Download pdf - ELECTROCHEMICAL IMPEDANCE SPECTROSCOPIC … · ) telah digunakan bagi menyelidik setiap lapisan modifikasi self-assembly pada elektrod emas, struktur monolayer dan penelapan ion menerusinya

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

iii

To my beloved mother and father

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


after 1 hour immersion

80


after 1 hour immersion

80


after 3 hours immersion

81



81



82



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


attachment) After 1 hour immersion

85


attachment) After 1 hour immersion

86


attachment) After 3 hours immersion

87



87



88

4.21 Bode plot of Au-MUA-pArg-pLys (pLys 88

xv


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


mM Of Isoeugenol

100


mM of Isoeugenol

100

xvi


mM Of Maleic anhydride

101


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


pLys In 40 mM of Isoeugenol

104


In 40 mM of Isoeugenol

105


pLys In 40 mM of Maleic anhydride

106


In 40 mM of Maleic anhydride

106

4.41 Impedance spectrums of haptenation between

pArg and pLys monolayers and Glycerol

107


pArg and pLys monolayers and Isoeugenol

108


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


MUA-pLys/Au-MUA-pArg-pLys with

Isoeugenol (moderate sensitizer)

112


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).

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