shown to fall between the HAuCl4 reduction
potential and the Au0 oxidation potential and were therefore allowed under
thermodynamic arguments. Follow-up experiments on those amines not
thermodynamically anticipated to form AuNPs verifies that AuNPs do not form
spontaneously. Of the amines studied here, only 3-amino-1-propanol, l-methylindole,

and pyridine are thermodynamically disallowed (Table 2.1).

36

 

Concentration Oxidation

 

Amine Solvent (M) Potential (V)
3-aminophenol MeCN 0.1 1 .02 1
4-aminophenol MeCN 0.1 1 .062
Triethylamine MeCN 0. 1 1 .079
Glycine H20 0.1 1.112
4-bromoani1ine MeCN 0. 1 1 .1 22
Indole MeCN 0.1 1 .1 29
l ,4-phenylenediamine MeCN 0. 1 1 .1 60
Aniline MeCN 0.1 1.1 83
DL-tryptophan H20 .005 1 .283
3-indolepropionic acid MeCN 0.1 1.308
3-amino- l -propanol MeCN 0. 1 l .589
1 -methy1indole MeCN 0. 1 l .641
Pyridine MeCN 0.1 1 .980

Table 2.1. Oxidation potentials of amines in a 0.5 M LiClO4 electrolyte solution

There are, however, several amines which are thermodynamically predicted to reduce
HAuCl4 to AuNPs which do not, experimentally, function as reducing agents.
Specifically, these are 3-aminophenol (oxidation potential 1.012 V vs SHE), aniline
(oxidation potential 1.183 vs SHE), and 4-bromoaniline (1.112 V vs SHE). The reason
for the failure of these amines to function as reducing agents is due to competitive

polymerization effects and will be discussed in more detail later in this chapter.

37

It is clear from this data that the oxidation potential of an amine, or any potential
reducing agent, can be used to quickly screen out compounds which will not, on
thermodynamic grounds, function as agents to reduce HAuC14 to AuNPs. However,
control of the redox reaction needs to also be understood from a kinetics standpoint to be
able to gain control over the formation of AuNP colloids.

AuNP Growth Kinetics. The presence of a characteristic PR band in the visible
spectrum is indicative of the presence of AuNPs in a solution. As such, the growth rate
of the PR band can be used to examine the growth of the AuNPs in a given system. In
these studies, the amine and HAuCl4 were added as 0.005 M solutions in either MeCN or
water with the amine in 100-fold excess. As noted previously, the position and shape of
the PR band are dependent on the particle size, degree of particle aggregation, and

solvent system'7

. In these experiments, AuNP-forming systems which demonstrated a
PR showed an increase in the absorbance but did not show significant variation in either
position or shape with time. Depending on the system the PR centered at either 530 nm
or 560 nm but did not vary significantly in time. While the reduction process involves
both the reduction of Au3+ and the formation of AuNPs, the spectral data used for growth
determination is strictly probing the presence of AuNPs in the system.

The initial rates were determined from the linear portion of a maximum PR

absorbance versus time plot. The initial rates calculated ranged across several orders of

magnitude (Table 2.2).

38

 

Oxidation

 

Reducing Agent Potential (Agirtrin)
(V)

4-aminophenol 1.062 3.4x10'3

Triethyl amine (TEA) 1.079 6.6x10“
Glycine 1.112 2.511105
Indole 1.129 5.9;110‘6
1,4-pheny1enediamine 1.160 2.201103
Tryptophan 1.283 8.2x10“
Sodium citrate 1.271 6.1x10‘5

Table 2.2. Summary of kinetic data from U V-visible spectroscopy.

Within groups of amines with the same number of nitrogens, there is a general trend
toward an increasing oxidation potential corresponding to a decrease in the AuNP PR
growth rate. However, due to limited data for most multiple nitrogen systems, the single
amine data will be the only data set more closely examined. In the instance of amines
with a single nitrogen per reducing agent molecule, the rate of AuNP formation was
found to decrease exponentially with increasing oxidation potential (Table 2.2 and Figure

2.3).

39

ln(PR Growth Rate) (AU/min)

 

 

 

_l l n l n L + l n l 1 l L l 1 J n
1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13
Oxidation Potential (V)

Figure 2.3. Relationship between oxidation potential and initial grth rate when one
nitrogen is present per mole reducing agent.
This rate decrease with increasing difference between the reduction potential of HAuCl4
and the oxidation potential of the amine is indicative of the system operating in the
Marcus inverted region as understood in Marcus electron transfer theory's’z'.
Marcus electron transfer theory operates under the assumption that an electron
transfer event is occurring on a much faster time scale than any structural rearrangement
which results from the reaction. To understand this, it is useful to think of the process in
terms of the F ranck-Condon principle, wherein an increase in the difference between the
potential energy minima of the reactant and product results in an increase of the electron

transfer rate. This relationship is expected to hold true until the product energy curve

passes through the energy minima of the reactant curve. This is the point where the

40

optimum electron transfer rate is achieved, and the reaction proceeds essentially without
barrier. After this point is passed, increasing the energy difference between the two
results in a decrease in the electron transfer rate. Within the context of a solvent
environment this decrease is caused by the necessity of a sufficiently large structural
difference necessitating a change in either the solvation environment or molecular
structure. These changes serve to slow down the electron transfer kinetics under these
electronic conditions. Here, we can consider the reactant and the product energy curves
to correspond to the oxidation potential of the amine and the reduction potential of the
HAuCl4, respectively. Using the aforementioned logic, the onset of reduction of HAuCl4
by an amine reducing agent occurs at the point when the amine oxidation and HAuC14
reduction potential are equal. The rate at this point is slow but increases with increasing
difference between the oxidation and reduction potentials. At some oxidation-reduction
potential combination, the optimum electron transfer rate is achieved. Beyond this point,
the kinetics of the reduction reaction decrease. This region is known as the Marcus
inverted region. The decrease continues until the amine oxidation potential exceeds the
Au0 oxidation potential. At this point, the reduction of HAuCl4 by the amine is no longer
thermodynamically viable. From the data obtained for the series of reducing agents
examined in this study, the decrease in PR grth rate with increasing oxidation potential
indicate that the HAuC14-amine redox system, and consequently the AuNP growth, is
operating in the Marcus inverted region.

Competitive Polymerization of the Reducing Agent. As previously noted, there are
several amines which are predicted on thermodynamic grounds to form AuNPs which do

not show significant, if any, spectral evidence of a PR. Specifically, the solutions of

41

anline, 3-aminophenol, and 4—bromoaniline do not react to form AuNPs, despite
oxidation potentials in the thermodynamically allowed region. In the instance of aniline
and 3-aminophenol, this is likely due to polymerization of the reducing agentzz‘ 23 which
competes with HAuCl4 reduction. This does not mean that no Au0 is formed, but rather
that no significant nucleation to AuNPs occurs, and therefore no PR is observed. In these
systems, AuNPs may be formed during amine monomer oxidation and then oxidized
during the polymerization process, if the reaction rates of HAuCl4 reduction and
polymerization are significantly different, or Au0 may be formed but never undergo
nucleation due to similarity in rates of the the Au3+ reduction and reducing agent
polymerization.

Aniline and 3-aminophenol show similar time-resolved spectral profiles. The CV
obtained for aniline shows an oxidation peak at 1.183 V (vs SHE) with a second broad

peak after the initial oxidation (Figure 2.4).

42

2.0x10'3 1-

1.51103

j

1.0x10'3

current (A)

5.0x10‘4 F

0.0

 

 

L _n_ l 1 I n l n J n I

0.6 0.8 1.0 1.2 1.4 1.6
potential vs. SHE (V)

Figure 2.4. CV of aniline in 0.5 M LiClO4 (MeCN).

The second broad peak is indicative of a radical initiated polymerization resultant from
aniline oxidation. This oxidative polymerization has been reported in the literature22
when aniline is present in solution with an oxidizing agent. The 1H NMR of the aniline
sample after CV was conducted, shows evidence of a downfield shifi and slight peak

broadening (Figure 2.5), which is typical of oligomerization or polymerization events.

43

 

 

 

l

11T1 11 1W1111111111Ti1rr111TT1111111111111111111111111111111 11fl 1m 1171 1111111111
7.[4 751870 618 6'6 614 6.2 6T0 5.8 5.6 5I4 5l2 510 18416114 412 410 3W316 314 3‘2

 

 

Figure 2.5. . 1H NMR in CDC13 for a) aniline after completion of electrochemistry and
b) prior to electrochemical oxidation.

Additionally, the time-resolved UV-visible spectrum indicates the development of a peak
in the 410 nm range (Figure 2.6) which is consistent with literature reports for the

spectrum of polymerized aniline.24

44

absorbance (a.u.)

   

0. mitialscan
4H ‘0 C. . 0.. I. .0 C :0.

wavelength (nm)

Figure 2.6. Time resolved US-visible spectrum of 100:1 0.005 M aniline-0.005 M
HAuCl4 conversion to poly(aniline).

Similar features are observed in the CV and time-resolved UV-visible spectrum of 3-
aminophenol (Figure 2.7 a and b, respectively), indicating a similar competitive

polymerization mechanism for this reducing agent as well.

45

absorbance (a.u.)

 

 

 

 

 

700 800
. wavelength (nm)
4.0x10“- b
3 3.0m“-
‘a’ .l
g 2.07.10“-
4
1.01110“-
0'0 1 ' 1 T 1 ' l ' 1 fl 1 ' 1
0.6 0.8 1.0 1.2 1.4 1.6 1.8

Potential vs. SHE (V)

Figure 2.7. a) CV and b) UV-vis of 3-aminophenol

4-aminophenol is a case where both AuNP formation and polymerization are
observed. This is due to the kinetics of HAuCl4 being different from the polymerization
kinetics. The PR growth rate determined for AuNPs reduced and stabilized with 4-
aminophenol was the fastest for any reducing agent studied. However, the AuNPs
resulting were the least stable of any successful system. As enzymatic and
electrochemical polymerization of 4-aminophenol has literature precedent” 25’ 26 with the
resulting poly(4-aminophenol) being electroactive, the polymerization and resulting
polymer are likely both the source of the AuNP formation and the instability of the

resulting colloid. The initial reduction of HAuCl4 and subsequent nucleation and growth

46

of AuNPs occurs on a faster time scale than the radical initiated polymerization of 4-
aminophenol. However, the polymeric reducing agent exists in both oxidized and
reduced form. Thus, the reduction of the polymer in solution results in the oxidation of
the AuNPs. As Au1+ is the most stable Au ion, the AuNPs are in equilibrium in this
system with Au”, not Au“. To examine this system in greater detail Au was added to 4-
aminophenol in the form of both HAuCl4 (Any) and Auo. 4-aminophenol demonstrates
spectral growth characteristic of polymerization (Figure 2.8, panels a and b), with the
band around 500nm (Figure 2.8, panel a) indicative of AuNP formation and the band at

approximately 375 being associated with polymer growth.

47

 

 

0~00ir1iti§189ar1.........rr...
350 400 450 500 550 600 650 700 750 800

t wavelength (nm)

.09!"
came

absorbance (a.u.)
.9
&

 

.9
N

 

 

.9
o

l n l n I n l l l n l 1 l n l L I

350 400 450 500 550 600 650 700 750 800

wavelength (nm)

Figure 2.8. UV-visible spectrum of a) 100:1 0.005 M 4-aminophenol-0.005 M HAuCl4
and b) excess Au0 and 0.005 M 4-aminophenol conversion to poly(4-aminophenol).

The disappearance of the AuNP peak in the case of the HAuCl4—4-aminophenol system is
due to the oxidized and reduced forms of poly(4-aminophenol) reaching an equilibrium

with Au0 and Au” in the solution.
Conclusion

The oxidation potential of a potential amine reducing agent can be compared to

the reduction and oxidation potentials of HAuCl4 and Auo, respectively, to determine the

48

thermodynamic feasibility of the amine functioning as a reducing agent for the formation
of AuNPs from HAuCl4. This provides a first line of predictability to determine the
ability of a given compound to reduce HAuCl4 to Auo with subsequent formation of
AuNPs. Amines that have oxidation potential that lies between the reduction potential of
HAuCl4 to Au0 and the oxidation potential of Au0 to Au”, are candidate reducing agents.
This method can be used for initial screening of potential reducing agents. Amines with
oxidation potentials outside this range will not react with the HAuCl4 to form AuNPs. In
some cases, however, the amines may undergo polymerization which competes with
AuNP formation, so the electrochemical measurement can only be used to determine
thermodynamic feasibility.

The kinetics of the oxidation and reduction reactions also play a large role in
AuNP formation in amine-HAuCl4 systems. At room temperature, thermodynamically
predicted amines with the same number of nitrogens per reducing agent unit, where
AuNPs are observed in the UV-visible spectrum, show an exponential decrease in the PR
growth rate with increasing oxidation potential. This can be understood in the context of
Marcus electron transfer theory where the kinetics indicate the redox reactions are taking
place in the Marcus inverted region. In the instance of amines which undergo
polymerization under the reaction conditions the relative kinetics of the reducing agent
and HAuCl4 determine whether AuNPs will be observed. In the case of aniline and 3-
aminophenol, the polymerization of the reducing agents takes place faster than the
formation of AuNPs and therefore no AuNPs are observed spectroscopically. In the case

of 4-aminophenol, the kinetics of the reduction of HAuCl4 to AuNPs and the

49

polymerization of the amine are similar and both AuNPs and poly(4-aminophenol) are
observed in the time-resolved spectrum.

Taken together the thermodynamic information gained from electrochemical
measurements and the kinetic information from the time-resolved UV-visible
spectroscopy can be useful to make some predictions about reasonable reducing agents
for HAuCl4 and to begin to understand how control can be gained over the reduction

process.

50

References

1.

IO.

11.

Aslam M, Fu L, Su M, Vijayamohanan K, Dravid VP. Novel one-step synthesis
of amine-stabilized aqueous colloidal gold nanoparticles. Journal of Materials
Chemistry. 2004;14(12):1795-1797.

Bhargava SK, Booth JM, Agrawal S, Coloe P, Kar G. Gold Nanoparticle
Formation during Bromoaurate Reduction by Amino Acids. Langmuir.
2005;21(13):5949-5956.

Iwamoto M, Kuroda K, Zaporojtchenko V, Hayashi S, Faupel F. Production of
gold nanoparticles-polymer composite by quite simple method. European
Physical Journal D: Atomic, Molecular and Optical Physics. 2003;24(1-3):365-
367.

Leff DV, Brandt L, Heath JR. Synthesis and Characterization of Hydrophobic,
Organically Soluble Gold Nanocrystals Functionalized with Primary Amines.
Langmuir. 1996; l 2(20):4723-4730.

Selvakannan PR, Mandal S, Phadtare S, et a1. Water-dispersible tryptophan-
protected gold nanoparticles prepared by the spontaneous reduction of aqueous

chloroaurate ions by the amino acid. Journal of Colloid and Interface Science.
2004;269(1):97-102.

Subramaniam C, Tom RT, Pradeep T. On the formation of protected gold
nanoparticles from AuCl-4 by the reduction using aromatic amines. Journal of
Nanoparticle Research. 2005;7(2-3):209-217.

Selvakannan PR, Mandal S, Phadtare S, Pasricha R, Sastry M. Capping of Gold
Nanoparticles by the Amino Acid Lysine Renders Them Water-Dispersible.
Langmuir. 2003; 1 9(8):3 545-3549.

Turkevich J, Stevenson PC, Hillier J. The nucleation and growth processes in the
synthesis of colloidal gold. Discussions of the Faraday Society. 1951;No. 11:55-
75.

Turkevich J, Stevenson PC, Hillier J. The formation of colloidal gold. Journal of
Physical Chemistry. 1953;57:670-673.

Enustun BV, Turkevich J. Coagulation of colloidal gold. Journal of the American
Chemical Society. l963;85(21):3317-3328.

Fry FH, Hamilton GA, Turkevich J. Kinetics and mechanism of hydrolysis of
tetrachloroaurate(III). Inorganic Chemistry. 1966;5(11):1943-1946.

51

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Brust M, Fink J, Bethell D, Schiffrin DJ, Kiely C. Synthesis and reactions of
functionalized gold nanoparticles. Journal of the Chemical Society, Chemical
Communications. 1995(16):1655-1656.

Brust M, Walker M, Bethell D, Schiffi‘in DJ, Whyman R. Synthesis of thiol-
derivatized gold nanoparticles in a two-phase liquid-liquid system. Journal of the
Chemical Society, Chemical Communications. 1994(7):801-802.

Pong BK, Lee JY, Trout BL. First principles computational study for
understanding the interactions between ssDNA and gold nanoparticles:
Adsorption of methylamine on gold nanoparticulate surfaces. Langmuir. Dec
2005;21(25):11599-11603.

Dubois LH, Nuzzo RG. Synthesis, Structure, and Properties of Model Organic-
Surfaces. Annual Review of Physical Chemistry. 1992;43:437-463.

Kuo P-L, Chen C-C. Generation of gold thread from Au(III) and triethylamine.
Langmuir. 2006;22(18):7902-7906.

Leff DV, Ohara PC, Heath JR, Gelbart WM. Thermodynamic Control of Gold
Nanocrystal Size: Experiment and Theory. Journal of Physical Chemistry.
1995;99(18):7036-7041.

Marcus RA. The theory of oxidation-reduction reactions involving electron
transfer. 1. Journal of Chemical Physics. 1956;24:966-978.

Marcus RA. Electrostatic free energy and other properties of states having
nonequilibrium polarization. 1. Journal of Chemical Physics. 1956;24:979-989.

Marcus RA. The theory of oxidation-reduction reactions involving electron
transfer. 111. Applications to data on the rates of organic redox reactions. Journal
of Chemical Physics. 1957;26:872-877.

Marcus RA. The theory of oxidation-reduction reactions involving electron
transfer. 11. Applications to data on the rates of isotopic exchange reactions.
Journal of Chemical Physics. 1957;26:867-871.

Mallick K, Witcomb MJ, Dinsmore A, Scurrell MS. Polymerization of aniline by
auric acid: formation of gold decorated polyaniline nanoballs. Macromolecular
Rapid Communications. 2005;26(4):232-235.

Shan J, Han L, Bai F, Cao S. Enzymatic polymerization of aniline and phenol
derivatives catalyzed by horseradish peroxidase in dioxane(II). Polymers for
Advanced Technologies. 2003;14(3-5):330-336.

52

24.

25.

26.

Kogan YL, Davidova GI, Knerelman EI, et a1. Kinetic peculiarities of chemical
aniline polymerization. Synthetic Metals. 1991;41(3):887-890.

Salavagione HJ, Arias-Pardilla J, Perez JM, et a1. Study of redox mechanism of
poly(o-aminophenol) using in situ techniques: evidence of two redox processes.
Journal of Electroanalytical Chemistry. 2005;576(1): 139-145.

Taj S, Ahmed MF, Sankarapapavinasam S. Poly(para—aminophenol): a new
soluble, electroactive conducting polymer. Journal of Electroanalytical
Chemistry. 1992;338(1-2):347-352.

53

Chapter 3

Polymeric Amine Reducing Agent

Introduction

The unique properties of AuNPs make them of interest to materials chemistry.
However, in order to harness their optical and electronic properties, it is necessary to
incorporate AuNPs into a stable and usable matrix. AuNPs have previously been
incorporated into a variety of materials used for chemical sensing and separations”! and
medical applications”ls including as potential tools in the diagnosis and treatment of

- 9
some types of tumors!“ .

Use of AuNPs in the medical field requires not only that
AuNPs be stable and easy to incorporate into a matrix, it also requires that matrix be bio.-
compatible. As such it would be useful to have AuNPs which are reduced, stabilized and
incorporated into a matrix in a single step.

To date, AuNPs have been incorporated into matrices in several ways. AuNPs
have been suspended in solutions and then physically deposited onto substrates of
interest4‘ 9. There are several problems with this type of substrate fabrication. First, the
AuNPs are not actually dissolved in solution but are instead suspended. This requires the
AuNPs meet two requirements. First, the particles must be able to be stably suspended in
the necessary solvent. Secondly, the suspension in the solvent must not result in any

undesired particle aggregation. Additionally, simply physisorbing the particles to the

substrate is not ideal due to the weak interactions holding the particles to the surface. A

54

more promising way to create AuNP materials is to directly incorporate the AuNPs into a
polymer matrix. This has been previously carried out in limited ways where the AuNPs

7' 8’ ‘0. The post reduction

are imbedded into polymer films subsequent to formation
addition of AuNPs to polymer films has some problems, especially the polymer matrix
itself providing a stable but accessible environment for the AuNPs. A better method of
incorporation of AuNPs in a polymer matrix is to utilize the matrix to reduce HAuCl4 to
AuNPs in situ. This study addresses the reduction of HAuCl4 by the amine moiety in
poly(allylarnine hydrochloride) (PAH) as well as the ability to gain control over the PR
resonance of the AuNPs in the resulting composite materials.

As discussed in the previous chapter, since the year 2000 amines, and especially
amino acids, have been reported as functioning as reducing and stabilizing agents in
AuNP forming systemszo’ 21 leading to stable water soluble AuNP suspensions. This
methodology has been extended to use larger macromolecules, dendrimers, and
polymeric systems for reduction of HAuC14 and stabilization of the resulting AuNPszz'3 1.
AuNPs have also begun to be formed directly in polymer matrices30’ 32'34. Work by
Iwamoto33 showed that the amine functionality in an amine-terminated poly(ethylene
oxide) system was the moiety responsible for reduction of HAuCl4. However, this study
did not address the potential control over the PR position of the resulting matrices and
required extensive heating as the polymer itself was also the solvent matrix.

This study addresses the benefits of growth of AuNPs in a PAH matrix. These

benefits may include enhanced nanoparticle stability, control over growth rate, and

control over resulting PR. The structurally simple PAH is a useful analog for more

55

complex systems and can also be used to begin to understand, in greater detail, the

mechanism of the reduction of HAuCl4 by amines.

Materials and Methods

Chemicals. Hydrogen tetrachloroaurate trihydrate (HAuCl4-3H20) and poly(allylarnine
hydrochloride (PAH) were obtained from Sigma-Aldrich (Milwaukee, WI) and used as
received.

PAH-AuNP Synthesis. PAH-AuNP composites were made by combining an aqueous
solution of the polymer with an aqueous solution of HAuC14 under temperature
conditions dictated by either the kinetics experiments or the equilibrium plamson
resonance experiments. Room temperature kinetics studies were carried out in aqueous
solution in molar ratios ranging between 50:1 and 1000:] PAH-HAuCl4. Concentration
dependence studies were carried out on aqueous solutions in the concentration range of
0.001M to 0.1M PAH and HAuCl4. For elevated temperature studies PAH and HAuCl4
were combined in mass ratios ranging from ~50:1 PAHzHAuC14 to ~775:1 PAHzHAuCl4,
corresponding to mole ratios between ~l.3:l and ~20.4:1. Solutions were diluted with
distilled water to make them ~10"3 M in the polymer. The polymer concentration was
made the same for all solutions in order to maintain approximately constant viscosity.
These solutions were stirred in air at 95°C for 45 minutes. Following the reduction of
HAuC14, the solutions were cooled and stored at room temperature in clear glass vials.
The solutions were stable at room temperature for more than one month, with no visible

evidence of AuNP precipitation.

56

Spectroscopy of PAH-AuNP composite. UV—visible spectra of the solutions for kinetics
studies were obtained undiluted. UV-visible spectra of the aqueous polymer-AuNP
composites were obtained on samples that had been diluted to 1/3 of their original
concentration. Absorbance spectra were acquired from 700 nm to 300 nm a Cary 300
UV-visible spectrometer (Varian). 1H NMR spectra were obtained in D20 using a Varian
Inova-300 MHz NMR spectrometer.

T EM Imaging. TEM samples were prepared by drying a drop of the aqueous composite
solution on TEM grids in air. Images of the resulting samples were collected using a
J EM-100CX transmission electron microscope operated at 100kV accelerating potential.

The images were collected digitally using Sofi-Image-System MegaViewIII.

Results and Discussion
Room temperature kinetics and growth. As discussed previously, amine moieties can be
used as agents for the reduction of HAuCl4 into AuNPs. Here, PAH is used as the
reducing agent and while the kinetics of this formation process are expected to differ
from monomeric amine reducing agents, this approach to AuNP formation is important
because it allows direct incorporation of AuNPs into a water-soluble polymer matrix.
This can be taken advantage of fiom a materials chemistry standpoint because the
polymer can then be utilized within the fiamework of well-developed layer deposition
methodologies.

If the reduction of HAuC14 to Au0 occurs by the proposed mechanism (Scheme 1),
the reduction requires the presence of three nitrogen atoms to reduce a single Au3+ ion.

HAuCl4 + 3 NR3 —> Au° + 3 NR3+' + H“ + 4 (21' (Scheme 1)

57

The resulting AuNP imbedded in PAH may appear as seen in Figure 3.1, although the

exact nature of the composite is unknown.

 

Figure 3.1 AuNP imbedded in PAH matrix

When the ratio of polymer to HAuCl4 is small, while there is still an excess of amine
groups present, there is a comparatively low concentration of reducing agent as compared
to higher polymerzHAuCl4 ratio systems. This would be expected to manifest itself in
formation of many smaller AuNPs as the nucleation and grth processes occur
competitively. When the polymer:HAuCl4 ratio increases, the growth processes become
more efficient, leading to larger particles at the completion of the reaction. This is
consistent with other groups which have found the initial ratio of stabilizer-to-Au3+ can
be manipulated to effect changes in the final AuNP size33‘ 35’ 36.

To probe the behavior of this system, the concentration of the PAH was held

relatively constant to control the systems viscosity, while the HAuC14 concentration was

varied. Molar ratios of 50:1 to 1000:] PAHzHAuCl4 were used. The solutions were

58

mixed immediately before beginning UV-visible absorbance scans. At these ratios,
increasing the concentration of HAuCl4 in the reaction mixture led to an increase in the

PR growth rate. The kinetics of this system was shown to be first order in [HAuCl4]

(Figure 3.2).

#####9#
OOGANOOOOx

NO

 

 

9)!
4;
l '

l l l i l I l L J 1 l 1 l l l

-5.4 -5.2 -5.0 -4.8 -4.6 -4.4 -4.2 -4.0

 

log(plasmon resonance band growth rate growth rate)

log [HAuCl4]

Figure 3.2. First order plot of the dependence of the plasmon resonance band growth rate
on the concentration of HAuCl4 for nominally constant PAH concentration. The line is a
calculated best-fit, with the slope being 0.86 log(a.u.)flog(M HAuC14) (1.39 a.u./M
HAuCl4).

The growth kinetics of this system was also explored with respect to changing the
concentration of PAH. In this case PAH was prepared in solutions ranging from 0.001-
0.05 M. The 0.05 M solution was the upper limit of PAH solubility in water at room

temperature. The molar ratio of PAHzHAuCl4 was maintained throughout at 100:1. The

PR growth rate scaled to the third power in the range of 0.001-0.03 M PAH (Figure 3.3).

59

2.5x10'3

2.0x10‘3 r

1.5x10‘3

I

1.0x10‘3

I

5.0x104

.'

.
.
.
‘O
O"
-
---

.. .............

.O
O
I

 

llllllllJllllg

0.000 0.005 0.010 0.015 0.020 0.025 0.030
PAH Concentration (M)

plasmon resonance band growth rate (a.u./min)

Figure 3.3. Concentration effect on plasmon resonance band growth rate at 100:1
PAHzHAuCl4. The dashed line is a fit of the data to a third order grth curve.

At concentrations exceeding 0.03 M, the reduction proceeds rapidly enough that instead
of the formation of colloidal gold particles, bulk gold is observed on both the top of the
solution and the sides of the cuvette. These data indicate that within the solubility limits
the room-temperature reaction rate can be controlled by simply controlling the reagent
concentrations. It is also interesting to note that over the concentration ranges of PAH
and HAuCl4 used in these studies, there was no significant variation in the PR
wavelength. This suggests that the size of the AuNPs formed remained approximately

constant throughout. Overall these kinetic studies have findings that are consistent with

60

the proposed reduction mechanism where three nitrogens were required to reduce one
Au3+

Equilibrium PR Control. The kinetics of the PAH-HAuCl4 system are predictable at high
reducing agent-HAuCl4 ratios, the kinetics are also quite slow. At room temperature, a

molar ratio of 100:1 PAH-HAuCl4 has a PR growth rate of 4.75 absorbance units/min

(Figure 3.4).

0.08 h-

0.07 ~ .9.

O
O
O‘
I
'1

.O .
O
M

V I I
’$

O

O

W
I

absorbance (a.u)
O
i
'r
\

 

I l I I I I I j I L I I I I I

0 200 400 600 800 1000 1200 1400 1600

time after mixing (min)

Figure 3.4. Plasmon resonance band spectral growth for a 100:1 PAHzHAuC14 mixture
at room temperature. Solution concentrations were 0.005 M for both PAH and HAuC14.

The onset of the PR is not seen until approximately 3 hours after the initial mixing. In
order to increase the reaction rate in order to facilitate a more practical investigation of
the equilibrium behavior of the system, the composites used for equilibrium
measurements were synthesized at 95°C. Under these conditions the color of the solution

changed from light yellow, characteristic of aqueous HAuCl4, to pink-purple,

61

characteristic of AuNP presence, in less than 15 minutes. The reaction was allowed to
continue to a total reaction time of 45 minutes, but no firrther color change was observed
after the first 15 minutes. The reactant molar ratios were maintained in ratios where the
PR remained concentration dependent. In total, five composites were synthesized with
ratios ranging from 1.3:1 to 20.411 PAH-HAuC14. The solutions of each of the

composites were different in color, ranging from light pink to purple-blue (Figure 3.5).

 

Figure 3.5. Polymer-AuNP composites with mass ratio increasing from left to right.
A) Sample 13:]; B) Sample 4.5:1; C) Sample 9.221; D) Sample 13.1:1; E) Sample
20.421 (This image is presented in color.)

Spectroscopically, these composites were characterized by different plamson resonances.

The absorbance spectrum of the polymer itself has no absorbance in the region of interest

(Figure 3.6).

62

0.45
0.40
0.35 h
0.30 .
0.25 -
0.20 h
0.15 b
0.10 h
0.05 h
0.00 I l
300 n 400 l 500 l 600 I 700
Wavelength (um)

I I 1

Absorbance (a.u.)

 

 

 

 

Figure 3.6. UV-visible spectra of the five polymer-AuNP composites and the aqueous
PAH. Mass ratios for the spectra are (a) 1.3:] PAH:HAuCl4, (b) 4.5:], (c) 9.2:1, (d)
13.1:1, and (e) 204:]. Baseline offset for highest polymer concentrations is due to light
scattering.

When HAuCl4 is added to the polymer solution, the absorption band in the region of 530
nm is prominent (Figure 3.6, traces a-e). The presence of this band is consistent literature
reports of the characteristic absorbance for AuNPs and is hereafter assigned as the PR
band35’ 37’ 38. It can be seen that as the ratio increases, the observed PR band red-shifts
and the band broadens. This is consistent with the macroscopically observed color
changes. Because the wavelength observed for the PAH-AuNP composites is expected to

be dependent on both the size of the AuNPs and the dielectric environment of the

surrounding medium, the solution phase polymer concentration was maintained

63

thoughout and the ratio was controlled by changing the amount of HAuCl4 solution

added. The PR maximum varies linearly with the polymer-gold ratio (Figure 3.7).

 

 

E 570

E

T) 560

E3

3

El 550

.g ,

g 540

§

8

g 530

8

§

CB

3'. 52 0 5 10 15 20
mole ratio PAH:HAuCl4

Figure 3.7. Linear relationship between polymer-to-gold mass ratio and plasmon
resonance maximum. The line through the data is a best-fit line.
Because the dielectric constant of the AuNP environment is essentially constant at these
ratios, the variations in the PR resonance are due to variances in the AuNP size. This will
be discussed in more detail later in this chapter.

It is important to understand what impact, if any, the oxidation of the PAH has on
the polymer structure in order to appreciate the nature of the final composite material.
The 1H-NMR spectra obtained for the polymer before and after the reduction of HAuCl4

are, as expected, identical (Figure 3.8).

64

 

 

 

 

 

 

 

 

a M
J L

h II I I7 fiITIfi Flj I Tfl ij I—[I Tfi IITIITI I Ifi FITI I Ij II

5'0 4T5 4.0 3.5 3'0 2.5 2.0 1.5 1.0 0 5

b L W
I I I I I I I I I I I I I I I I I I Tj—I I I I I I I I I I I I T I OI I I I OIS I I

50 4'5 40. 3'5 30l 25' 20' 1'5

chemical shift (ppm)

Figure 3.8. 1H NMR spectra of a) poly(allylamine hydrochloride) in D20 and b) AuNPs
in a poly(allylarnine hydrochloride) (from a 1:1 molar ratio) matrix in D20.
The peak areas integrate as calculated and the linewidths are characteristic of a polymer.
Three peaks are observed which correspond to PAH. The broad singlets located at 1.419,
1.931, and 2.964 result from the secondary, tertiary, and amine protons, respectively.
There is no evidence of any stable oxidized nitrogen species present in the post-oxidation
NMR spectrum. This is not surprising as even at a molar ratio of 1:1, there is still an
approximately 160:1 excess of N:Au3+.

The PR maxium has been reported to be related to the AuNP size 35‘ 37’ 38 so the
linear relationship between the PR band position and molar PAH-HAuCl4 is significant
because it indicates that there is a relationship between reactant ratio and the size of the

resultant AuNPs. Particularly significant in this case is that simple variations in the

65

amount of aqueous reagents can allow for tenability of the optical properties of the
composite material. This can be important in applications which require control of the
optical response of AuNPs.23

Imaging. Transmission electron microscopy (TEM) images of the composite materials
can be used to a) verify the presence of AuNPs in the composite and b) examine the
relationship between particle size and the reaction conditions. In all composite materials,
AuNP were observed as approximately spherical particles. Non-aggregated particles

were then used to determine the size and approximate distribution of the particles (Figure

3.9).

 

Figure 3.9. TEM images of a) a sample made from 4.521 polymer-to-gold ratio solution,
with particle sizes ranging from 5-15nm in diameter (measurement bar indicates 100 nm)
and b) a sample made from 20.4:1 polymer-to-gold ratio solution, with particle sizes
ranging from 35-50nm in diameter (measurement bar indicates 50 run).

As the mole ratio of the reagents increases fi'om 1.3:1 to 13.1 :1, there was an increase in

the diameter of the non-aggregated particles from 13 nm to 22 nm (Figure 3.10).

66

 

 

 

7 r +50_1 ---....-175_1 --r--350_1 +500_1

 

 

Particle Count

 

 

 

0 10 2O 3O 40
Particle size (nm)

Figure 3.10. Particle size distributions recovered from TEM micrographs for AuNPs
synthesized and imbedded in PAH. For each distribution, the PAHzHAuC14 ratio is
indicated in the legend.

The observed trend of red-shifting particle size with increasing PAHzHAuCl4 ratio can
correlated in this range to increasing particle size. At 20.4:1, the degree of particle
aggregation was too great to determine the size of a non-aggregated particle. The mean
particle size for the 9.2:] and 13.1:1 ratios was the same. However, there were more
particles observed in the 13.1: ratio that were larger than the mean. This accounts for the
overall red-shift for this composite, making the PR of this composite longer in
wavelength than the 9.2:] composite. While it should be noted that a limited number of
measured particles were used to draw these conclusions, taken collectively, these imaging

data support the absorption data.

67

Conclusion

The kinetics of PR growth, and by association AuNP growth, can be readily and
predictably controlled through variation in the ratio or overall concentrations of
PAHzHAuCl4 in aqueous solution. The kinetics of the system are first order in HAuCl4 at
constant PAH concentration. When the PAH concentration is varied, the system kinetics
vary to the third power. This validates the postulated reduction mechanism where one
Au3+ ion is reduced by three amine groups. These data can be used to optimize the
efficiency of room temperature reactions resulting in AuNP-polymer composites.

While the kinetics of the system at room temperature can be readily predicted, the
reaction rate is also quite slow. In order to speed up the reaction it is possible to add heat
to the system and still have a measure of control over the optical behavior of the resulting
system. At small molar ratios of PAHzHAuCl4, the position of the PR band can be both
controlled and correlated to AuNP size. A linear relationship exists between the position
of the PR band and the ratio of the reactants in solution. Additionally, a general increase
is seen in the size of non-aggregated particles in the composite material with an increase
in the reactant ratio. This data can be considered together to conclude that the increase in
particle size is responsible for the red-shift in the PR band maximum.

Formation of AuNPs using this method provides a simple means of formation
polymer-AuNP composite materials with control over the optical properties of the
composite. This could be significant when designing polymer films for sensing
applications and biomedical applications. It could also be important for understanding

potential in situ AuNP formation for use as therapeutic agents.

68

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2951.

72

Chapter 4
Investigations of Alkanethiol Self-Assembled Monolayer Organization and Viscoelastic

Properties using Impedance Spectroscopy

Introduction

Alkanethiol monolayers on gold have been studied extensively because of their
facile growth properties and the resulting well organized molecular assemblies.
Following initial work in this area in the 1980’s,"8 the field has grown enormously and
thiol/gold monolayers find use in a variety of technological applications today. Much is
understood about the fundamental structural and growth pr0perties of alkanethiol
monolayers, but a persistent issue in the field remains the separation of the various events
that occur during monolayer growth. The initial step in monolayer growth is the gold-
sulfur interaction?” in which the thiol hydrogen is lost, being sequestered as either H2 or
H202 on the Au surface, depending on the amount of water present.'3"6 The gold-sulfur
bond is on the order of 5-6 kcal/mol, a value that represents a balance between a modest
gold-sulfur bond enthalpy (ca. 20 kcal/mol) and a large entropic penalty (ca. 50 cal/mol-
K) that results from the formation of a two-dimensional quasi-crystalline assembly fi'om
solution.'0’ '3’ '7 The modest net driving force for monolayer formation implies that the
monolayer will likely be labile, and AFM data on alkanethiol islands on Au has shown

this to be the case.18 Subsequent to the initial mass deposition of the monolayer, which

73

occurs on the timescale of seconds to minutesz, there is a more subtle structural
rearrangement of the monolayer, sometimes termed “annealing”, which serves to
organize the aliphatic chains of the monolayer, producing a well organized, hydrophobic
surface.2’ '9 The details of this annealing process remain to be understood in detail,
despite extensive examination. Infrared spectroscopic data have shown that the CH
stretching resonances of the aliphatic chains go from being liquid-like initially to quasi-
crystalline over a timescale of hours to days, depending on the monolayer chain length,
temperature and environment.” 20 This information addresses average structure, but
there remain open issues regarding interchain interactions and the evolution of
organization within the film. We are interested in examining this issue from the
standpoint of how the viscoelastic properties of the monolayers change following initial
deposition. Viscosity, in the traditional sense, is a measure of the strength of
intermolecular interactions, a phenomenon that will depend sensitively on monolayer
chain organization. In order to evaluate the monolayer viscoelastic properties, quartz
crystal microbalances (QCMs) with Au electrodes have been used as the growth
substrates for alkanethiol monolayers. By measuring the complex impedance response of
the QCM in solution as a firnction of time after thiol introduction, and modeling changes
in this response in the context of an equivalent circuit model, information can be
extracted on what factors change most during monolayer structural evolution. This study
demonstrates that the structural evolution of the monolayer depends on both the thiol
aliphatic chain length and on the annealing time. For alkanethiols in the range of C6-C16,
initial deposition and organization occurs on roughly the same time scale. However, the

C6 monolayer, which is expected to be relatively less organized at equilibrium, has

74

different viscoelastic properties than C9-C16 SAMs, which are essentially
indistinguishable from one another in terms of their viscoelastic properties. Solubility of
the alkanethiols will also be shown to play a large role in determining the functional form
of impedance spectral data, as C13 monolayers demonstrated bulk adsorption under the
deposition conditions used. This bulk deposition could be observed visually, and it was
also evident in the evolution of the C13 SAM impedance spectra. We consider first the
QCM response and the information content of our measurements.

Background. Quartz crystal microbalance (QCM) gravimetry is a highly sensitive
technique capable of quantitating nanogram mass changes. The measurements are based
on the piezoelectric behavior of crystalline quartz under the influence of an AC electric
field. In the case of the QCM, the oscillations induced by the electric field are parallel to
the surface of the crystal and occur at a frequency determined by the properties of the
quartz, including thickness and shear modulus. In the simplest application, changes in
the thickness of the quartz crystal determine changes in its resonance frequency. In the
limit that the mass added to the QCM surface is rigid, as in many vapor phase deposition
applications, the QCM resonance frequency changes in direct proportion to the mass

added to the device, and this frequency change is described by the Sauerbrey equation,21

_ -2Amnf02

Af (1)
A(#.p.)%

where n is the harmonic of fo, the resonance frequency of the unperturbed QCM crystal, A
is the area of the QCM crystal, [1,, is the quartz shear modulus, pq is the quartz density, Af
is the fi'equency change upon deposition, and Am is the mass change upon deposition.

This relationship has been used extensively to interpret QCM resonance frequency shift

75

data, and in applications such as measuring the formation kinetics of self-assembled
monolayers.22

The most common measurement method for QCMs is to monitor the frequency
change of the device as a function of some deposition parameter. This measurement
scheme records only the center frequency of the crystal resonance, but does not provide
direct information on changes in the shape of the resonance associated with the addition
of non-rigid materials to the face(s) of the device. Measurement of the complex device
impedance as a function of frequency provides information on changes in resonance line
shape as a function of experimental variables, and this information can be related to the
viscoelastic properties of the adsorbed film(s). To measure the complex impedance
response of the QCM as a function of frequency, we use an impedance analyzer which
senses phase shift and standing wave ratio of the reflected signal that is applied to the
crystal. The quantities |Y|, the magnitude of the admittance, and 0, the phase angle, are
measured experimentally and are related to the real and imaginary parts of the device

3'28 is used to model the frequency-dependence Of these

response. An equivalent circuit2
data, and relate the quantities in the model to the properties of the QCM.

The equivalent circuit approach to the analysis of QCM data has proven to be
remarkably useful. The Butterworth-Van Dyke (BVD) circuit model (Figure 4.1a) is an
effective model for QCM impedance data on mass loaded QCM crystals.27’ 29' 30 This
model is useful for understanding a gas phase moiety adsorbing onto a QCM. However,
in more complex cases the BVD circuit can be modified (Figure 4.1b) to account for both

the effect of liquid viscous drag and mass loading on the QCM surface. The modification

lies not in the functional form, but in making the correspondence between the model

76

circuit components and the properties of the QCM adsorbate. Using the model circuit

shown in Figure 4.1b, the frequency-dependent complex response of the QCM is given

 

byEq. 2,
R—j[arL———-]
YzjaXCO-l-Cp)+ 1 '2
R2+[arL———]
(”Ci
|Y|= (1’3“?) (2)

Where YR is the real part of the admittance and Y1 is the imaginary part.

3 He

 

1— 0‘0 0‘0
9
1

a:

U

i."
|_0000—

 

 

\I
.0
/

\I
.0

 

 

 

 

 

 

Figure 4.1. (a) BVD equivalent circuit where the left arm of the circuit is the static
capacitance (Co) and the right arm is the motional arm where Lm is the motional
inductance, Cm is the motional capacitance, and Rm is the motional resistance. (b)
Equivalent circuit describing the unperturbed (L1, C1, R1), liquid loaded (R2, L2) and
mass loaded (L3) portions of the QCM crystal

In this model, the inductance L is comprised of three inductors in series, L = L1 + L2 +

L3, and R likewise represents two resistors in series, R = R1 + R2, as shown in Figure

77

4.1b. The key to this model is the correspondence between the equivalent circuit
components and the physical quantities of interest. Martin has made this connection,
describing the relationship between the quantities C, L and R, the properties of the QCM,
and the characteristics of the liquid medium and the adsorbed layer.23 For the data
reported here, two circuit components provide the most useful information on the
properties of the SAMs; L2 and R2, the inductance and resistance associated with the

mass loading of the crystal. Eqs. 3 and 4 show the relationship between each of these

23

 

terms,
(00 21077 %

L2=11sz[ai,,p,] (3)
at 2601077 y,

R2 zl‘Wl 566/). i (4)

Where (no is the resonance frequency, L 1 is the inductance associated with the
unperturbed QCM crystal, pq is the density of the quartz comprising the QCM, (0 is the
resonance frequency of the QCM after SAM adsorption, and C66 is the piezoelectrically
stiffened quartz constant.23 Most of the quantities that describe the equivalent circuit
components are constant properties of the materials used, and their values are known.
The quantity 000 is measured and it is the terms p and n, the adlayer density and viscosity,
respectively, which reflect the properties of the adsorbed SAM layer. It is these latter
two quantities that are of primary interest in this work and, in particular, how these
adlayer properties depend on the identity of the thiol aliphatic chain length and on the

time allowed for SAM annealing after initial deposition.

78

Materials and Methods

Materials. l-Hexanethiol (Cs-SH), 1-nonanethiol (Cg-SH), 1-dodecanethiol (Cu-SH),
and l-octadecanethiol (Clg-SH) were obtained from Sigma-Aldrich (Milwaukee, WI) and
used as received. QCM crystals were obtained from McCoy Crystals/Coming Frequency
Control (Mount Holly Springs, PA). The crystals were cleaned with Piranha solution, a
3:1 solution of sulfuric acid-hydrogen peroxide (caution! Strong oxidizer) for 30 seconds,
rinsed with copious amounts of distilled water, and dried thoroughly with nitrogen prior
to use.

Monolayer Deposition. SAMs were grown in situ on clean QCM crystals at 14°C,
without stirring, from 1 mM ethanolic thiol solutions. The temperature was controlled
using a circulating water bath and a jacketed 100 mL beaker. The beaker and oscillator
were covered to minimize evaporative losses.

Impedance spectroscopy. Impedance spectra were collected using a QCM driven by an
oscillator circuit (Maxtek, Inc, Cypress, CA). The phase angle and reflected signal from
the oscillator-QCM circuit was measured as a function of frequency using a HP 4192A
LF Impedance Analyzer (Hewlett Packard, Palo Alto, CA). The impedance analyzer was
interfaced to a personal computer and controlled using LabViewTM v. 7.0 code (National
Instruments, Austin, TX). Impedance spectra were collected at 30 minute intervals. The
frequency was scanned from 5.92 MHz to 5.95 MHz with 50 Hz resolution. Time zero
for each experiment was established by immersion of the QCM crystal in the thiol

solution.

79

Results and Discussion

The central goal of this work is to understand how the viscoelastic properties of
the alkanethiol monolayer adsorbed on the QCM electrodes evolves over time. It is
interesting to understnad how the viscoelastic evolution process of the SAM depends on
the length of the thiol aliphatic chain and on the time the SAM is immersed in solution.
The SAM properties measured in this work are related to alkane chain annealing; the
initial mass deposition reaction, dominated by interactions between the thiol S and the Au
electrode, is essentially complete within less than a minute after immersion of the QCM
in solution. In order to gain information on the SAM viscoelastic properties, the
impedance data is fit to the BVD model circuit and monitor how specific model circuit
elements change with time. It is the changes in these model parameters that relate to the
physical characteristics of the system through Eqs. 3 and 4. To extract the information of
interest, we must first determine the appropriate values for the other circuit components
in this model, and this matter is considered next.
Modeling Impedance Response. We note at the outset that the equivalent circuit model is
not capable of distinguishing the individual circuit elements, and treats the inductive and
resistive components as single elements. For these experiments, the interpretation hinges
on understanding which of the experimental quantities is varying and thus which model
circuit elements are changing and which are being held comparatively constant. The
spectrum obtained for each impedance measurement was fit to a corresponding model
system. A blank spectrum (Figure 4.2) and a spectrum for a QCM with an alkanethiol

adlayer (Figure 4.3) were fit for each data point.

80

 

3000

 

 

 

 

 

- 10000
2500 -
4 8000
2000 - .
[Ti
_ - 6000 .3;
a: 1500 - a.
s 1 El
--1 =3
E 1000 r _ 4000 g
r ‘ 3
500 __ '1 2000
0 0
I n I n I I

 

 

5.930x10" 5.933x10" 5.935x106 5.938x106 5.940x106
Frequency(Hz)

Figure 4.2. Comparison of experimental data (-) and model fit (—) for a blank QCM
crystal

81

 

 

 

 

 

800
700
- 1'11
600 >4
, R
500 .3,
:3
‘ .53.
400 Q
300
0 P
- 200
L J_ I 1 I
5.932x10° 5.934x106 5.936x10°
Frequency (Hz)

Figure 4.3. Comparison of experimental data (-) and model fit (—) for a C16-SH
monolayer on QCM crystal

For a blank QCM, where there is no adlayer but only the solvent overlayer, the quantity
L3 (Figure 4.1b) is identically zero. The quantity Cp, the parasitic capacitance determined
by the physical connection between the QCM and the oscillator circuit, and the quantity
Co, the intrinsic capacitance of the QCM, cannot be separated experimentally, and these
quantities are treated as a single capacitance. The intrinsic device quantities C), L1, and
R1 were determined by fitting the spectral lineshape of a bare QCM (Figure 4.2), and
these values will remain constant throughout a measurement. There is no discernible
information related to the viscoelastic properties of the adlayer in these terms. With the
addition of a monolayer to the QCM, the quantities L2, R2 and L3 can all change because

the adlayer will add mass and alter the coupling between the QCM device and the

82

solution. The quantity L3 will change by a fixed amount within the first minute after

1,22 an amount which can be estimated based on the QCM surface

introduction of the thio
area, but was seen experimentally to remain essentially unchanged throughout all
experiments. It is the terms L2 and R2 that contain viscoelastic information on the SAM,
and these quantities will vary most significantly with time. To fit the SAM spectra, C1,
L), R], and L3 are held constant and R2 and L2 are used as the experimental variables
(Figure 4.3).

Interestingly, we find the value of Cp plays a primary role in determining the
dispersive nature of the observed lineshape. This parameter changes upon QCM
immersion in solution, which is expected, and differs from Cp for the blank QCM, likely
because of the presence of the SAM between the QCM electrode and the oscillator
connections. The change in going from the QCM in air to the QCM in solution was
similar for all samples, consistent with Cp being dominated by the contact between the
QCM electrode and the oscillator circuitry. For all of the SAMs studied here, the spectra
exhibited a time-dependent evolution that could. be accounted for by changes in L2 and
R2. The dominant effect of L2 is to change the resonance center frequency in a manner
consistent with the Sauerbrey equation, i. e. as the value of L2 increases, the resonance
center fiequency decreases. This finding is expected based on Martin’s treatment of the
BVD model, where L2 is related to the liquid mass loading of the QCM. Because the
adlayer mass loading inductance term (L3) is nominally constant over the 12 hour
duration of the experiments, the observed changes can be attributed to inductive effects

must be associated with changes in the evolution of the SAM viscoelastic properties. The

effect of the quantity R2, which depends on the viscoelastic properties of the adlayer in a

83

manner similar to L2 (see Eqs. 3 and 4), is primarily on the resonance linewidth.
Increasing R2 results in a broadening of the resonance and a slight increase in the center
frequency. This latter effect is expected based on the contribution of L1 to R2. The goal
of the following discussion is to extract chemically useful information on SAMs from
these QCM impedance data.

SAM viscoelastic properties. As noted above, the inductive impedance term L2 in the
BVD model has a similar functional form to R2, and in terms of applying this model, we
find that the term L2 dominates the impedance resonance frequency while R2 is related
most closely to the linewidth of the resonance. We focus on the inductive term, but it

should be noted that the temporal behavior of L2 and R2 are functionally the same (Figure

 

 

 

 

4.4).
- 0.16
4 - 1:1
2.50x10 . .
r- " 0.14
.4
A 2.00XIO '- _ 0.12 N”
' Q
% 150x104 - ' 0'10 °
7; ’ « é
a; r - 0.08 pa
M 4 j 9’-
? 1.00x10 F - 006 3;
i. r ' O
8 00.. E
N 5.00x10" - D ‘ ' ”’
r—I D a ' E
- 0.02 V
I o R
0.00 . 0 i
- 0.00
I I I L j 4 I I I I I I I
6 8 10 12 14 16 18
Carbon Chain Length

Figure 4.4. Comparison of growth behavior of L2 (I) and R2 (0).

84

Figures 4.5a and 4.5b show a series of impedance spectra taken over a period of 12 hours,

to provide a sense of how these spectra evolve in time.

 

 

“N

 

 

SEN
s: E “PIE-4%

 

 

 

6 6 6 6 200 6 ' 6 fl 6 ' 6
5.92x10 5.93x10 5.94x10 5.95x10 5.92x10 5.93x10 5.94x10 5.95x10
Frequency (Hz) Frequency (Hz)

Figure 4.5. Time-dependent impedance responses of a) Cg—SH and b) Cig—SH SAMs
on Au-coated QCM. Spectra were acquired starting at time zero, at 1 hour intervals for a
period of 12 hours.

Because the adlayer mass loading (L3) is constant, it must be changes in L2 and R2,
specifically the terms p and n in Eqs. 3 and 4, account for the spectral evolution (Figure
4.5). Examining the term L2, we see that this quantity varies in a nominally linear

manner over the 12 hour observation time (Figure 4.6).

85

1.00132
1.00130
1.00128
1.00126
A 1.00124
E 1.00122
.4" -
1.00120

 

 

 

1.00118

1.00116

1.00114 I 4 I A I r l l I I I I I
0 2 4 6 8 10 12

immersion time (hr)

Figure 4.6. Q; monolayer growth from ethanolic solution

There are several interesting features contained in the L2 data. In Figure 4.6, it can be
seen that the structural evolution of the SAM proceeds at a nominally constant rate. The
slope of the temporal growth in inductance is the same, to within our experimental

uncertainty, for all of the SAMs examined (Figure 4.7).

86

5.0x10"

 

 

 

 

4.0x10‘S -
g r
-5 L
V 3.0x10 .
2
g ti
g 2.0x10'5 -
'4 1.0x10s -
L
00 I n I I l . L L J n I
6 3 10 12 14 16

Carbon Chain Length

Figure 4.7. Initial growth rate of L2 for alkanethiol SAMs of varying carbon chain
length.

The growth rate is modest, on the order of a 2% change in L2 per hour, and the nominal
value of L2 is 1 mH. The value of L2 after 12 hours of annealing is nominally

independent of SAM aliphatic chain length (Figure 4.8).

87

1.0035

 

 

 

 

 

1.0030 -
fl 1
1.0025 -
i t '
v i
,2" 1.0020 - —
. ~~ 1
1.0015 -
in
1.0010 J I I n I 4 I L I L I

 

6 8 10 12 14 16 18
CarbonChainLength

Figure 4.8. Inductance of alkanethiol SAMs of varying carbon chain length

These interesting findings provide some insight into the evolution of SAM viscoelastic
properties. The data reported here indicate that the aliphatic chain length, at least in the
range of C9 — C16, does not play a dominant role in determining the viscoelastic properties
of alkanethiol SAMs. There are two possible contributions to the structural evolution of
the SAMs; rearrangement or adsorption and desorption of the thiol head groups on the Au
surface to form a well organized monolayer, and the evolution of the aliphatic chains. It
is clear that these two processes cannot be separated cleanly because the formation of
predominantly all-trans aliphatic chains is a cooperative effect, requiring the presence of
neighboring chains. Our data appear to implicate head group mediated organization as

the dominant process, consistent with what is known about SAMs. While the initial mass

88

deposition is fast for SAMs, the organization of the headgroups into a well-ordered array
may take substantially longer, depending on the experimental conditions. For the
interaction of aliphatic chains with one another, the characteristic energy of interaction is
ca. 300 cal/mol-CH2. For a C16 system, the energetic driving force for chain annealing
will be ca. 4.8 kcal/mol. In contrast, the enthalpy of the Au-S bond formation has been
measured to be on the order of 20 kcal/mol.13 The relative energies of these processes
indicate that the limiting factor in achieving an organized monolayer is the equilibrium
adsorption and desorption of the thiol groups on the Au surface, consistent with our
findings.

From the L2 values, it is possible to extract the quantity p11 (Eq. 3), and the

dependence of pr] on thiol aliphatic chain length is presented in Figure 4.9.

89

5.40x10‘

 

 

 

5.38x10“—
3?
a
O
E 5.35x10‘-
1';
”a
O
E I
r: 4 O
o. 5.33x10 -
5.30x104‘t““'t 1'
6 8 10 12 14 16
alkanethiol aliphatic chain length

Figure 4.9. Viscoelastic response (p11) for alkanethiol SAMs of varying carbon chain
length

These data indicate that the shortest aliphatic chain examined, C6-SH, exhibits a p11 value
slightly higher than that of the other thiols, and for chain lengths C9 — C16, there is no
discernible chain length dependence for p11. This finding is interesting in that the higher
the pn value, the more “rigid” the adlayer appears to the QCM. For sufficiently short
SAM aliphatic chains, there is limited opportunity for viscous interactions with the
solvent. It is known that for the longer aliphatic chains examined, there is a break in the
extent of SAM organization at C9. Once the CH2 attractive interactions become
sufficiently strong, the SAM is capable of creating a predominantly all-trans aliphatic
chain structure. For such an organized structure, it would appear that the chain length of

the SAM would matter little, because in all cases the structure of the SAM in contact with

90

the solvent is the same. Our experimental data lend credence to this explanation, as the
pn values are approximately constant for chain lengths between C; and C16. In the above
discussion, the term p11 has been considered, without attempting to separate the density
and viscosity contributions. The density of thiol head groups will be determined by the
Au substrate, and should be the same for all of the SAMs. Changes in aliphatic chain
length will not affect the density because the thickness of the film scales with number of
carbons, so the primary experimental variable in the term p11 is the adlayer viscosity, 1].
Therefore, evaluation of the L2 variable, as determined thorough model fitting of
experimental data, can be used to investigate the relative viscosities of the SAMs as a
function of aliphatic chain length.

C18 SAMs. In the discussion to this point, the focus has been on the results for
alkanethiols C6-SH through C16-SH. The results of that work indicate that the
viscoelastic properties of the SAMs in this aliphatic chain length range are remarkably
similar. In contrast, the data recorded for (313-311 is fundamentally different. These data
are presented below and the uniqueness of Clg-SH may be attributed to the fact that the
measurements are performed at 14°C, and the solubility of Clg-SH in ethanol at this
temperature is limited. Figure 4.5b shows the time-dependent impedance response of
QCMs exposed to a Cls-SH solution. These data stand in sharp contrast to the spectra
shown in Figure 4.5a for C9, (which are the same functional form for C16 and shorter
aliphatic chains) and demonstrate that the deposition and structural evolution mechanism
is fundamentally different for Clg-SH. Coincident with this spectral evolution, we

observe bulk deposition of material on the QCM surface (Figure 4.10).

91

Figure 4.10. QCM crystal with a) C16-SH monolayer and b) Clg-SH monolayer. Both
SAMs were deposited from ethanolic solution.

The formation of physical deposits on the QCM electrodes perturbs the response of the
device significantly, leading to values obtained for both growth rate and pi) which are
significantly different from the C6-C16 examples. The C13 grth rate is an order of
magnitude faster than the corresponding grth rates for the other SAMs (growth rate =
(3.0:t2.0) x 104 mH/hr for C13 versus ~10'5 mH/hr for C6-C16). The observed pr] value
for Clg-SH is 25% lower than those obtained for the C9-C16 SAMs. Further examination
of the Clg-SH monolayer deposition at elevated temperatures would be required to
elucidate more information on the deposition kinetics and viscoelastic properties of

SAMs with C13 and longer aliphatic chains.

92

Conclusions

The time evolution of the impedance spectra of QCM devices that have been
modified by the presence of an alkanethiol SAM adlayer have been measured. This study
addresses the issue of how the viscoelastic properties of the SAM vary with aliphatic
chain length and time after initial formation. Impedance spectra can be interpreted in the
context of an equivalent circuit model used by Martin to understand QCM response in

liquids.23

In the equivalent circuit, two components dominate the physical and chemical
contributions of the adlayer to the QCM response. These quantities, L2 and R2 in Figure
4.1b, have similar time-courses for all of the alkanethiols examined in this study, and
from these quantities information can be extracted about the quantity pr], which is related
to the viscoelastic properties of the SAM adlayer. SAMs having between 9 and 16
carbons in their aliphatic chains are characterized by nominally the same value of p11
indicating similar solvent-monolayer interfacial environments. The C6SH monolayer has
a slightly higher prg value, a finding which is ascribed to the absence of aliphatic chain
organization and thus a stronger coupling to the solvent. In other words, the shorter
aliphatic chain gives rise to a different adlayer-solvent coupling than is seen for the
longer alkanethiols. This difference is coupling is manifested in this model by a higher
viscosity term. We believe the viscosity term to dominate changes in p11 because both the
monolayer density and bulk thiol density are constant for the systems reported here. It
was found that deposition of Clg-SH differed from that of the shorter alkanethiols, likely

a consequence of the limited solubility of this alkanethiol. The anomalous behavior of

C13 SAMs can be attributed to the bulk deposition of the thiol on the QCM surface.

93

This study shows that simple measurements of the impedance spectrum of a QCM
crystal during alkanethiol monolayer deposition can be used in conjunction with
modeling of the response to provide information on the evolution of monolayer
organization. Our findings indicate that the viscoelastic properties of alkanethiol SAMs
change little with aliphatic chain length for systems where the aliphatic chains are long

enough to exhibit organization due to attractive interchain interactions.

94

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Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J .; Whitesides, G. M.; Nuzzo, R.
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Bain, C. D.; Evall, J .; Whitesides, G. M., Formation of Monolayers by the
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Bain, C. D.; Whitesides, G. M., Formation of Monolayers by the Coadsorption of
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Bain, C. D.; Biebuyck, H. A.; Whitesides, G. M., Comparison of Self-Assernbled
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Nuzzo, R. G.; Fusco, F. A.; Allara, D. L., Spontaneously Organized Molecular
Assemblies .3. Preparation and Properties of Solution Adsorbed Monolayers of
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Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H., Fundamental-Studies of the
Chemisorption of Organosulfur Compounds on Au(111) - Implications for

Molecular Self-Assembly on Gold Surfaces. Journal of the American Chemical
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Bain, C. D.; Whitesides, G. M., Modeling Organic-Surfaces with Self-Assembled
Monolayers. Angewandte Chemie-Intemational Edition in English 1989, 28, (4),
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Dubois, L. H.; Nuzzo, R. G., Synthesis, Structure, and Properties of Model
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Designing Ordered Molecular Arrays in 2 and 3 Dimensions. Acs Symposium
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Lee, T. R.; Laibinis, P. E.; Folkers, J. P.; Whitesides, G. M., Heterogeneous
Catalysis on Platinum and Self-Assembled Monolayers on Metal and Metal-
Oxide Surfaces. Pure and Applied Chemistry 1991, 63, (6), 821-828.

Schessler, H. M.; Karpovich, D. S.; Blanchard, G. J., Quantitating the balance
between enthalpic and entropic forces in alkanethiol/gold monolayer self-
assembly. Journal of the American Chemical Society 1996, 118, 9645-9651.

Brust, M.; Walker, M.; Bethell, D.; Schiffiin, D. J.; Whyman, R., Synthesis of
Thiol-Derivatized Gold Nanoparticles in a 2-Phase Liquid-Liquid System.
Journal of the Chemical Society-Chemical Communications 1994, (7), 801-802.

Chailapakul, 0.; Sun, L.; Xu, C. J .; Crooks, R. M., Interactions between
Organized, Surface-Confined Monolayers and Vapor-Phase Probe Molecules .7.
Comparison of Self-Assembling N-Alkanethiol Monolayers Deposited on Gold
from Liquid and Vapor-Phases. Journal of the American Chemical Society 1993,
115, (26), 12459-12467.

Thomas, R. C.; Sun, L.; Crooks, R. M.; Ricco, A. J ., Real-Time Measurements of
the Gas-Phase Adsorption of Normal-Alkylthiol Monolayers and Multilayers on
Gold. Langmuir 1991, 7, (4), 620-622.

Schlenoff, J. B.; Li, M.; Ly, H., Stability and self-exchange in alkanethiol
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McCarley, R. L.; Dunaway, D. J .; Willicut, R. J ., Mobility of the alkanethiol-gold
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Ulman, A., Formation and structure of self-assernbled monolayers. Chemical
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Poirier, G. E.; Tarlov, M. J .; Rushmeier, H. E., 2-Dimensional Liquid-Phase and
the Px-Root-3-Phase of Alkanethiol Self-Assembled Monolayers on Au(111).
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Martin, S. J.; Granstaff, V. E.; Frye, G. 0, Characterization of a quartz crystal
microbalance with simultaneous mass and liquid loading. Analytical Chemistry
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Martin, S. J.; Bandey, H. L.; Cemosek, R. W.; Hillman, A. R.; Brown, M. J.,
Equivalent-Circuit Model for the Thickness-Shear Mode Resonator with a
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Responses of Thickness-Shear Mode Resonators under Various Loading
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97

Chapter 5
Optical Organophosphate Sensor Based upon Gold Nanoparticle Functionalized Fumed

Silica Gel

Introduction

Over the past several years, organophosphates and phosphonates (OPPs) have
become increasingly important as analytes for several reasons. Many nerve agents and
pesticides are organophosphates or organophosphonates, so detecting small quantities
and/or low concentrations of these compounds critically important for human health and
safety. In certain cases, sequestration is desirable owing to the toxicity of the analyte.
Ratification of the Chemical Warfare Convention (CWC) has underscored the need to
develop rapid, sensitive, and selective detectors for chemical warfare agents and other
organophosphate/phosphonate compounds in order to verify compliance with the
provisions of the treaty.1 Contamination of groundwater and agricultural products
entering the human food chain by organophosphate/phosphonate pesticides has also led
to the need for sensitive detection and sequestration methods.2'9 We report here on the
development of a high-surface area material based upon silica microparticles. The use of
covalently attached gold nanoparticles (AuNPs) as a surface area enhancing scaffold and
a foundation for surface modification chemistry allows for the creation of a high affinity

material that is selective primarily for OPP compounds'o'l3 Once the analyte(s) have

98

been sorbed onto this novel material, the coated silica particles can be suspended in a
solvent and the AuNP plasmon resonance can be monitored with relatively high signal to
noise ratio, depending on the choice of solvent. While there are technical challenges
associated with this approach, we have chosen to use fumed silica in conjunction with
AuNPs because the fumed silica can be suspended in aqueous and other solutions without
aggregation for extended periods of time, unlike AuNPs, because silica microparticles are
more amenable to physical separation methodologies, such as filtering, than AuNPs, and
because this structural motif provides a relatively high surface-to-volume ratio to enhance
the adsorption and sequestration of OPP species.

Current methods for detection of OPP compounds include chromatographic

14-19 15, 17 3, 6, 7, 20, 2]

techniques, mass spectrometric methods, electrochemical detection,

. .9 0
brosensors,3’ 7 ’ 2 ’ 2'

and fluorescent beads.22 While these techniques have many
advantages, including sensitivity and selectivity, they are often time consuming and labor
intensive. The ideal OPP sensor would possess the sensitivity and selectivity of the
already-established methods and would reduce the dependence on expensive
instrumentation. Robustness, simplicity, the ability to sequester the analyte of interest,
and possible incorporation into existing air filtration schemes would be beneficial, and
there are several recent examples of OPP sensors based on nanoparticles which begin to
satisfy these requirements.3' 6’ 8' 22 Both AuNPs and zirconium oxide (Zr02) nanoparticles
have shown promise in the detection of OPPs. Pavlov, Xiao and Willner have
demonstrated the utility of AuNP plasmon resonance measurements for the optical

determination of OPPs23 and Liu and coworkers6 have demonstrated an OPP sensor based

on the affinity of the analyte for Zr02 nanoparticles. In that work, the binding of the

99

OPPs to the nanoparticles was determined electrochemically. In this work, we combine
the chemical advantages of Liu’s sensor with the simple detection scheme of Pavlov’s
sensor to create a high-surface area optical sensor and sequestration medium for OPPs.
The sensor / dosimeter methodology we report here utilizes several well established
chemical reactions to achieve relatively low level detection and OPP sequestration
simultaneously.

The material and measurement methodology is based on a series of well
established silica and gold surface modification reactions. First, the silica gel is
functionalized with a mercaptosilanez‘l'27 followed by attachment of AuNPs by stirring
the functionalized silica gel in an aqueous citrate-stabilized AuNP colloidal solution.l " 28'
30 Following the attachment of AuNPs, the citrate stabilizer is displaced by exposing the
silica-bound AuNPs to an ethanolic solution of an c0-hydroxythiol.31 The hydroxyl
termini on the AuNPs are then reacted with P0C13, H20 and Zr4+ to form a zirconium-
phosphate (ZP) terminated surface. This chemistry was pioneered by the Mallouk,

Thompson and Katz groups,” 25. 32. 33

and the Blanchard group has made extensive use of
ZP chemistry to grow robust interfacial films efficiently.“37 Because the ZP chemistry
operates by using Zr4+ to link phosphate and/or phosphonate groups, the terminal Zr“
ions on our modified AuNPs have a high affinity for OPPs.6’ 35 The ZP chemistry could
be used directly on the silica gel to produce a surface with high affinity for OPPs, and it
is possible that the chemistry applied to the AuNPs could also react with any residual
surface silanol groups on the silica gel. Here the AuNPs are used as optical reporting

agents, where the plasmon resonance band of the AuNP is sensitive to the condition of

the ZP linkage bound to its surface.

100

Both the material and the associated plasmon resonance detection methodology
reported on here are useful in several regards. First, the covalent nature of the attachment
chemistry makes it robust and able to be stored under ambient conditions. Second, the
incorporation of AuNPs provides access to the plasmon resonance, a band that is
remarkably sensitive to changes in the local dielectric environment of the AuNPs. The
plasmon resonance band is observed to blue-shift when OPPs bind to the ZP-modified
particles, and this data may be interpreted in the context of two spectrally overlapped
bands, rendering an apparent blue-shift. The magnitude of the observed spectral shift is
independent of OPP concentration after its initial appearance at a threshold OPP
concentration in the microgram/liter range, and we understand this behavior through a

simple complexation model.

Materials and Methods

Materials. Silica gel (Grade 633, 200-425 mesh, average surface area 480 mz/g, with an
average particle diameter of 63.6 urn, as determined by scanning electron microscopy)
was purchased from Spectrum and used as received. Sodium citrate, hydrogen
tetrachloroaurate (HAuCl4-3H2O, 99.9+%), methylphosphonic acid (MPA) (Figure 5.1a),
diethylchlorophosphate (DECP) (Figure 5.1b), and 3-mercaptopropylt1imethoxysilane

(MPTMS) were purchased from Sigma-Aldrich and used as received.

Figure 5.1. OPP analytes a) methylphosphonic acid and b) diethylchlorophosphate

101

6-Mercapto-l-hexanol and phosphorous oxychloride (POC13) were purchased from Fluka
and used as received. Collidine was purchased from Spectrum and used as received.
Zirconium(IV) oxychloride octahydrate (ZrOCl2'8H20) was purchased from Allied and
used as received.

Preparation of AuNP Colloid. AuNPs were synthesized by reduction and stabilization
with sodium citrate according to a modification of a published procedure.38 A 1% (w/w)
solution of HAuCl4°3H2O was added to a vigorously stirred aqueous solution of sodium
citrate. The solutions were combined for an overall molar ratio of 3:1 sodium
citratezHAuCl4-3H2O. The plasmon resonance band of the resulting colloid was centered
at ca. 530 nm. SEM images of particles made using this method indicate an average

particle size of 20 nm (Figure 5.2).

  

Figure 5.2. SEM image of AuNPs prepared by the citrate reduction method. Average
particle size is 20 nm.

Silica Gel F unctionalization. Silica gel (0.5 g) was placed into a round bottom flask with

an oval stir bar. A 5% (v/v) solution of MPTMS in toluene (10 mL) was added to the

102

round bottom flask and stirred at room temperature for 24 hours. The silica gel was
filtered, rinsed with copious amounts of toluene and ethanol, and returned to a dry round
bottom flask. The aqueous solution of AuNPs (50 mL) was added to this round bottom
flask and stirred for 24 hours at room temperature, filtered, then rinsed with water and
ethanol. These reaction conditions provide a AuNP:Silica particle ratio of ca. 109. The
silica gel coated with AuNPs was returned to a round bottom flask and a 10 mM
ethanolic solution of 6-mercapto-1-hexanol (10 mL) was added. The slurry was stirred at
room temperature overnight. The product was filtered, rinsed with ethanol and ethyl
acetate and dried under vacuum in a round bottom flask for a minimum of two hours.
The flask was purged with argon, then placed under vacuum again. To this round bottom
flask was added a solution of 3.5% (v/v) POCl3/5% (v/v) collidine in anhydrous
acetonitrile. The resulting slurry was kept under an argon atmosphere and stirred
overnight. The silica gel slurry was filtered, rinsed with acetonitrile, then acetone, and
distilled water. The silica gel was returned to a dry round bottom flask. To this flask was
added a solution of 5 mM ZrOCl2 in 60:40 ethanol-water and the slurry was stirred at
room temperature overnight. The functionalized silica gel was filtered, rinsed with
ethanol and dried under vacuum for a minimum of 24 hours prior to use. For measures of
model analytes, the analytes were introduced as ethanolic solutions and the resulting
slurry stirred at room temperature overnight prior to spectroscopic analysis.

UV-visible spectroscopy. UV-visible spectroscopy was performed in transmission mode
using a Cary model 300 UV-visible spectrometer (Varian), with 1 nm resolution. Spectra
over the range of 400 nm to 800 nm were acquired at a scan rate of 600 nrn/min.

Measurements were made using 3 mL of ethanolic solution (0.50 g silica gel/5 mL

103

ethanol) for each measurement. The maximum absorbance for the PR was determined by
integration of the spectrum.

Scanning Electron Microscopy (SEM). The samples were imaged using a JEOL J SM-
6300F Scanning Electron Microscope at 25kV accelerating voltage. The sample was

placed on a SEM stub and was sputter coated with 3 nm of gold prior to analysis.

Results and Discussion

Four central issues are considered in this work. These are the chemical selectivity
of the AuNP-coated silica gel, the morphology of this system, the mechanism by which
the interaction of OPPs with the AuNP-coated silica gel are observed, and the sensitivity
of this material to the presence of OPPs. These issues will be considered separately.
Chemical selectivity. The chemistry used to achieve selective adsorption of the OPPs
onto the AuNPs is, as noted above, zirconium bisphosphonate (ZP) chemistry. This

394' in the solid state and

chemistry has been studied extensively by the Clearfield group
by the Mallouk, Thompson and Katz groups”’ '3' 25’ 32’ 314245 for binding at surfaces. It is
difficult in many instances to determine the strength of the ZP bond because the
equilibrium for bond formation lies far to the right, but indirect estimates of the strength
of the ZP bond indicate that it is ca. 60 kcal/mol or larger, under favorable conditions.37
The issue is thus what other compounds can complex with Zr4+ and whether or not Zr“,
once in place can be displaced by other metal ions. Bakiamoh and Blanchard have found
that Zr(RPO3)2+ can form complexes with RS03' and RCO2', but these complexes are not

as strong as the Zr(RPO3)2 system.46 While not quantitative, the formation constant for

OPPs with the Zr(RPO3)2+ functionalized surface should be sufficiently more favorable

104

than for complexation with sulfonates and carboxylates and that OPPs will displace these
compounds in a competitive system. It is also important to note that while there may be
slight differences in binding efficiency for the different OPPs, in all cases the binding to
Zr4+ is sufficiently strong that preferential binding phenomena are not expected. These
AuNP-coated silica particles are thus selective for the entire class of OPP compounds
rather than for a specific OPP.

Also of concern in the design of this sensor is the use of Zr4+ as opposed to other
metal ions. There is a body of literature on the use of other metals in bisphosphonate
structures and the conclusion of that work is that most other metal ions, especially with a
formal charge lower than that of Zr“, will not bind as strongly to phosphates or
phosphonates.“7 The only other metal ion that is known to bind to phosphates or
phosphonates as strongly is Hfl+.48'5°
Morphology. We show in Figure 5.3 a scanning electron microscope (SEM) image of

silica gel particles prior to their reaction with the AuNP-containing solution.

 

Figure 5.3. SEM image of a) unfirnctionalized silica gel beads with average particle
size of approximately 63.6 um and b) AuNP-functionalized silica gel beads with average
particle size of approximately 10 um. Measurement bar indicates 100 um.

105

The silica particles are not regular in shape and are characterized experimentally as
having an average diameter of 63.6 um (Figure 5.3a). The treatment to which the silica
particles are subjected appears to have a significant influence on their morphology. As
can be seen in Figures 5.3a and 5.3b, the average particle size is substantially smaller
following reaction of the silica particles with silane and AuNPs. While this may be due
in part to the harsh nature of the functionalization chemistry, it is more likely a result of
milling caused by the rigorous physical agitation required for the functionalization of the
silica gel. This size reduction is advantageous as the resulting slurrys remain suspended
longer than the non-functionalized silica gel particles. The SEM images reveal an
apparent spatially heterogeneous distribution of AuNPs, implying that the surface AuNP
coverage is not characterized by a homogeneous monolayer. Despite the limited
resolution of our images, it is clear that the functionalized surface (Figure 5.4b) exhibits

more roughness than the non-functionalized surface (Figure 5.4a).

     

Figure 5.4. SEM image of the surface of a) unfunctionalized silica gel beads (as seen
in Figure 5.3a) and b) AuNP-functionalized silica gel beads (as seen in Figure 5.3b). The
measurement bar represents 200 nm in each image.

106

This rougher surface, combined with the macroscopic observation of the purple color of
the silica gel, is indicative of the binding of AuNPs to the silica gel. Further attempts to
characterize this apparent roughness using atomic force microscopy were unsuccessful
due to the large difference in the size domains of the silica gel and AuNPs. A significant
issue lies in understanding the reaction efficiency and resulting optical properties of the
AuNPs that are bound to the silica particles, and this issue is considered next.
Characterization of the AuNP-coated silica. Because the optical properties of AuNPs are
comparatively well understood, we use absorption spectroscopy as the basis for
understanding AuNP surface coverage of the silica particles. We use absorption
spectroscopy to determine the amount of AuNP present and estimate the silica particle
concentration and surface area based on particle size and mass used. Because of the
change in silica particle size seen upon deposition of AuNPs, we provide a AuNP surface
coverage range.

Using a 0.5 g sample of modified silica particles in 10 mL of ethanol, we observe
the plasmon resonance band with a peak centered near 528 nm, and a background-

corrected absorbance of 3.27 (Figure 5.5, black trace).

107

3.5- r' 1.50

3.0-

2.5 -

2.0 -

silica gel-AuNP absorbance (a.u.)
.c'>
d
(we) ooueqrosqa dNnv

 

 

 

         

 

' I V I Y I '
550 600 650

wavelength (nm)

' I ' I I
400 450 500 700 750 800

Figure 5.5. UV-visible spectrum of AuNP-functionalized silica gel beads with the
spectrum of unfunctionalized silica gel beads subtracted in ethanol (black line) and citrate
stabilized AuNPs (gray line).

Despite the adsorption of the AuNPs onto the silica gel substrate, there was not a
significant plasmon band shift compared to the citrate stabilized AuNPs in the same
solvent (Figure 5.5, gray trace). The active surface of the bound AuNPs apparently
experiences an environment similar to that of the unbound AuNPs. There are clearly
some differences, however, as can be seen in the different band profiles for the free and
surface bound AuNPs. The added width of the bound AuNP plasmon band could be due
to a range of binding sites on the silica particles. It should also be noted here that despite
the high absorbance of 3.27, we remain well within the linear response range of the
instrument used in these measurements, which has an upper limit of 5 absorbance units.
The literature value for the extinction coefficient for 30 nm AuNPs is 4.7 x 109 L/mol-

51-53

cm, and this value is used for the nanoparticles in this study. Using a 1 cm pathlength

cuvette, a concentration of 6.96 x 10"0 M is calculated. This corresponds to a density of

108

4.19 x 1011 AuNP/cm3. With this AuNP loading density, we consider next the number of
silica particles present, and their surface area.

There are subtle features in the plasmon resonance spectrum for the surface bound
AuNPs at ca. 450 nm and ca. 580 nm (Figure 5.5). While the limited S/N ratio of the
data and the breadth of the plasmon resonance make any detailed assignment difficult, we
can speculate that there are several types of coupling of the AuNP to the silica, and the
580 nm band reflects a subgroup of AuNPs bound in a different manner. This band

(Figure 5.6) does not exhibit a positional-dependence on OPP concentration.

 

 

 

 

 

1.00 _
1.0 ..
”I? i) V.“
3 0.9 p. 'v "’t 0.95
E 08 I?
. "£1.
52 \iz'sa
V 0.7 ji-LQ' :
8 ‘3, 0.90 . 1- - _.
'8 0.6 V“ 510 520 530 540
.5]. I!!! l. ‘
"‘ 0.5 / \
8 0.4 ' --------- 5x109 M
g 0-3 --------- 5x10'8M
E 0-2 -----5x10‘7M
8 0.1 ----—--- 5x10'6M
0.0 , . 1 . r . 1 . "“"”"" SXIOOSM
450 500 550 600 ~— 5x104M
-3
wavelength (nm) " ' ' SXIO M

 

 

 

Figure 5.6. UV-visible spectra of silica gel sensor exposed to varying concentrations
of DECP. Absorbance has been normalized to 450 nm absorbance.

Further, more detailed spectroscopic studies could perhaps shed some light on the origin

of the 580 nm band.

109

Several assumptions are made for the purposes of simplicity in calculations. First
an upper bound of 63.6 um was placed on silica particle size (from the experimental
diameter). Also, spherical particles are assumed as well. While this is clearly not a
highly accurate estimate, the concern here is the establishment of a range of surface
coverage rather than determining a precise value. The volume of a 63.6 um diameter

3. A density for the silica particle of 2.0 g/cm3 is

spherical particle is 1.35 x 10'7 cm
assumed, recognizing that this is somewhat less than that of fused silica (2.2 g/cm3). This
estimate is made under the assumption that the porosity of the silica gel likely reduces its
density compared to the corresponding solid material. Using a value of p = 2.0 g/cm3, the
mass of a silica particle is estimated to be 2.69 x 10'7 g. In a 0.5 g sample, there are thus
1.86 x 106 particles in a 10 mL sample volume. From this estimate of 4.19 x 10H
AuNP/cm3 and 1.86 x 105 silica particles/cm3, we obtain 2.25 x 106 AuNP/silica particle.
The size of the AuNPs used in this work is ca. 10 nm radius, so the corresponding
“footprint” of an AuNP is 3.14 x 10'12 cmz/AuNP. If the silica particle were covered with
a uniform monolayer of AuNPs, 4.04 x 107 AuNP would be on each silica particle.
Comparing the absorbance-based estimate of 2.25 x 106 AuNP/particle to a maximum
coverage of 4.04 x 107 AuNP/silica particle yields a surface coverage of 5.6 % of a
monolayer. If we make the same estimate as above, but using 10 um diameter silica
particles, to more accurately reflect the reacted particle size distribution shown in Figure
5.3b, a silica particle density in solution of 4.77 x 107 particles/cm3 is assumed, and a
surface area of 3.14 x 10'6 cmz/particle is calculated. For these smaller silica particles,

the absorbance data indicate the presence of 8784 AuNP per silica particle and a

theoretical maximum coverage of 1.00 x 106 AuNP per silica particle, corresponding to a

110

0.8% AuNP surface coverage. From these two silica particle size limits, we see that the
surface coverage ranges from 0.8% to 5.6%, depending on particle size. While it may be
tempting to make a closer estimate of AuNP coverage, we believe that this would not be
a useful exercise given the particle size distribution present in the samples. This range of
coverage probably provides a realistic estimate of the coverage range that exists within a
given sample. With this coverage range in hand, we now turn to understanding the
mechanism for the AuNP plasmon band shift that is seen upon exposure to OPPs.

A spectral blue-shift is observed on complexation of the surface-modified AuNPs
with OPPs. The plasmon resonance is well understood and generally the band position
and linewidth are expected to vary with particle size and dielectric constant of the local

environment (Eq. 1).54

0(60)

abs

_ 2 % 82(4))
_ 9 c 8“ V0 [6,(ar)+em]2 +.€,(ar)2 (1)

where am is the dielectric constant of the medium surrounding the nanoparticle, V0 is the
particle volume, and 81 and 82 are the real and imaginary components, respectively, of the
frequency-dependent dielectric response of metallic gold.55 For the experimental
conditions described here, neither the particle volume nor the dielectric response of the
metal are influenced to any significant extent by the binding of the OPP to the particle
surface. The plasmon resonance frequency changes observed here are influenced most
significantly by the dielectric constant of the AuNP immediate environment. The blue-
shift indicates OPP binding causes a decrease in the dielectric response of AuNP
immediate environment. While the position of the pre-exposure band is influenced by the
size of the nanoparticles and the degree of nanoparticle binding it is likely that the AuNP

surface coverage would influence the absorbance but not the magnitude of the spectral

111

blue-shift. Based on this information, the blue band in the overlapped band model
presented below is used to represent the complexed form of the ZP-coated AuNP.
Interpreting the band shift. The measured plasmon resonance band can be treated as two
spectrally overlapped bands, one associated with the fi'ee form of the AuNP and one
associated with an OPP complexed to an AuNP through a ZP linkage. In this model, the
spectral shift associated with complexation is due to the modulation of the dielectric
response of the environment immediately surrounding the AuNP. Within the framework
of this model, we begin by assuming that only one complexation event per AuNP is
required to produce the observed spectral shift. If multiple complexation events were
required to produce the observed band shift, the OPP concentration dependence of this
effect would have a different functional form than is seen experimentally (vide infra).
The complexation reaction can be described by
OPP + ZP-AuNP —r OPP-ZP-AuNP (2)
K = [0PP-ZP-AuNP]/[ZP-AuNP][OPP] (3)

Where OPP-ZP-AuNP is the complex between the OPP analyte and the ZP-
functionalized AuNP. The extinction coefficients of the free and complexed AuNPs are
taken to be the same owing to the slight perturbation that the complexation event causes
to the AuNP electronic transitions. Under this condition, the ratio of the free and
complexed absorption bands is proportional to [ZP-AuNP]/[OPP-ZP-AuNP]. In this
model, when [ZP-AuNP]/[OPP-ZP-AuNP] S 0.1 or Z 10, the AuNP plasmon band will
appear to be either that of the fully complexed form (blue band) or the free form (red
band), respectively. For spectrally overlapped bands, the apparent center wavelength will

correspond to the weighted average of the band maxima for the free and complexed

112

forms. When [ZP-AuNP]/[OPP-ZP-AuNP] = 1, giving rise to a plasmon band
intermediate between the free and complexed forms, K = [OPP]", and this point is
observed experimentally for [DECP] = 5 x 10'7 M. With the value of K in hand, [ZP-
AuNP]/[OPP-ZP-AuNP] can be calculated as a function of [OPP], and thus estimate the
experimental plasmon band center wavelength.

Sensor response to OPP Compounds. To determine the response of the silica gel to
OPPs, we prepared 2 mM solutions of MPA and DECP in ethanol. Ten mL of either
MPA or DECP solution was added to a clean 20 mL scintillation vial containing
approximately 0.5 g of functionalized silica gel and a stir bar. Ten mL ethanol with no
analyte was added to separate vials containing 0.5g firnctionalized silica gel as a blank.
For all of these measurements, the portions of functionalized silica gel used came from
the same batch. The resulting slurries were stirred overnight prior to analysis to ensure
completion of the ZP complexation reaction. In practice, the kinetics of ZP complex
formation are fast, with Mallouk previously reporting indistinguishable results from

32

incubation times of one hour and 24 hours. The sample containing no analyte

demonstrated a plasmon resonance maximum at 519 nm :1: 3 nm (10') (Figure 5.7, trace a,

Table 5.1).

113

normalized absorbance (a.u.)

 

0.91 , .

 

500 520 540 560
wavelength (nm)

Figure 5.7. Response of MPTMS functionalized silica gel sensor to 24 hour exposure
to a) ethanol, b) 2 mM MPA, and c) 2 mM DECP

 

Analyte PR (nm) Blue Shift (nm)

 

2mM MPA 516i6 3
2mM DECP 51434 4
EtOH (blank) 519 a: 2

Table 5.1. Response of sensor to OPP compounds compared to the ethanol control
sample

Both samples containing OPPs exhibited a plasmon resonance blue-shift relative to the
reference samples. The silica gel slurry with MPA analyte exhibited a plasmon
resonance at 514 nm d: 4 nm (Figure 5.7, trace b) and the silica gel slurry with DECP
analyte exhibited a plasmon resonance at 516 nm i 7 nm (Figure 5.7, trace c),
corresponding to blue-shifts of 5 nm and 3 nm, respectively. The uncertainties, as

determined through multiple measurements of the same sample, appear relatively large,

114

owing to the width of the plasmon resonance bands and the limited S/N available with
these data due to background scattering.

The sensitivity of our coated silica particles to OPPs was evaluated using DECP
as a model OPP for concentrations ranging fi'om 5x10'9 M to 5x10'3 M (Figure 5.6). The
plasmon resonance band maximum of the analyte-exposed silica gel was compared to
that of non-exposed silica gel to determine presence and magnitude of the plasmon -
resonance band shift. At DECP concentrations lower than 5x10'7 M, the plasmon
resonance band does not manifest a spectral shift outside the spectral resolution of the

measurement (:5 1 nm) (Table 5.2).

 

 

DECP . Blue Shift from
Concentration PR (nm)
(M) 0 mM (nm)
0 528 --
5 x 10'9 531 -—
5 x 10‘8 529 --
5 x 10‘7 525 3
5 x 10'6 520 8
5 x 10'5 523 5
5 x 10“ 518 10
1 x 10‘3 526
5 x 10'3 524

Table 5.2. Sensitivity of sensor to DECP

For DECP concentrations higher than 5x10'7 M (86.3 ug/L) the modified AuNP plasmon
resonance exhibits a blue-shift relative to the unexposed AuNP-modified silica particles.

The errors associated with these measurements was < 1 nm for replicate measurements

115

and further analysis was conducted using a larger i 3 nm error associated with
experimental error due to expected scattering due to the silica gel. It should be noted that
the blue-shift does not appear to reach a plateau for high OPP concentration extremes,
despite the expectation of sensor saturation. The reason for this finding is likely due to
the uncertainty attributed to the measurement, resulting from the background scattering
from the silica particles in ethanol solution. The sensor response can be viewed as a
digital response, with there being either a shift or not, making it most useful as a
presumptive test for the presence or absence of OPP compounds.

' We show the comparison of the experimental data to the model in Figure 5.8,
where the solid lines are the model calculations for 1:1 OPP:AuNP (black trace) 2:1
OPP:AuNP (dark gray trace) and 3:1 OPP:AuNP (light gray trace), and the individual

data points are the experimental band positions as a function of [OPP].

116

534- 4
5324
530-

 

L

 

 

 

 

Wavelength (nm)
Ur LII kit kit til
N N N N N
O N A O\ 00

I n J 4 I I I I I
P
I

 

518-

5164 I

OPP Concentration (nM)

 

 

Figure 5.8. Comparison of observed PR bands to 1:1 free-complexed AuNP model
(black trace), 2:1 free-complexed AuNP model (dark gray trace) and 3:1 model (light
gray trace).

While there may be some contribution of 2:1 and 3:1 complexation the correspondence
between the 1:1 OPP:AuNP model and the experimental data in the most variable range
follows the functional form of the data more closely, especially in terms of the
concentration-dependence. This correspondence between model and experimental data
suggests that the assumption of a 1:1 OPP:AuNP complexation stoichiometry is valid. It
should be noted that there is little distinction between 2:1 and 3:1 complexation, a finding
that supports the assertion that the most noticeable change in the AuNP optical response

occurs for the first complexation event. While further complexation may occur between

the AuNPs and the OPP, it appears that it will not have a significant effect on the data.

117

We also believe that this approach to studying complexation processes holds significant
promise for a number of other systems that are adaptable to surface-modification
chemistry on AuNPs.

S/N Enhancement. The observed signal-to-noise ratio (S/N) for the plasmon resonance
spectra of the silica gel-AuNP suspensions in ethanol prior to their exposure to an OPP
was 5.8, and, significantly, there was a background signal of ca. 2.5 absorbance units.
The attenuation of the transmitted light due to scattering loss gives rise to the large
background. One way to reduce the scattering background is to use a solvent system that
is better matched to the refractive index of the silica particles. The refractive index of
silica gel is approximately 1.46.56 By approaching the refractive index of the immersion
solvent to that of the silica gel, the S/N was enhanced because of a decrease in the

observed background absorbance (Table 5.3).

 

 

Solvent Ba k ound
Solvent Refi'active S/N c gr
Abs (a.u.)
Index

EtOH 1.3614 5.8 2.5

DMF 1.4305 23.1 1.75
CHC13 1.4458 36.2 1.75
DMSO 1.4783 42.0 0.37

Table 5.3. Influence of solvent refractive index on S/N ratio and background absorbance

We found that by using DMSO as the solvent, (11 = 1.48), we could produce a seven-fold

increase in S/N compared to ethanol (n = 1.36). This increase in S/N is associated with

118

the decreased background scattering observed from the DMSO solution to a level of 0.37

absorbance units (Table 5.3, Figure 5.9).

 

0.81

absorbance normalized to 400nm

 

-:1 1'1 1 ”1 .1 :1 600 620 .-1
wavelength(nm)

Figure 5.9. S/N of sensor in a) DMSO (S/N = 42), b) DMF (S/N = 23.1), c) ethanol
(S/N= 5.8), and d) chloroform (S/N = 36.2)
We expect that this sort of increase in S/N and reduction in background signal will be

especially beneficial in studies that focus on small concentrations of OPPs.

Conclusion

The detection of OPPs has become increasingly important due to concerns about
organophosphate/phosphonate based chemical warfare agents and pesticides. While
sensitive and class-selective detection is vital, the construction of such detection systems
using simple and robust detection methodology is also of importance. The material
presented here, hybrid silica gel-AuNP-ZP particles, is robust and the plasmon optical
response is capable of detecting comparatively low levels of OPPs complexed to the ZP

termini of the particles. These materials are synthesized simply and can be made on a

119

large scale, if required. The AuNP plasmon resonance shift observed is independent of
OPP concentration beyond a threshold concentration of ca. 5x10'7 M for DECP,
corresponding to a K ~ 2 x 10'5 M". From the functional form of the plasmon resonance
maximum concentration-dependence, that the stoichiometry on the AuNP-ZP-OPP
complex is estimated to be 1:1, consistent with the estimated silica gel surface loading of
1.00 x 106 to 4.04 x 107 AuNPs per silica gel particle. The sensitivity of this
methodology may be improved by using a solvent system where its refractive index
matches more closely the refractive index of the silica. The S/N ratio of the AuNP
plasmon resonance band is improved seven-fold from 5.8 in ethanol to 42 in DMSO.

This composite material is simple to synthesize construct and utilize, and its use
in the detection and sequestration of OPPs makes it useful for a host of applications in
chemical warfare and pesticide analysis and remediation. The simple optical detection
method is amenable to the incorporation of these materials in field-portable instruments.
Continued improvements in the sensitivity and chemical selectivity of this novel material

will afford its use as a sensor for presumptive testing for OPPs.

120

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Wang, Z.; Heising, J. M.; Clearfield, A., Sulfonated Microporous Organic-
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Yang, C. Y.; Clearfield, A., The preparation and ion-exchange properties of
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Ruvarac, A.; Milonjic, S.; Clearfield, A.; Garces, J. M., On the mechanism of ion
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exchange of a-zirconium phosphate. Journal of Inorganic and Nuclear Chemistry
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Hong, H. G.; Sackett, D. D.; Mallouk, T. E., Adsorption of Well-Ordered

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Ionic-Covalent Strategy for Self-Assembly of Inorganic Multilayer Films. Journal
of the American Chemical Society 1997, 119, (50), 12184-12191.

Katz, H. E., Multilayer Deposition of Novel Organophosphonates with
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126

Chapter 6
Optical Organophosphate/Phosphonate Sensor Based upon Gold Nanoparticle

Functionalized Quartz

Introduction

As discussed in the previous chapter, concerns about release and disposal of
chemical warfare agents (CWAs) and environmental contamination from pesticides has
lead to an increased need to detect organophosphorous compounds sensitively and
selectively. Organophosphate/phosphonate compounds (OPPs) can have devastating
physical effects including muscular paralysis leading to death.1 The Chemical Warfare
Convention (CWC), ratified in 1997, cited an urgent need to develop detectors for CWAs
and other OPP compounds in order to verify compliance with the provisions of the treaty
that prohibits the use of chemical warfare agents as well as the continued development,
production and storage thereof.2 Additionally, concerns surrounding the impact of
organophosphorous pesticides on the environment and on the food chain has lead to the
need for rapid detection these compounds as well.3'lo This chapter discusses an OPP
sensor based on monitoring the plamson resonance (PR) of gold nanoparticles (AuNPs)
covalently attached to a planar solid substrate. This device can be used to qualitatively

detect the presence of OPP compounds using simple optical spectroscopic measurements

127

as an inexpensive, robust, presumptive test for the presence of these compounds in a
variety of matrices.
There are a number of techniques currently in use for the detection of CWAs,

CWA metabolites, and other OPP compounds including chromatography,"'19 mass

12, 14 4, 8-10, 20, 21

spectrometry, electrobhemistryf’ 7’ 8‘ 20'” biosensors, and others.23 Recent
work in the field has focused on the use of nanoparticles of various compositions and
their use in the detection of OPPs4’ 7’ 9' 23. Both AuNPs and zirconium oxide (Zr02)
nanoparticles have shown promise for use in the detection of OPP compounds. In the
case of AuNPs, Pavlov and coworkers24 have demonstrated that in the absence of
thiocholine, a product of the decomposition of acetylthiocholine by acetocholine esterase
(AChE), AuNPs do not form. Therefore, if AChE is inhibited and cannot break down
acetylthiocholine, a decrease in the plasmon resonance intensity will occur. Additionally,
they bound 2-3 nm diameter AuNP “seeds” to a planar surface using an
aminopropylsilane film and created a solid-phase sensing platform based on the same
chemistry which was also analyzed by UV-visible spectroscopy. Liu and coworkers7 also
demonstrated a planar solid-phase sensing platform for OPPs based upon the affinity of
OPP compounds for zirconium. In this case, they electrochemically deposited Zr02
nanoparticles on a gold electrode and probed for the OPP methyl parathion
electrochemically. Both these instances demonstrate the potential of a planar substrate
format for chemical sensing. Ideally, however, the chemical specificity of the zirconium
based sensor could be combined with the simple design of the} bound AuNP sensor to

create a robust sensor that is simple to construct and utilize with optical rather than

electrochemical detection. It is this type of sensor we describe here.

128

The sensing platform we have developed is based on a series of well established
surface chemistries. First, quartz surfaces are functionalized with either amino- or
mercaptosilane compounds.”28 The AuNPs are attached through simple deposition from
the solution phase by soaking in an AuNP colloid.”32 The AuNPs are necessary for
optical sensing due to the characteristic PR in the visible spectrum associated with these
particles. Finally the AuNPs are functionalized through displacement of the citrate
stabilizers by a m-hydroxythiol compound.33 The sensing platform is completed using
zirconiurn-phosphate/phosphonate (ZP) chemistry previously described by our group.”37
Many CWAs and OPP pesticides contain phosphoesters. Because of this, such analytes
demonstrate good affinity for ZP-functionalized surfaces.7' 35

While these sensors are less sensitive than some other techniques, including one
demonstrated by our research group for OPP detection,38 they provide a very simple use
platform. These sensors are robust and their analysis is inexpensive and simple and
shows promise for use as a presumptive sensor of OPP compounds in a variety of

matrices including liquids, liquid extracts of solid matrices, and perhaps biological

tissues.

Materials and Methods

Materials. Sodium citrate, hydrogen tetrachloroaurate (HAuC14°3H2O, 99.9+%),
methylphosphonic acid (MPA), diethylchlorophosphate (DECP), 3-aminopropyl-
triethoxysilane (APTES), and 3-mercaptopropyltrimethoxysilane (MPTMS) were
purchased from Sigma-Aldrich and used as received. 3-Aminopropyldimethylethoxy-

silane (APDMES) was purchased from Gelest and used as received. 6-Mercapto-1-

129

hexanol and phosphorous oxychloride (POCl3) were purchased fi'om Fluka and used as
received. Collidine was purchased from Spectrum and used as received. Zirconium(IV)
oxychloride octahydrate (ZrOC12-8H2O) was purchased from Allied and used as received.
Preparation of AuNP Colloid. AuNPs were synthesized from HAuC14 by reduction and
stabilization with sodium citrate according to a modified published procedure.39 Briefly,
an aqueous solution of sodium citrate was brought to a boil. While stirring vigorously, an
aqueous solution of HAuCl4-3H2O was added. For these procedures a 1% (w/w) solution
of HAuCl4-3H2O was used. The solutions were combined for an overall molar ratio of
3:1 sodium citratezHAuCl4-3H2O. The plasmon resonance band of the resulting colloid in
water was approximately 530 nm.

Preparation of sensor. Quartz substrates were soaked in piranha solution (3:1 sulfuric
acidzhydrogen peroxide) for at least 30 minutes. The substrates were then rinsed with
copious amounts of distilled water and ethyl acetate, and dried with N2. For surface
activation with an amine terminus, a 5% solution of 3-aminopropyltriethoxysilane or 3-
aminopropyldimethylethoxysilane in toluene was used. For surface activation with a
thiol terminus a 5% solution in toluene of 3-mercaptopropyltrimethoxysilane was used.
This step will be referred to hereafter as the linking step and the compound used as the
linker. The substrates were soaked overnight at room temperature, then rinsed with ethyl
acetate and dried with N2. The substrates were then immersed in the AuNP colloid
overnight at room temperature, then rinsed with ethyl acetate and dried with N2.
Following attachment of AuNPs to the quartz surface, the citrate stabilizer was displaced
by soaking the sample in a 10 mM 6-mercapto-1-hexanol solution in ethanol overnight at

room temperature, then rinsed with ethyl acetate and dried with N2. After this step the

130

sample was dried in an oven for five minutes. The sample was then placed into a round
bottom flask, put under vacuum, then purged with Ar for at least 2 hours. To the round
bottom flask under vacuum was added a 5% collidine/3.5% P0C13 solution in anhydrous
acetonitrile. The sample was soaked for a minimum of two hours, and then rinsed with
anhydrous acetonitrile, acetone and water. The sample was then rinsed with ethyl acetate
and dried with N2. To the phosphorylated sample was added a 5 mM ZrOCl2 solution in
60:40 ethanol-water. The sample was soaked overnight at room temperature, then rinsed
with ethyl acetate and dried with N2. A representation of the AuNP functionalized quartz

sensor can be seen in Figure 6.1.

Si O
\ 0\
R0 \ / ,Zr“+
\ H2 H2 H2 . 0"
Si\—O—/Si—'C —C "C "'S AUNP 8M0

R0
81))

Figure 6.1. Representation of the AuNP-functionalized quartz sensor

Following this step, a baseline UV-visible spectrum was taken to obtain the blank PR for
the sensor substrate. The analytes were introduced as ethanolic solutions of MPA or
DECP and soaked overnight. Additionally, ethanol was used as a control analyte for all
analyses. The samples were thoroughly rinsed with ethanol and dried with N2 prior to
spectroscopic analysis.

SEM Imaging. Substrates for SEM imaging were prepared on indium tin oxide (ITO)
substrates under conditions identical to those discussed for the quartz substrates up to the

attachment of AuNPs. This was the final step for the ITO samples. The samples were

131

imaged using a JEOL J SM-6300F Scanning Electron Microscope at 25kV accelerating
voltage.

U V-visible Spectroscopy. The samples were examined prior to and following exposure
to analytes. The sensors were analyzed in air. The spectra were acquired in transmission
mode from 300 to 800 nm using a Cary model 300 UV-visible spectrometer (Varian) and
acquisition time was ca. 1 minute per spectrum.

Sensitivity Studies. Ethanolic solutions of the DECP analyte were prepared to
concentrations of 5mM to 5nM. Blank UV-visible spectra were acquired for the sensor
substrates in air prior to immersion in the analyte. The sensors were immersed in analyte
solution for 24 hours, rinsed with ethyl acetate, and dried with N2. The after-immersion

U V-visible spectra were again acquired in air.

Results and Discussion
SEM Imaging. APTES was explored first as the quartz-AuNP linker. Despite efforts to
make the surface visually homogeneous, the sensors retained an uneven distribution of

AuNPs on the surface (6. 2a). AuNPs on the surface (6. 2a).

132

 

  

 

‘.1'n

Figure 6.2. SEM images of AuNPs on ITO substrates at 10,000 times magnification
A) APTES linker, B) APDMES linker C) MPTMS. Measurement bar indicates 2 pm.

It has been suggested that the cause of this visual surface heterogeneity is polymerization
of the linker on the surface leading to local areas of increased concentration of both linker
and subsequently AuNPs.26’ 27’ 29 Multilayer coverage of the linker would in turn lead to a
greater degree of heterogeneity in the AuNP coverage. SEM images at 10,000 X
magnification verified that the sensors prepared with APTES did have a greater degree of
aggregation on the surface than either of the other two sensors (Figure 6.2). In an effort
to make the surface more uniform, APDMES was used as the quartz AuNP linker.
Literature precedent suggests that the presence of a single ethoxy in APDMES leads to a

monolayer coverage of the silane on quartz surfaces.” 27 Monolayer coverage should, in

133

turn, lead to better uniformity of AuNP coverage on the surface. In this case, the
substrates were more visually homogeneous, although with lower apparent AuNP density
(Figure 6.2b). To achieve a more robust sensor, MPTMS was used as the linker. This
linker takes advantage of the strong S-Au bond that has been exploited in self-assembled
monolayer chemistry40’ 4' as well as methods to attach various forms of gold to silicon

38’ 42' 43 Despite the presence of a trimethoxy species in

substrates of various geometries.
MPTMS, which was implicated in the case of APTES to create multilayer coverage, the
resulting sensors had a macroscopically visually regular surface (Figure 6.2c).

The SEM images showed aggregates in the approximately 10pm size regime in
the case of APTES, while aggregates in the lum range were observed in the cases of
APDMES and MPTMS. Additionally, there were a greater number of aggregates
observed in the APTES sensors. At 100,000 X magnification, both amine terminated

linkers showed the majority of AuNPs on the surface present in the form of aggregates

(Figure 6.3 panels a and b).

134

 

Figure 6.3. SEM images of AuNPs on ITO substrates at 100,000 times magnification
A) APTES linker, B) APDMES linker C) MPTMS. Measurement bar indicates 200 nm.
In some places on these substrates, there were no observable non-aggregated AuNPs. On
the substrate prepared with MPTMS, the AuNPs were much more evenly dispersed on
the surface. While some aggregates were present (Figure 6.3, panel c), there was a large
population of either isolated AuNPs or aggregates of a very small number of particles.

U V- Visible Spectroscopy. Sensors were prepared using all three quartz-AuNP linkers to
explore the effect of the three linkers on the spectroscopic response. When APTES was
used as the linker, a blue shift of 4 nm was observed in the spectrum following exposure

to MPA (Figure 6.4).

135

0.20

0.15

0.10

absorbance (a.u.)

0.05

 

 

0.00 ' ‘ ‘
300 400 500 600 700 800

wavelength (nm)

 

Figure 6.4. APTES linker PR before (dashed line) and after (solid line) exposure to 2
mM MPA (~200ppm). PR blue shifted 4 nm upon exposure.
While this result was consistent with the results discussed in the previous chapter for
AuNPs on a silica gel substrate, the other linkers were explored to determine if similar or
better results could be seen while achieving a more homogenous sensor surface.

As seen in the imaging study, APDMES produced a similar, though slightly more
homogenous, substrate to APTES. The spectra obtained for the substrates before and
after exposure to MPA, DECP demonstrated blue shifted plasmon resonance bands of 17

nm and 5 nm, respectively (Table 6.1).

136

 

Before/After

 

Analyte Exposure PR (11111) APR (nm)
before 593
Methylphosphonic acid -17
after 576
before 555
Triethylchlorophosphate -5
after 550
before 568
Ethanol (blank) 8
after 57 6

 

Table 6.1. Plasmon resonance band shifts for APDMES sensor exposed to analytes.

Additionally, when exposed to ethanol the sensor demonstrated a red shifted plasmon
resonance of 8 nm (Table 6.1), allowing for differentiation of the response in the presence
of the solvent. This response showed the same trend as observed with the APTES linker
but was greater in magnitude indicating the more uniform surface served to better
elucidate the diagnostic blue shift.

While the attachment chemistry is efficient and the sensor effective when an
amine group is used in the linker, MPTMS was explored as an alternative as the MPTMS
substrates appeared to be more homogeneous when viewed microscopically. The
behavior of the sensors, however, was consistent with the observations made for the
APTES sensors demonstrating blue shifted plasmon resonances of 13 nm and 10 nm for

MPA and DECP, respectively (Figure 6.5 panels a and b, Table 6.2).

137

    
  

 

 

 

 

 

    
    

030- 030-
025- ;”K, 025-
5 l : . ”i 1
83 (120-2‘ E; (120-.
8 ' 8
g 0.15- g 0.15-
8 ' g -
'3 010- _g 010-
a q a
(105- (105-
0-00 ' I ' I r T ' I ‘ I 0.00 ' r r I ' I ' I ‘i I
300 400 500 600 700 800 300 400 500 600 700 800
0.30- wavelength (urn) wavelength (nm)
0.253
5 .
83 1120-:
8
8 0151 “in
.e .
_§ 010-
a 4
(105-
. C
000 - . . .

 

 

300 1' 400 ' 500 600 700 ' 800

wavelength (nm)

Figure 6.5. a) MPTMS PR before (dashed line) and after (solid line) exposure to 2
mM MPA. PR blue shifted 13 nm upon exposure.

b) MPTMS PR before (dashed line) and after (solid line) exposure to 2 mM DECP. PR
blue shifted 10 nm upon exposure.

0) MPTMS PR before (dashed line) and after (solid line) exposure to ethanol. No blue
shift observed.

138

 

Before/After

Analyte PR (11 m) APR (nm)

 

Exposure
before 570
Methylphosphonic acid -13
after 557
before 564
Triethylchlorophosphate - 1 0
after 554
before 554
Ethanol 3
after 557

 

Table 6.2. Plasmon resonance band shifts for MPTMS sensor exposed to analytes.

Ethanol was again seen to red shift the plasmon resonance position, by 3 nm in this
instance (Figure 6.5c, Table 6.2).

The major influence on the absorbance of AuNPs comes from the dependence of
the absorption cross section and the PR wavelength on the dielectric response of the
nanoparticle and the medium (Eq. 1)44

2473185? 6(a))
1 (E.(w)+2€m). +(s'(w>)’

 

0(0)).n. ={ (1)

where am is the dielectric constant of the medium surrounding the nanoparticle, R is the
particle radius, and e’(00) and e”(0)) are the real and imaginary components, respectively,
of the fi'equency-dependent dielectric response of metallic gold. Under the condition that
8”(00) is small, the resonance occurs where 8’(0)) = -28m, and the primary contribution to
the PR is in the first term of equation 1. Thus, the major influences on changes in the
position of the plamson resonance can be attributed to the size of the AuNP and the local

dielectric environment it experiences (em). Typically, the plasmon resonance wavelength

139

of AuNPs is most greatly affected by changes in AuNP size of the AuNPs.“ 45 When
increases in the size of the AuNPs are the greatest influence, red shifts and broadening of
the plasmon resonance peak are observed. This is due to quantum size effects as well as
the particle size distribution. Because increasing the size of the particles is expected to
produce a red shift in the position of the plasmon resonance band, it is unlikely that the
small size change caused by OPP binding has an influence on the plasmon resonance
position in this case. Therefore, the plasmon resonance blue shift in the presence of OPP
compounds is due to the influence of the local dielectric change when OPP compounds
have been chemisorbed to the sensor substrate. This dielectric change is not seen when
the sensor is soaked in ethanol. The slight red shift observed when the sensor is exposed
to ethanol is likely due to either minor aggregation effects arising from the mobility of
the AuNPs on the surface or residual ethanol causing a dielectric change, relative to air,
near the AuNP surface. Because the analytes were prepared in ethanol solution, it is most
likely that the effect of the ethanol is incorporated into the effect observed with the OPP
analytes and the blue shift observed in these cases is actually great enough to overcome
the influence of the solvent. Additionally, the blue-shift is believed to be the
consequence of the change in the local dielectric environment associated with OPP
binding. Because binding of the OPP analytes to the AuNP surface would be expected to
slightly enlarge the particles, if the effect was due to size changes, a red-shift would be
expected. The most significant other parameter associated with PR position is the
medium dielectric constant. As seen in equation 1, under resonance conditions the PR
wavelength is proportional to 8,1,3 ’2. Therefore, a decrease in em would result in a blue-

shift. We believe that this is the case for this sensor.

140

Sensitivity. It is important to note here that this sensor is class-specific and not able to
differentiate between individual OPP compounds. As this sensor design is intended to be
a presumptive test for the presence of OPP compounds, we do not view this as a
limitation of the sensor. The sensitivity of the sensor was explored using concentrations

of DECP ranging from 5 x 10'3 to 5 x 10'9 M (Table 6.3).

 

 

Concentration Average
(M) Blue Shift (nm)
5 x 10“” 16.00 a 5.66
5 x 10'04 15.00 a: 5.67
5 x 10*” 4.00 n 1.00
5 x 104’6 2.00 i 0
5 x 10m 5.00 :1: 2.65
5 x 10'08 2.00 n 0
5 x 10°9 2.00 r 6.78

 

Table 6.3. Sensitivity to DECP

A blue shift of 2 nm was considered a negative result because the spectral resolution of
the instrument was 1 nm and the experimental data are characterized by a modest signal-
to-noise ratio. At concentrations greater than or equal to 5 x 10'5 M, a blue shift in the
plasmon resonance band of the sensor was observed, characteristic of the presence of
OPP compounds. The magnitude of the blue-shift was in the range of 4 - 16 nm (Figure

6.6), well outside the uncertainty of the measurement. The level of uncertainty associated

141

with these measurements suggest they are useful only as a qualitative determinant of OPP

presence and are not, in their present form, capable of quantitative analysis.

Blue Shift (nm)

Figure 6.6.

25.00 7

20.00 -

15.00 A

10.00 i

5.00 _

 

 

 

'—

 

0.00
1.E

-5.00 -

.09 l

 

 

I l l I l l I l

.E-08 l.E-07 l.E-06 l.E-05 l.E-O4 l.E-O3 l.E-02 l.E-Ol

-- DECP Concentration (M)

Observed PR response of the sensor to variations in DECP concentration.

The relatively large variation in band shift is due to the degree of scattering imparted by

the sensor itself. While not as sensitive as methods based on mass spectrometric

detection or our previously reported silica gel-based sensor,38 this limit of detection is on

the same order of magnitude as reported for other methods based on UV-visible

detection.

46—49

At high analyte concentrations, the magnitude of the blue shift is seen to be

independent of DECP concentration due to rapid saturation of binding sites. Due to this

142

rapid saturation, there is limited dynamic range associated with this sensor and it exhibits
a digital response associated with DECP binding.

The sensor response can be explained by considering that there are two spectrally
overlapped bands, with one band attributed to AuNPs terminated with uncomplexed Zr4+
and the other attributed to Zr4+ complexed to an analyte OPP compound. In chapter 5,
the ability to model this spectral response was demonstrated. In this case, the response
has been attributed to the dominance of a 1:1 OPP:ZP-AuNP binding event.38 There may
be contributions from 2:1 and 3:1 OPP:ZP-AuNP binding, but these contributions are
minor. In the case of these sensors the position of the plasmon resonance changes with
variations in the degree of AuNP surface coverage achieved during sensor construction.
The blanks had plasmon resonance bands ranging from 519 nm to 577 nm. Therefore,
the same rigorous mathematical interpretation could not be made. However, the sensor
behavior is consistent with the previous study. The blue shift obtained OPP attachment
was independent of the blank plasmon resonance band position for the sensor and the
magnitude of the blue shift is more important as a diagnostic tool than the absolute

position of the band after exposure in the case of these planar OPP sensors.

Conclusions

Concerns about the impact of organophosphate/phosphonate compounds, used as
pesticides or chemical warfare agents, have made the detection of these compounds
increasingly a priority. When developing sensors for these compounds, it is important to
consider the simplicity of construction and durability of the sensors in addition to the

vital standards being sensitivity and selectivity for OPP compounds in a variety of

143

matrices. We have presented an OPP sensor based on optical detection of ZP modified
AuNPs bound covalently to a planar quartz substrate. These sensors are simple to
construct, rugged, and utilize simple detection with a UV-visible spectrometer. We have
shown through SEM imaging that while APTES and APDMES both serve to bind large
numbers of AuNPs to the quartz surface, using MPTMS as the quartz-AuNP linker
results in the sensor substrate with the most uniform surface coverage. After
displacement of citrate stabilizing agents on the AuNP surface with a mercaptoalcohol
and subsequent ZP chemistry, the completed sensors were tested for their ability to bind
OPP compounds. These sensors demonstrate a blue shift in the plasmon resonance band
when exposed to the OPP compounds MPA and DECP, but display a more typical red
shift in the presence of ethanol.

Sensitivity studies conducted using DECP as the OPP analyte show that the
sensor is able to detect OPP analytes at concentrations > 5 x 10'5 M. Below this level, the
response of the sensor does not produce a band shift in excess of the instrumental
uncertainty. At DECP concentrations of Z 5 x 10'5 M, the sensor response is independent
of the analyte concentration and a characteristic blue-shift is observed in the spectrum.
This can be explained by the spectrum of the sensor being dominated by the bands
associated with the complexed AuNP-ZP-OPP species. While further improvements are
necessary to decrease the detection limit of this type of optical OPP sensor, the current
sensor provides a simple and robust qualitative method of detecting OPPs above the limit

of detection and has potential for use as a presumptive test for OPP presence.

144

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Chapter 7

Conclusions and Future Work

Due to their unique properties gold nanoparticles (AuNPs) have become a widely
studied area in many areas of chemistry. AuNPs are finely divided particles of gold
which have properties which fall between those observed for atomic and bulk gold.
These properties, both optical and electronic, have been studied with the goal of utilizing
AuNPs in areas ranging from signal-enhancing substrates, to sensing technologies, to
medical technologies. This thesis has covered three areas which are useful in the study of
AuNPs; formation, characterization, and application of AuNPs and surface-modified
gold.

Formation. The thermodynamic potential for formation of AuNPs using amines as the
reducing and stabilizing agent can be predicted though electrochemical interrogation of
HAuC14 and the potential reducing agent using cyclic voltammetry. Comparison of the
oxidation potential of an amine to the reduction and oxidation potentials of the HAuCl4 in
a given solvent system can be the first line of predictability for the potential of a given
HAuCl4-amine system to result in the formation of a gold colloid. If the oxidation
potential of the amine lies between the reduction potential of HAuCl4 to Au0 and the

oxidation potential of Au0 to Au”, the amine can be considered a viable reducing agent.

150

This initial screening of the reducing agent is useful for both monomeric and polymeric
amines and may help to determine which systems bear further investigation.

In addition to the thermodynamic considerations, the kinetics of the redox
reaction also plays a role in the formation of AuNPs. The kinetics of AuNP formation
was probed using time-resolved ultra-violet-visible spectrometry. It was found that
amines which were thermodynamically predicted not to function as reducing agents did
not show any evidence of AuNP formation as determined through a lack of PR in the
visible spectrum. When thermodynamically predicted amines did successfirlly reduce
Au3+ to AuNPs, it was found that, among reducing agents with the same number of
amines per mole, an increase in the oxidation potential resulted in a decrease in the rate
of PR evolution. This indicated that the system was operating in the Marcus inverted
region, as explained by Marcus electron transfer theory.

Several amines which were predicted on thermodynamic grounds to function as
reducing agents either showed limited evidence of AuNP formation, as determined by the
presence of a small PR band, or no PR band at all. These exceptions were understood in
terms of competitive polymerization events which kinetically precluded AuNP formation.
In the case of aniline and 3-aminophenol, the polymerization of the reducing agents takes
place faster than the formation of AuNPs and therefore no AuNPs are observed
spectroscopically. In the case of 4-aminophenol, the kinetics of the reduction of HAuC14
to AuNPs and the polymerization of the amine are similar and both AuNPs and poly(4-
aminophenol) are observed in the time-resolved spectrum.

Poly(allylamine hydrochloride) (PAH) was investigated in further detail as a

potential polymeric reducing and stabilizing agent. It was predicted to function as a

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reducing agent under the thermodynamic considerations and it was found that the kinetics
of the system are first order in HAuClr at constant PAH concentration. When the PAH
concentration is varied, the system kinetics vary to the third power. This validates the
postulated reduction mechanism where one Au3+ ion is reduced by three amine groups.
Knowledge of the concentration dependence helps provide information which can help to
optimize the formation of AuNP-PAH composites. This information could also be
applied to other polymeric reducing agents as well.

While the kinetics of the system at room temperature can be readily predicted, the
reaction rate is also quite slow. In order to speed up the reaction it is possible to add heat
energy into the system and maintain a degree of control over the optical behavior of the
resulting colloid. With sufficiently small molar ratios of PAHzHAuClr, the position of
the PR band can be both controlled and roughly correlated to AuNP size. A linear
relationship exists between the position of the PR band and the ratio of the reactants in
solution. Additionally, a general increase is seen in the size of non-aggregated particles
in the composite material with an increase in the reactant ratio. This data can be
considered together to conclude that the increase in particle size is responsible for the
red-shift in the PR band maximum.

Taken together the electrochemical and spectroscopic investigations of these
HAuCl4-amine systems can provide predictive information about their potential to result
in AuNP colloids. The thermodynamic and kinetic information gained is important to
understanding the degree of control achievable in a given system in terms not only of
final outcome but also of growth rate. Predictive knowledge about the formation of

AuNPs using amines as the reducing and stabilizing agent has a great deal of potential.

152

The ubiquitous nature of amines in biological systems indicates this knowledge could be
of great use when designing polymer films for sensing applications and biomedical
applications. It could also be important for understanding potential in situ AuNP
formation for use as therapeutic agents. Interrogation of poly-peptides, for example poly-
tryptophan, would be quite useful for these types of investigations. In initial studies of
poly-tryptophan as a reducing agent, solubility was found to be a limitation in both
electrochemical and kinetic studies. Other poly-amino acids or poly-peptides may not
have the same limited solubility and could therefore be more useful.

Characterization. Impedance spectroscopy conducted on QCM crystals is a
characterization method for surface-modified gold which can be useful in probing the
viscoelastic properties of adlayers in addition to the more typical mass uptake studies.
The study discussed here addressed the issue of how the viscoelastic properties of
alkanethiol SAMS vary with aliphatic chain length and time after initial formation. The
collected impedance spectra were interpreted in the context of an equivalent circuit model
used to understand QCM response in liquids. It was found that the quantities L2 and R2
in the equivalent circuit to denote the inductance and resistance of liquid loading,
respectively, dominate the physical and chemical contributions of the adlayer in the QCM
response. Both have similar temporal behavior for all of the alkanethiols examined in
this study. The quantity L2 was used to extract information about the value of pr], which
is related to the viscoelastic properties of the SAM adlayer. The p11 value for the C6$H
monolayer was found to have a higher value than that observed for SAMS having
between 9 and 16 carbons in the aliphatic chain. This is indicative of the C6SH

monolayer having a different solvent-monolayer interface than the SAMS formed longer

153

carbon chain alkanethiol. This is likely due to the lack of organization in short chain
alkanethiol SAMs. Monolayers formed from C9-C16SH had similar values of p11,
indicating the similarity of the interfaces between these alkanethiol SAMS and the
solvent. The viscosity is believed to dominate changes in p11 because both the monolayer
density and bulk thiol density are constant for the systems reported here. The deposition
of Clg-SH was also studied and differed from that of the shorter alkanethiols, likely a
consequence of its limited solubility. The anomalous behavior of C13 SAMS can be
attributed to the bulk deposition of the thiol on the QCM surface. These studies were
conducted at 14°C and it would be useful to expand this work to higher temperatures.
This would serve the purpose of both examining the changes which occur in the behavior
of the SAMS at elevated temperature along with increasing the aliphatic chain lengths
able to be probed by increasing the range of soluble alkanethiols. Also interesting to
study would be the physical behavior of other surface modifications to the Au electrodes
on the QCM crystal. These may include the Chemisorption of polymers and polymer
multi-layers or the behavior of a composite material such as the PAH-AuNP discussed in
chapter 3.

Application. Due to environmental and health concerns surrounding the use of
organophosphate/phosphonate (OPP) based compounds, their detection has become
increasingly important. While sensitive and class-selective detection is fundamental, it is
also important to develop sensors which are simple and robust. The sensors described
here are based on zirconium phosphonate (ZP)-functionalized AuNPs which are

immobilized on a silica platform. These sensors are capable of detecting relatively low

154

levels of OPPs which complex irreversibly to the ZP termini of the AuNPs. The
detection is based on simple UV-visible detection of shifts in the PR position.

First, a hybrid silica gel-AuNP-ZP sensor was developed which was simple to
synthesize and could be produced on a large scale if required. These silica-gel based
sensors were found to exhibit a characteristic blue-shift in the presence of OPP
compounds. Additionally they have a limit of detection of ca. 5x10'7 M for
diethylchlorophosphate (DECP). This corresponds to a K ~ 2 x 106 M". The functional
form of the PR maximum concentration-dependence can be used to model the
stoichiometry of the AuNP-ZP-OPP complex, which is estimated to be 1:1. This is
consistent with the calculated silica gel surface loading of 1.00 x 106 to 4.04 x 107 AuNPs
per silica gel particle. Additionally, because sensing is conducted in a solvent system for
this type of sensing platform, the sensitivity can be further improved from a signal-to-
noise (S/N) ratio of 5.8 in ethanol by selecting a solvent with a refiactive index closer to
that of silica in order to reduce scattering. A S/N of 42 was achieved in DMSO.

Another sensor was created based on the same detection chemistry but where the
ZP-modified AuNPs were covalently bound to a planar quartz substrate. The detection
methodology for these also utilized optical detection based on UV-visible spectrometry.
For this sensor platform, the influence of different quartz-AuNP linkers was explored and
it was found through scanning electron microscopy that 3-
mercaptopropyltrimethoxysilane (MPTMS) was the quartz modifier which created the
surface most uniformly covered with AuNPs when compared to 3-
aminopropyltriethoxysilane and 3-aminoproplydimethylethoxysilane. Sensors created

with all three linkers demonstrated the characteristic blue-shift associated with OPP

155

binding to the ZP-modified nanoparticles, but MPTMS was used due to the relative visual
and microscopic uniformity of the sensors. Sensitivity studies conducted on this sensor
geometry, again using DECP as the analyte, show that the sensor is capable of detection
of DECP at concentrations 2 5 x 10‘5 M. Below this concentration, no characteristic band
shift if observed which occurs at a level greater than the uncertainty provided by the
instrument. The behavior of this system is consistent with the behavior observed in the
case of the silica gel based sensor. While the current sensor provides a simple and robust
qualitative method of detecting OPPs above the limit of detection, and has potential for
use as a presumptive test for OPP presence, improvements are still needed to decrease the
limit of detection. This may be achieved by improving the surface coverage of AuNPs on
the quartz surface or by immersion of the substrate in an index of refraction matching
solvent in order to decrease scattering associated with the quartz substrate. Also, it would
be important to further study the response of these sensors to DECP more complex
matrices such as water or soil extracts. It would also be important to determine the
sensor response to other analytes including structurally similar but non-OPP compounds
to determine any possible interferences which may also result in a spectral blue-shift.

The three areas of study presented here encompass three areas of interest in AuNP
research; formation, characterization, and application. The formation and stabilization of
AuNPs thorough reduction of HAuC14 by amines has the potential to be useful in the
preparation of bio-compatible AuNP materials. This could have significant impact in the
use of AuNPs in therapeutics. The preparation of polymeric-amine-AuNP composites,
such as the PAH-AuNP described, could be important in the development of stable AuNP

preparations. The technique of impedance spectroscopy was described and the

156

information gained from comparison to the equivalent circuit model was utilized to
provide information about the viscoelastic properties of SAMS adsorbed onto a QCM
with Au electrodes. While applied in this instance to simple adlayers adsorbed on the
QCM, the information gained from using this technique could also be applied to the study
of AuNP composite materials to elucidate their mechanical properties. Finally, the
application of AuNPs in a sensory device was described. This sensor was based on the
surface-modification of AuNPs using ZP chemistry to create a class-selective
presumptive sensor for OPP compounds. This variety of studies indicates the breadth of

potential in the area of AuNP research.

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