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ELECTROCHEMICAL IMPEDANCE SPECTROSCOPIC ANALYSIS OF
BINDING INTERACTIONS BETWEEN HAPTENS AND POLY L-ARGININE
AND POLY L-LYSINE
MOHAMMAD SOLTANI
A thesis submitted in fulfillment of the
requirements for the award of the degree of
Master of Engineering (Bioprocess)
Faculty of Chemical Engineering
Universiti Teknologi Malaysia
MARCH 2013
iv
ACKNOWLEDGEMENT
In preparing this thesis, I was in contact with many people, researchers,
academicians, and practitioners. They have contributed towards my understanding
and thoughts. In particular, I wish to express my sincere appreciation to my main
thesis supervisor, Professor Dr. Azila Abd Aziz, for encouragement, guidance, critics
and friendship. Without her continued support and interest, this thesis would not
have been the same as presented here.
I am also indebted to Universiti Teknologi Malaysia (UTM) for funding my
Master course study. My sincere appreciation also extends to all my colleagues and
others who have provided assistance at various occasions. Their views and tips are
useful indeed. Unfortunately, it is not possible to list all of them in this limited space.
I am grateful to all my family members.
v
ABSTRACT
Fabrication of an 11-mercaptoundecanoic acid (MUA) self-assembled
monolayer (SAM) modified gold electrode in-situ functionalized with
poly L-Arginine (pArg) and poly L-Lysine (pLys) is presented and described. The
fabricated electrode was used for highly selective and sensitive accumulation and the
determination of hapten molecules (Glycerol, Isoeugenol and Maleic anhydride) in a
nanomolar concentration. Techniques like cyclic voltammetry (CV) and
electrochemical impedance spectroscopy (EIS) with an external redox probe
(FeCN6 (3-/4-)) were used to investigate the layer-by-layer self assembly modification
on a gold electrode, monolayer structure and the ion permeation through it. For CV,
Results indicated considerable decrease in current due to immobilizations of amino
acid monolayers. In addition, increase in Charge Transfer Resistance (Rct) as main
EIS output and Constant Phase Element (CPE) illustrated the quality and the
accuracy of Layer-by-Layer (LBL) assembly of SAM monolayers on the surface of
gold. EIS study showed the stable readings of Rct relating to pArg and pLys
attachment, which was obtained after at least 20 min of immobilization indicating the
minimum stability time for amino acid monolayer formation on the gold. In terms of
haptenation, increase in Rct showed the direct relation of surface resistance and the
degree of hapten sensitization (from weak to strong sensitizers). Eventually,
increases in Rct values after attachment of poly l-Arginine SAM monolayer (three vs.
two monolayers) illustrated more resistance on the surface of the electrode and more
haptenation between probes and haptens. This study can be further used on the
development of impedance-based biosensor especially for skin sensitization studies
in cosmetic products.
vi
ABSTRAK
Rekaan sebuah 11-mercaptoundecanoic acid (MUA) self-assembled
monolayer (SAM) modified gold in-situ elektrod yang berfungsi bersama poly L-
Arginine (pArg) dan poly L-Lysine (pLys) telah dibentang dan dihuraikan. Rekaan
elekrod ini digunakan untuk pemilihan yang tinggi dan pengumpulan secara sensitif
dan penentuan molekul hapten (Glycerol, Isoeugenol dan Maleic anhydride) didalam
sebuah kepekatan nanomolar. Kaedah seperti cyclic voltammetry (CV) dan
electrochemical impedance spectroscopy (EIS) dengan external redox probe
(FeCN6
(3-/4-)
) telah digunakan bagi menyelidik setiap lapisan modifikasi self-
assembly pada elektrod emas, struktur monolayer dan penelapan ion menerusinya.
Bagi CV, hasil menunjukkan penurunan arus disebabkan oleh penetapan monolayer
asid amino. Disamping itu, peningkatan Charge Transfer Resistance (Rct) sebagai
output utama EIS dan Constant Phase Element (CPE) menggambarkan kualiti dan
ketepatan penyusunan monolayer SAM secara Layer-by-Layer (LBL) di atas
permukaan emas. Kajian EIS menunjukkan bacaan Rct yang stabil, berkait dengan
penambahan pArg dan pLys, yang mana diperoleh selepas sekurang-kurangnya 20
minit penetapan menunjukkan masa kestabilan minimum bagi pembentukan
monolayer asid amino pada emas. Dalam terma haptenation, peningkatan Rct
menunjukkan hubungan terus rintangan permukaan dan darjah pemekaan hapten
(dari pemekaan lemah hingga kuat). Seterusnya, peningkatan pada nilai Rct selepas
penambahan poly l-Arginine SAM monolayer (tiga vs. dua monolayer)
menggambarkan lebih rintangan pada permukaan elekrod dan lebih haptenation di
antara prob dan haptens. Hasil kajian ini boleh diteruskan dalam pembangunan
impedance-based biosensor terutamanya bagi kajian pemekaan kulit pada produk
kosmetik.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF SYMBOLS xviii
1 INTRODUCTION 1
1.1 Background of Study 1
1.2 What is the haptenation? 2
1.2.1 How does haptenation occur and change the
impedance of the reaction?
3
1.3 Electrochemical Impedance Spectroscopy (EIS)
method using Self-assembled monolayers (SAMs)
4
1.4 Problem Statements 5
1.5 Objectives 6
1.6 Scopes of Research 6
2 LITERATURE REVIEW 7
2.1 Skin sensitization (haptenation) mechanism 7
viii
2.1.1 Concepts and Equations 10
2.2 Faradaic vs. non-Faradiac 13
2.3 What causes an impedance change (on faradaic
sensors)?
13
2.4 Data Fitting 14
2.5 Selectivity 15
2.6 What does limit the impedance biosensor
performance?
15
2.7 Impedance biosensors for Pathogen detection 16
2.8 Optical biosensors for skin sensitization 19
2.9 Detection of Glucose using impedance biosensor 19
2.10 Impedance biosensors for ethanol 20
2.11 DNA detection using impedance-based biosensors 20
2.12 Organic Coating methods used on SPR and
Impedance Biosensors
22
3 RESEARCH METHODOLOGY 65
3.1 Process Flowchart 65
3.2 Materials 66
3.3 Instrumentation 66
3.4 Buffer Preparation 67
3.5 Cleaning Method 68
3.6 Surface Modification Process 68
3.7 Immobilization Method (Electrostatic Adsorption) 68
3.8 Sample preparation 70
3.9 Cyclic Voltammetry (CV) Measurement 70
3.10 Electrochemical Impedance Spectroscopy (EIS)
Measurement
70
4 RESULTS AND DISCUSSION 71
4.1 Introduction 71
4.2 Characterization 72
4.2.1 Cyclic Voltammetry (CV) 72
ix
4.2.2 Electrochemical Impedance Spectroscopy 75
4.3 Haptenation Analysis using Electrochemical
Impedance Spectroscopy (EIS)
95
4.3.1 Circuit Model 95
4.3.2 Analysis of results of two vs. three
immobilized SAM monolayers in
haptenation with haptens
97
4.3.3 Comparison of haptenation between
immobilized amino acid monolayers
and Glycerol (weak) and Maleic anhydride
(Strong) haptens
113
CONCLUSION 117
REFERENCES 119
x
LIST OF TABLES
TABLE NO. TITLE PAGE
4.1 Oxidation and Reduction points extracted using
GPES software
74
4.2 Au-MUA-pArg Immobilization Data 91
4.3 Au-MUA-pArg-pLys Immobilization Data 91
4.4 Results of Haptenation between amino acids and Glycerol (weak), (Buffer: 5 mM FeCN6 in Na2SO4 0.1 M) (Mean ± SD)
109
4.5 Results of Haptenation between amino acids and Isoeugenol (moderate) (Mean ± SD)
110
4.6 Results of Haptenation between amino acids and Maleic anhydride (strong), (Buffer: 5 mM FeCN6 in Na2SO4 0.1 M) (Mean ± SD)
111
xi
LIST OF FIGURES
FIGURE NO. TITLE PAGE
2.1 Mechanism of skin sensitization and related
reactions in different stages
8
2.2 Gold electrode (Au) / 11-mercaptoundecanoic acid
(MUA) / Poly l-Lysine (PL)
23
2.3 PM-FTIR spectra (a) modified gold with MUA
(b) Coated Au-MUA with PL and (c) Differences
between (a) and (b) as (c)
25
2.4 The various spectrum of the PM-FTIR between the
MUA-PL and MUA-PL-FePc on the gold
electrode
25
2.5 SPR reflectivity curves for bare gold electrode
(Au) / Au-MUA / Au-MUA-FePc self-assembled
monolayer
26
2.6 MUA-PL + FePc Scheme shows the feasibility of
the robust connection
26
2.7 Diagram of specific adsorption of avidin onto
modified gold electrode with Bi-PL bilayers
28
2.8 SPR reflectivity curves for bare gold surface and
after modification with MUA, Bi-PL and avidin
29
2.9 SPR angle shifts and calculated layer thicknesses 30
2.10 SPR image observed for photo-patterned film on
the Gold electrode surface
30
xii
2.11 Reaction of the (Au) with MUA, (Au-MUA) +
NH3/PL
32
2.12 PMFT-IRRAS diagram shows the slight
conversion of MUA into amides
33
2.13 PM-FTIRRAS spectra of NHSS ester layers 35
2.14 PMFT-IRRAS of PL layers for (a) covalently
attachment And (b) electrostatically adsorbed to
the modified Au-MUA
36
2.15 Reaction of malemide attachment onto PL-coated
gold Surface
36
2.16 Structures of MUA, UDT and MUD 37
2.17 FT-IRRAS spectra (Gold substrate) in 2500-3500
1/cm
39
2.18 FT-IRRAS spectra (Gold substrate) in 1250-22500
1/cm
39
2.19 Spectrum of UDT before and after exposure of
Co-PL
41
2.20 FT-IRRAS Spectra of adsorption of pArg (solid
line) Onto carboxylic acid-terminated MUA gold
surfaces
46
2.21 Au + MUA + pArg 47
2.22 FT-IRRAS Spectra of the LBL assembly of
(pTyr/pArg) Multilayer film
47
2.23 FT-IRRAS Spectra that show reaction of
MUA/pArg with α-dicarbonyl surfaces
48
2.24 FT-IRRAS Spectra showed the reaction of
MUA/pArg or MPSA/pArg with different
reactants (pSS, APG)
48
2.25 FT-IRRAS Spectra of photoimmobilization of
various Proteins onto pArg-coated slides
49
2.26 Chemical structures of pSS, pHis 54
2.27 Different conditions of diffusion through the
surface (Open spots/capillaries)
55
xiii
2.28 FT-IRRAS spectra of amino acids linked with pSS
attached On cysteamine modified gold electrode
55
2.29 CV diagram of [Fe(CN)6] (3-/4-) (0.5 M in 0.1 M
Na2SO4
56
2.30 Nyquist diagrams based on above figures
illustrated Diffusion impedances
56
2.31 Impedance spectra of pHis/pSS 57
2.32 SAM assembly layer-by-layer onto gold electrode
and Copper II ion adsorption on modified gold
electrode
61
2.33 CV diagrams for different adsorption of SAM
Monolayers and copper II ions
62
2.34 CV diagrams for different adsorption of SAM
Monolayers and copper II ions
62
2.35 Complex plots gained for faradaic impedance
Measurements
63
2.36 Response of Au modified electrode (Au-CA-GA-
Lys) as purpose of accumulation
63
2.37 Response of Au modified electrode (Au-CA-GA-
Lys) As purpose of pH
64
2.38 DPV diagram gained on Au-CA-GA-Lys modified
Electrode as function of copper II ion
concentration
64
3.1 General view of the experiment presented in eight
steps
65
3.2 Layer-By-Layer (LBL) assemblies of Au-MUA-
pArg-pLys
69
4.1 CV plots of various attached SAM monolayers on
the Surface of the gold disk electrode
73
4.2 Nyquist plot for bare gold electrode (Au) 76
4.3 Bode plot for bare gold electrode (Au) 76
4.4 Nyquist plot for MUA modified gold electrode
(Au-MUA)
77
xiv
4.5 Bode plot for MUA modified gold electrode (Au-
MUA)
77
4.6 Nyquist plot of Au-MUA-pArg (pArg attachment)
After 20 min immersion
78
4.7 Bode plot of Au-MUA-pArg (pArg attachment)
after 20 min immersion
79
4.8 Nyquist plot of Au-MUA-pArg (pArg attachment)
after 1 hour immersion
80
4.9 Bode plot of Au-MUA-pArg (pArg attachment)
after 1 hour immersion
80
4.10 Nyquist plot of Au-MUA-pArg (pArg attachment)
after 3 hours immersion
81
4.11 Bode plot of Au-MUA-pArg (pArg attachment)
after 3 hours immersion
81
4.12 Nyquist plot of Au-MUA-pArg (pArg attachment)
after 16 hours immersion
82
4.13 Bode plot of Au-MUA-pArg (pArg attachment)
after 16 hours immersion
82
4.14 Nyquist plot of Au-MUA-pArg-pLys (pLys
attachment) After 20 min immersion
84
4.15 Bode plot of Au-MUA-pArg-pLys (pLys
attachment) After 20 min immersion
84
4.16 Nyquist plot of Au-MUA-pArg-pLys (pLys
attachment) After 1 hour immersion
85
4.17 Bode plot of Au-MUA-pArg-pLys (pLys
attachment) After 1 hour immersion
86
4.18 Nyquist plot of Au-MUA-pArg-pLys (pLys
attachment) After 3 hours immersion
87
4.19 Bode plot of Au-MUA-pArg-pLys (pLys
attachment) After 3 hours immersion
87
4.20 Nyquist plot of Au-MUA-pArg-pLys (pLys
attachment) After 16 hours immersion
88
4.21 Bode plot of Au-MUA-pArg-pLys (pLys 88
xv
attachment) After 16 hours immersion
4.22 Collapsed electrochemical impedance
spectroscopy plots of poly l-Arginine (pArg)
immobilization onto MUA-modified gold
electrode (20min, 1h, 3h and 16h of immersion
time)
89
4.23 Collapsed electrochemical impedance
spectroscopy plots of poly l-Lysine (pLys)
immobilization onto MUA-pArg modified gold
electrode (20min, 1h, 3h and 16h of immersion
time)
90
4.24 Overall trend of Resistance of Charge Transfer
Resistance, (Rct) and Constant Phase Element
(CPE) parameters on immobilization of pArg
92
4.25
Overall trend of Charge Transfer Resistance, (Rct)
and Constant Phase Element (CPE) parameters on
immobilization of pArg-pLys
93
4.26 Collapsed Nyquist plots of different
immobilization steps onto gold electrode
93
4.27 CPE Model used for Electrochemical
Impedance Spectroscopy (EIS) experiment
97
4.28 Haptenation of three different haptens (Glycerol
(1), Isoeugenol (2) and Maleic anhydride (3))
with Au-MUA-pArg-pLys electrode
98
4.29 Nyquist plot of immersion of Au-MUA-pLys in 40
mM Of Glycerol
99
4.30 Bode plot of immersion of Au-MUA-pLys in 40
mM Of Glycerol
99
4.31 Nyquist plot of immersion of Au-MUA-pLys in 40
mM Of Isoeugenol
100
4.32 Bode plot of immersion of Au-MUA-pLys in 40
mM of Isoeugenol
100
xvi
4.33 Nyquist plot of immersion of Au-MUA-pLys in 40
mM Of Maleic anhydride
101
4.34 Bode plot of immersion of Au-MUA-pLys in 40
mM Of Maleic anhydride
102
4.35 Nyquist plot of immersion of Au-MUA-pArg-
pLys In 40 mM of Glycerol
103
4.36 Bode plot of immersion of Au-MUA-pArg-pLys
In 40 mM of Glycerol
103
4.37 Nyquist plot of immersion of Au-MUA-pArg-
pLys In 40 mM of Isoeugenol
104
4.38 Bode plot of immersion of Au-MUA-pArg-pLys
In 40 mM of Isoeugenol
105
4.39 Nyquist plot of immersion of Au-MUA-pArg-
pLys In 40 mM of Maleic anhydride
106
4.40 Bode plot of immersion of Au-MUA-pArg-pLys
In 40 mM of Maleic anhydride
106
4.41 Impedance spectrums of haptenation between
pArg and pLys monolayers and Glycerol
107
4.42 Impedance spectrums of haptenation between
pArg and pLys monolayers and Isoeugenol
108
4.43 Impedance spectrums of haptenation between
pArg and pLys monolayers and Maleic
anhydride
108
4.44 Rct and CPE trends of haptenation between Au-
MUA-pLys/Au-MUA-pArg-pLys with Glycerol
(weak sensitizer)
112
4.45 Rct and CPE trends of haptenation between Au-
MUA-pLys/Au-MUA-pArg-pLys with
Isoeugenol (moderate sensitizer)
112
4.46 Rct and CPE trends of haptenation between Au-
MUA-pLys/Au-MUA-pArg-pLys with Maleic
anhydride (strong sensitizer)
113
4.47 Rct results of haptenation interactions between 114
xvii
Au-MUA-pLys and Glycerol (weak sensitizer)
and Maleic anhydride (strong sensitizer)
4.48 Rct results of haptenation interactions between
Au-MUA-pArg-pLys and Glycerol (weak
sensitizer) and Maleic anhydride (strong
sensitizer)
114
4.49 CPE results of haptenation interactions between
Au-MUA-pLys and Glycerol (weak sensitizer)
and Maleic anhydride (strong sensitizer)
115
4.50 CPE results of haptenation interactions between
Au-MUA-pArg-pLys and Glycerol (weak
sensitizer) and Maleic anhydride (strong
sensitizer)
115
xviii
LIST OF SYMBOLS
ɠ - Nernst diffusion layer thickness
Б - Warburg Coefficient
A - surface area of the electrode
AC - Alternating Current
ACD - Allergic Contact Dermatitis
APG - ρ-azidophenyl glyoxal
Bi-PL - biotinylatyed-polylysine
CA - Cysteamine
Cf - Capacity
CMM - Capillary membrane model
CPE - Constant Phase Element
CV - Cyclic Voltammetry
DC - Direct Current
DMSO - Dimethyl sulfoxide
DNA - deoxyribonucleic acid
Do - diffusion coefficient of oxidant
DPV - Differential pulse voltammetry
Dr - diffusion coefficient of reductant
E.coli - Escherichia coli
ECIS - Electric Cell-based Impedance Change method
EDC - 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide
xix
EDTA - Ethylediaminetetraacetic acid
EIS - Electrochemical Impedance Spectroscopy
EtOH - Ethanol
FRA - Frequency Response Analyzer
FT-IRRAS - Fourier Transform Infrared Spectroscopy
GA - Glutaraldehyde
GGR - D-glucose/galactose
Gly - Glycine
GPES - General Purpose Electrochemical System
HIgG - High-G Solution
HMM - Homogeneous membrane model
IME - Interdigitated microelectrodes
ImT - Immersion Time
L.B - Lennox broth
LBL - Layer-by-Layer
MCT - Mercury cadmium telluride
MPA - 3-mercaptopropionic acid
MPSA - 3-mercaptopropanesulfonic acid sodium salt
MUA - 11-mercaptoundecanoic acid
MUD - 11-mercaptoundecanol
n - number of electrons
NHSS - N-hydroxysulfosuccinimide ester
NY - New York city
pArg - poly l-Arginine
PBQ - p-benzoquinone
PBS - Phosphate Buffer Saline
PCB - printed circuit board
PEM - polyelectrolyte multilayer modified
xx
pHis - poly l-Histidine
pLys/PL - poly l-Lysine
PM-FTIR - Polarization-modulation Fourier transform infrared
spectroscopy
pSS - poly sodium 4-styrenesulfonate
pTyr - poly l-tyrosine
Ra - Resistance
Rct - Charge Transfer Resistance
Rp - Polarization Resistance
Rs - Solution Resistance
SAM - self-assembled monolayer
SPR - Surface Plasmon Resonance
TEA - Thriethanolamine Buffer
UDT - 1-undecanethiol
UV - Ultraviolet
W - radial frequency
WI / Zw - Warburg Impedance
XPS - X-ray photoelectron spectroscopy
Yo - admittance
Zd - Diffusion impedance
CHAPTER 1
INTRODUCTION
1.1 Background of Study
Cosmetic product is one of the most demandable products among people
around the world. Products such as moisturizers, shampoos, deodorants, make-up,
colognes, and other cosmetics have become part of daily grooming habits. The
American Academy of Dermatology reports the average adult uses at least seven
different cosmetic products each day. Although cosmetics can help us feel more
beautiful, they can cause skin irritation or allergic reactions. Allergic Contact
Dermatitis (ACD) is the clinical result of skin contact with chemicals to which an
individual is sensitized (e.g. Eczema, burning, stinging or itching without visible skin
lesions). This occurs in people who are allergic to a specific ingredient or ingredients
in a product. In some cases, the skin becomes red and raw. The face, lips, eyes, ears,
and neck are the most common sites for cosmetic allergies, although reactions may
appear anywhere on the body. Allergic contact dermatitis occurs because the body's
immune system is reacting against a specific substance (haptens) that it considers
foreign and harmful. ACD is an important occupational and consumer health
problem.
An allergic reaction (happens because of haptenation) is the body's way of
responding to an "invader." When the body senses a foreign substance, called
2
an antigen, the immune system is triggered. The immune system normally protects
the body from harmful agents such as bacteria and toxins. Its overreaction to a
harmless substance (an allergen) is called a hypersensitivity reaction, or allergic
reaction. Certain ingredients used in cosmetics, such as fragrances and preservatives,
can act as allergens, substances that trigger an allergic reaction. Reactions may occur
in one spot, such as a small-localized skin rash, itchy eyes, face bumps, or all over, as
in a whole body rash or hives (urticaria). Allergic reactions are unique for each
person. Reaction time to allergens can vary widely. Some people will have an
allergic reaction immediately, for others it will take time to develop. Serious
cosmetic allergies are rare. However, it is not uncommon for a person to have a mild
reaction or irritation to an ingredient in a cosmetic product. Studies suggest that up to
10% of the population will have some type of reaction to a cosmetic over the course
of a lifetime. Reactions to cosmetics occur more often in women, most likely because
women tend to use more cosmetic products than men do. Thus, checking the
products’ sensitivity before distribution to the market is required. Some strategies are
based on dendritic cells for detecting sensitizing potential of chemicals and some
others are based on electrophilic assays. The reaction of nucleophiles with some
chemicals inside the product can be useful for detection of the allergens (Xiaobo Yu
2006).
1.2 What is the haptenation?
The term, “hapten,” is derived from the Greek “hapten”, meaning “to fasten.”
Haptens are low molecular weight (LMW; <1000 daltons) chemicals that must bind
to a carrier molecule to be antigenic. The carrier is usually an endogenous or
exogenous protein to which the LMW chemical is covalently bound. The hapten
hypothesis was originally proposed to explain both humoral and cellular immune
responses to LMW chemicals observed by Landsteiner and Jacobs in their research
(Landsteiner and Jacobs 1935). The absolute requirement for covalent binding of a
hapten to a protein for immune recognition in the development of all drug/LMW
(haptens) chemical allergies has been challenged in recent years, but substantial
3
evidence exists for this to be a prominent mechanism through which chemicals and
drugs or their metabolites become antigenic.
The role of chemical reactivity has been proposed to be one of the major
determinants in allergic contact dermatitis (ACD). In the context of occupational
health, predictive toxicology, and ensuring overall safety of manufactured products,
it is important that skin sensitization potential of new and existing chemicals be
assessed. The use of guinea pigs has been the experimental model of choice in
evaluating the skin sensitization potential of chemicals until about a decade ago
when the local lymph node assay (LLNA) was adopted after extensive
interlaboratory validation. Selective protein targets or sites on a protein may be
important and recent advances in protein mass spectrometric analysis now provide
the capability to better explore how and where such chemicals bind.
1.2.1 How does haptenation occur and change the impedance of the reaction?
The hapten hypothesis was developed from the interaction of nucleophilic
moieties on proteins with chemicals that are electrophilic. Approximately 40% of the
skin sensitizers have at least an electrophilic center that is amenable to nucleophilic
attack. From an organic chemistry perspective, formation of such adducts is via
covalent bonds and to a certain extent coordination bonds. This is chiefly because
covalent and coordination bonds have bond energies ranging from 200 to 420 kJ/mol
compared to hydrophobic, dipolar, and ionic interactions with bond energies <50
kJ/mol. The high bond energies enable covalent adducts to survive the intracellular
antigen processing of the haptenated protein into short peptides for cell surface
expression by major histocompatibility complexes (MHC). The hypothesis is the
haptenation of poly l-Arginine and poly l-Lysine residues with selected haptens due
to electrophile-nucleophile interactions. Based on the electrochemistry principles,
any interaction between components within solution on electrochemical condition
causes the change on the electrochemical equilibrium. Any modification on the
4
equilibrium changes the impedance property of the reaction and related parameters
such as Charge Transfer Resistance (Rct). Thus, it can be one of the useful techniques
to detect haptenation within electrochemical condition. In the following research,
haptenation within the skin is discussed, as most of the research knowledge gained
has been through examining the relationship between chemical reactivity and
allergenicity in this organ system.
1.3 Electrochemical Impedance Spectroscopy (EIS) method using Self-
assembled monolayers (SAMs)
What is the impedance? “Electrical impedance is defined as the ratio of an
incremental change in voltage to the resulting change in current.” (Jonathan S.
Daniels 2007). Electrochemical devices can be modified based on usage and
accuracy for the assessments. The main idea is to detect some changes based on
electrochemical properties inside the solution (Barreira and Silva 2003, Barreira et al.
2004). Electrochemical based impedance sensing coupled with self-assembled
monolayers (SAM) is quite advantageous due to possibility of label free and simple
operations. In this research work, poly l-arginine and poly l-lysine self-assembled
monolayer (SAM) modified gold electrode was used to detect binding between
haptens and poly l-arginine and poly l-lysine through impedance analysis (Barreira
et al. 2004). Haptenation of the haptens with poly l-Arginine or poly l-Lysine alters
the impedance property and electrochemical equilibrium and leads to create
complexes.
Self-assembled monolayers or SAMs is the promising term has been
introduced as unstructured way and chemical reaction-based links of integrated
molecular building blocks to produce a thickly packed monolayer, stable and highly
ordered of molecules from different environments such as gas phase or liquid phase
(solution) onto a metal structure (substrate) (Ulman 1996), (Love et al. 2005) and
(Denayer et al. 2009). High stability, multi usage (potential to use in various fields)
5
and simple process of formation on the substrates are the advantages of SAMs
method specially used in biosensors construction (Shervedani and Mozaffari 2006),
(Mozaffari et al. 2010) and (Chow and Gooding 2006), trace ion determination
(Yang et al. 2001), (Berchmans et al. 2000), (Shervedani and Mozaffari 2006),
(Shervedani et al. 2009) and (Liu et al. 1999), attachment of biocatalyst (Forzani et
al. 2000) and (Shervedani et al. 2006), charge transfer kinetics studies (Protsailo and
Fawcett 2000), drug delivery (Crisponi et al. 2010), biomolecules electronic devices
(Arya et al. 2009), resistance to corrosion (Laibinis and Whitesides 1992) and also
molecular electronics (Kitagawa et al. 2005) and (Chen et al. 1999).
Rapid response, antifouling effects, high sensitivity and even the in-situ
attachment of biological recognition elements such as enzymes are major profits of
the electrode modification using self-assembled monolayers (SAMs) technique
(Mandler and Turyan 1996), (Wink et al. 1997), (Mirsky 2002), (Postlethwaite et al.
1995), (Rahman et al. 2003), (Mohadesi and Taher 2007) and (Wang et al. 2009).
Another form of modification was introduced as mixed SAMs on the metal surface.
Surface modification by thiols, adsorption of single component and filling the holes
(defects) using shorter monolayers was used to overcome the limitations of synthesis
and testing of macrocyclic ligands with properties for selectivity of target metal ions
(Park et al. 2008).
1.4 Problem Statements
Lysine and Arginine can bind onto the surface of the alkanthiol modified gold
electrode because of the strong bonds between the gold and lysine or arginine.
However, the binding is just a physical reaction and the instability of the bindings
can be assumed. In addition, the revealing of haptens reactivity (haptenation) with
the skin proteins such as poly l-Arginine and poly l-Lysine and the possibility of
selective detection of the specific type of haptens in terms of sensitivity (weak,
6
moderate or strong) are the other concerns in this field. This study can lead to the
development of a skin sensitizer biosensor.
1.5 Objective
To assess the haptenation between poly l-Arginine and poly l-Lysine Self
Assembled Monolayers (SAMs) modified MUA-gold electrode with Glycerol
(weak), Isoeugenol (moderate) and Maleic anhydride (strong) skin sensitizers
(haptens).
1.6 Scopes of Research
1. Immobilization of 11-mercaptoundecanoic acid (MUA), poly l-Arginine
and poly l-Lysine onto gold disk electrode using self-assembled
monolayer (SAM) attachment technique.
2. Characterization of poly l-Arginine and poly l-Lysine SAMs modified
impedance biosensor using cyclic voltammetry (CV) and electrochemical
impedance spectroscopy method.
3. Haptenation analysis of binding interactions between poly l-Arginine and
poly l-Lysine and Glycerol (weak), Isoeugenol (moderate) and Maleic
anhydride (strong) skin sensitizers using Electrochemical Impedance
Spectroscopy (EIS).
REFERENCES
Achilleos, C., Tailhardat, M., Courtellemont, P., Le Varlet, B. and Dupont, D. (2008).
Investigation of surface plasmon resonance biosensor for skin sensitizers studies.
Elsevier. 308-318.
Alfonta, L., Katz, E. and Willner, I. (2000). Sensing of acetylcholine by a tricomponent-
enzyme layered electrode using faradaic impedance spectroscopy, cyclic
voltammetry, and microgravimetric quartz crystal microbalance transduction methods.
Anal. Chem. 72 927-935.
Arya, S. K., Solanki, P. R., Datta, M. and Malhotra, B. D. (2009). Recent advances in self-
assembled monolayers based biomolecular electronic devices. Biosens. Bioelectron. 24
2810-2817.
Barreira, S. V. P., García-Morales, V., Pereira, C. M., Manzanares, J. A. and Silva, F.
(2004). Electrochemical Impedance Spectroscopy of Polyelectrolyte Multilayer Modified
Electrodes. The Journal of Physical Chemistry B. 108 (46), 17973-17982.
Barreira, S. V. P. and Silva, F. (2003). Surface Modification Chemistry Based on the
Electrostatic Adsorption of Poly-l-arginine onto Alkanethiol Modified Gold Surfaces.
Langmuir. 19 (24), 10324-10331.
Berchmans, S., Arivukkodi, S. and Yegnaraman, V. (2000). Self-assembled monolayers of 2-
mercaptobenzimidazole on gold: stripping voltammetric determination of Hg(II).
Electrochem. Commun. 2 226-229.
Boukamp, B. A. (1980). Package for impedance/admittance data analysis. Solid State Ionics.
18, 19 136-140.
Boukamp, B. A. (1986). Nonlinear least squares fit procedure for analysis of immittance data
of electrochemical systems. Solid State Ionics. 20 31-44.
Chen, J., Rawlett, Z. M. A. and Tour, J. M. (1999). Large on-off ratios and negative
differential resistance in a molecular electronic device. Science. 286 1550-1552.
120
Chow, E. and Gooding, J. J. (2006). Peptide modified electrodes as electrochemical metal
ion sensors. Electroanalysis. 18 1437-1448.
Crisponi, G., Marina Nurchi, V., Fanni, D., Gerosa, C. and Nemolato, S. G. (2010). Copper-
related diseases: from chemistry to molecular pathology. Coordination Chem. Rev. 254
876-889.
Ďaďo, S. (2009). Tissue Morphology and Cell Impedance Based Biosensors for Toxicity
Testing. MEASUREMENT SCIENCE REVIEW. 9 (4), 105-108.
Daniel Berdat, A. M., Fernando Herrera, Martin A.M. Gijs (2006). DNA biosensor using
fluorescence microscopy and impedance spectroscopy. Elsevier. 53-59.
Denayer, J., Delhalle, J. and Mekhalif, Z. (2009). Comparative study of copper surface
treatment with self assembled monolayers of aliphatic thiol, dithiol and dithiocarboxylic
acid. Electroanal. Chem. 637 43-39.
Dubas, S. T. and Schlenoff, J. B. (2001). Swelling and Smoothing of Polyelectrolyte
Multilayers by Salt. Langmuir. 17 (25), 7725-7727.
Fischer, L. M., Tenje, M., Heiskanen, A. R., Masuda, N., Castillo, J., Bentien, A., Émneus,
J., Jakobsen, M. H. and Boisen, A. (2009). Gold cleaning methods for electrochemical
detection applications. Microelectronic Engineering. 86 (4–6), 1282-1285.
Flink, S., Boukamp, B. A., van den Berg, A., van Veggel, F. C. J. M. and Reinhoudt, D. N.
(1998). Electrochemical detection of electrochemically inactive cations by self-assembled
monolayers of crown ethers. Am. Chem. Soc. 120 4652-4657.
Forzani, E. S., Solis, V. M. and Calvo, E. J. (2000). Electrochemical behavior of polyphenol
oxidase immobilized in self-assembled structures layer by layer with cationic
polyallylamine. Anal. Chem. 72 5300-5307.
Frey, B. L. and Corn, R. M. (1996). Covalent attachment and derivatization of {poly(L-
lysine)} monolayers on gold surfaces as characterized by polarization-modulation {FT-
IR} spectroscopy. Analytical Chemistry. 68 (18), 3187-3193.
Frey, B. L., Jordan, C. E., Kornguth, S. and Corn, R. M. (1995). Control of the Specific
Adsorption of Proteins onto Gold Surfaces with Poly(L-lysine) Monolayers. Analytical
Chemistry. 67 (24), 4452-4457.
G Kim, M. M., B K Hahm, A Bhunia, J H Mun, and A S Om (2008). Interdigitated
microelectrode based impedance biosensor for detection of salmonella enteritidis in food
samples. IOPscience. 1-4.
121
Gamry Instruments, I. (2009). Basics of Electrochemical Impedance Spectroscopy.
Application Note. 17.
Hoare, J. P. (1984). A cyclic voltammetric study of the gold-oxygen system. Electrochem.
Soc. 131 1808-1815.
J. Wang, K. S. C., L. A. Luck, and I. I. Suni (2005). Electrochemical Impedance Biosensor
for Glucose Detection Utilizing a Periplasmic E. coli Receptor Protein. The
Electrochemical Society. 8 H61-H64.
Janek, R. P. and Fawcett, W. R. (1997). Impedance spectroscopy of self-assembled
monolayers on Au(111): evidence for complex double-layer structure in aqueous NaClO4
at the potential of zero charge. Phys. Chem. B. 101 8550-8558.
Jonathan S. Daniels, N. P. (2007). Label-Free Impedance Biosensors: Opportunities and
Challenges. NIH Public Access. 19 (12),
Jordon, C. E., Frey, B. L., Kornguth, S. and Corn, R. M. (1994). Characterization of poly-L-
lysine adsorption onto alkanethiol-modified gold surfaces with polarization-modulation
Fourier transform infrared spectroscopy and surface plasmon resonance measurements.
Langmuir. 10 (10), 3642-3648.
Jurczakowski, R., Hitz, C. and Lasia, A. (2004). Impedance of porous Au based electrodes.
Electroanal. Chem. Rev. 572 355-366.
Jurczakowski, R., Hitz, C. and Lasia, A. (2005). Impedance of porous gold electrodes in the
presence of electroactive species. Electroanal. Chem. 582 85-96.
Kitagawa, K., Morita, T. and Kimura, S. (2005). Electron transfer in metal−molecule−metal
Junction composed of self-assembled monolayers of helical peptides carrying redox-
active ferrocene units. Langmuir. 21 10624-10631.
Kropan, S. (1994). Impedance Biosensors equipped by Saccharomyces cerevisiae for ethanol
recognition. Elsevier. 1
Laibinis, P. E. and Whitesides, G. M. (1992). Self-assembled monolayers of n-
alkanethiolates on copper are barrier films that protect the metal against oxidation by air.
Am. Chem. Soc. 114 9022-9028.
Landsteiner, K. and Jacobs, J. (1935). STUDIES ON THE SENSITIZATION OF
ANIMALS WITH SIMPLE CHEMICAL COMPOUNDS. JEM. 61 (5), 643-656.
Lasia, A. and Rami, A. (1990). Kinetics of hydrogen evolution on nickel electrodes.
Electroanal. Chem. 294 123-141.
122
Liju Yang, R. B. (2008). Electrical/electrochemical impedance for rapid detection of
foodborne pathogenic bacteria. Elsevier. 135-150.
Liu, A. C., Chen, D. C., Lin, C. C., Chou, H. H. and Chen, C. H. (1999). Application of
cysteine monolayers for electrochemical determination of sub-ppb copper(II). Anal.
Chem. 71 1549.
Love, J. C., Estroff, L. A., Kriebel, J. K., Nuzzo, R. G. and Whitesides, G. M. (2005). Self-
assembled monolayers of thiolates on metals as a form of nanotechnology. Chem. Rev.
105 1103-1170.
Macdonalds, R. (1987). Impedance Spectroscopy, 1st ed. Wiley.
Mandler, D. and Turyan, I. (1996). Applications of self-assembled monolayers in
electroanalytical chemistry. Electroanalysis. 8 207-213.
Miroslav Pohanka, P. S. (2008). Electrochemical biosensors – principles and applications.
APPLIED BIOMEDICINE. 8.
Mirsky, V. M. (2002). New electroanalytical applications of self-assembled monolayers.
TrAC Trends Anal. Chem. 21 439-450.
Mohadesi, A. and Taher, M. A. (2007). Voltammetric determination of Cu(II) in natural
waters and human hair at a meso-2,3-dimercaptosuccinic acid self-assembled gold
electrode. Talanta. 72 95-100.
Mozaffari, S. A., Chang, T. and Park, S. M. (2010). Self-assembled monolayer as a pre-
concentrating receptor for selective serotonin sensing. Biosens. Bioelectron. 26 74-79.
Park, J. Y., Lee, Y. S., Kim, B. H. and Park, S. M. (2008). Label-free detection of
antibody−antigen interactions on (R)-lipo-diaza-18-crown-6 self-assembled monolayer
modified gold electrodes. Anal. Chem. 80 4986-4993.
Postlethwaite, T. A., Hutchison, J. E., Hathcock, K. W. and Murray, R. W. (1995). Optical,
electrochemical, and electrocatalytic properties of self-assembled thiol-derivatized
porphyrins on transparent gold films. Langmuir. 11 4109-4116.
Protsailo, L. V. and Fawcett, W. R. (2000). Studies of electron transfer through self-
assembled monolayers using impedance spectroscopy. Electrochim. Acta. 45 3497-3505.
R. SHABANI, S. A. M., S. W. HUSAIN AND M. SABER TEHRANI1 (2009).
SELECTIVE NANOSENSING OF COPPER (II) ION USING L-LYSINE
FUNCTIONALIZED GOLD CYSTEAMINE SELF-ASSEMBLED MONOLAYER.
Iranian Journal of Science & Technology. 33 (A4), 10.
123
Rahman, M. A., Won, M. S. and Shim, Y. B. (2003). Characterization of an EDTA bonded
conducting polymer modified electrode: its application for the simultaneous
determination of heavy metal ions. Anal. Chem. 75 1123-1129.
Shervedani, R. K., Bagherzadeh, M., Sabzyan, H. and Safari, R. (2009). One-impedance for
one-concentration impedimetry as an electrochemical method for determination of the
trace zirconium ion. Electroanal. Chem. 633 259-263.
Shervedani, R. K., Mehrjardi, A. H. and Zamiri, N. (2006). A novel method for glucose
determination based on electrochemical impedance spectroscopy using glucose oxidase
self-assembled biosensor. Bioelectrochemistry. 69 201-208.
Shervedani, R. K. and Mozaffari, S. A. (2006). Copper(II) nanosensor based on a gold
cysteamine self-assembled monolayer functionalized with salicylaldehyde. Anal. Chem.
78 4957-4963.
Shervedani, R. K. and Mozaffari, S. A. (2006). Impedimetric sensing of uranyl ion based on
phosphate functionalized cysteamine self-assembled monolayers. Anal. Chim. Acta. 562
223-228.
Smalley, J. F., Chalfant, K., Feldberg, S. W., Nahir, T. M. and Bowden, E. F. (1999). An
indirect laser-induced temperature jump determination of the surface pKa of 11-
mercaptoundecanoic acid monolayers self-assembled on gold. Phys. Chem. B. 103 1676-
1685.
T Gregory Drummond, M. G. H., Jacqueline K Braton (2003). Electrochemical DNA
sensors. nature biotechnology. 21 (10), 8.
Ulman, A. (1996). Formation and structure of self-assembled monolayers. Chem. Rev. 96
1533-1554.
Vivek Nandakumar, J. f. T. L. B., Justin Reed, Miti Shah, Douglas Cochran, Lokesh Joshi,
T.L. Alford (2008). A methodology for rapid detection of Salmonella typhimurium using
label-free electrochemical impedance spectroscopy. Elsevier. 1039-1042.
Wang, F., Liu, Q., Wu, Y. and Ye, B. (2009). Langmuir–Blodgett film of p-tert-
butylthiacalix[4]arene modified glassy carbon electrode as voltammetric sensor for the
determination of Ag+. Electroanal. Chem. 630 49-54.
Wink, T., van Zuilen, S. J., Bult, A. and van Bennekom, W. P. (1997). Self-assembled
monolayers for biosensors. Analyst. 122 43R-50R.
X. Huang, D. W. G., I. Nausieda, D. Nguyen, M.M. Domach (2009). Impedance-based
Biosensors. 7.
124
Xiao, Y., Ju, H.-X. and Chen, H. Y. (1999). Hydrogen peroxide sensor based on horseradish
peroxidase-labeled Au colloids immobilized on gold electrode surface by cysteamine
monolayer. Anal. Chim. Acta. 391 73-82.
Xiaobo Yu, D. X., Danke Xu, Renjie Lv, and Zhihong Liu (2006). An impedance biosensor
array for label-free detection of multiple antigen-antibody reactions. Frontiers in
Bioscience. 11 983-990.
Yam, C.-M., Zheng, L., Salmain, M., Pradier, C.-M., Marcus, P. and Jaouen, G. (2001).
Labelling and binding of poly-(l-lysine) to functionalised gold surfaces. Combined FT-
IRRAS and XPS characterisation. Colloids and Surfaces B: Biointerfaces. 21 (4), 317-
327.
Yang, W., Gooding, J. J. and Hibbert, D. J. B. (2001). Characterisation of gold electrodes
modified with self-assembled monolayers of L-cysteine for the adsorptive stripping
analysis of copper. Electroanal. Chem. 516 10-16.