Electrochemical synthesis of polypyrrole nanowires and application of biosensor

Three electrode setup for electrochemical synthesis composed of working electrode WE, counter electrode CE and Reference electrode RE.. PREFACES Recently, Polypyrrole PPy is one of the

HANOI UNIVERSITY OF SCIENCE AND TECHNOLOGY INTERNATIONAL TRAINING INSTITUTE FOR MATERIALS SCIENCE

BUI DAI NHAN

SUPERVISOR : Dr MAI ANH TUAN

HANOI - 2011

ACKNOWLEDGMENTS

I would like to express my appreciation to my supervisor, Dr Mai Anh Tuan

for his guidance patience, advice and support during the course at International

Training Institute for Materials Science (ITIMS)

I would like to express my sincere gratitude to Prof Tran Trung, Hung Yen

University of Education and Technology for giving me a chance to attend master

course in ITIMS and providing me the necessary facilities for my master thesis

My very special thanks goes to my co-supervisor M.Sc Luu Manh Quynh,

Institute of Materials Science, Hanoi University of Science, for his endless guidance

Without his advice and technical support, this thesis would never been written

I wish to thank to my friend Tran Thi Trang for her friendship and cooperation,

thank to Eng Phuong Trung Dung who has helped me in doing measurements

I am indebted to the teaching ITIMS for their motivation and support,

particularly the friendly and helpful manner of ITIMS staffs will remain in my mind,

especially the members of Biosensor group in ITIMS for sharing friendly research

environment

Many thanks to my friends who have encouraged me during the time of study

Above all, I am grateful to my beloved family, especially my father who

always be with me with endless encouragement, inspiration and love

ITIMS, Hanoi, November 2011

I hereby declare that all the result in this document has been obtained and presented in

accordance with academic rules and ethical conduct I also declare that, as required by

these rules and conduct, I have fully cited and referenced all material and results that

are not original to this work

The author of this thesis

Bui Dai Nhan

CONTENTS

Acknowledgement

Reassure words

Contents

List of Abbreviation

List of Table

List of Figure

Preface

Chapter1 INTRODUCTION 1

1.1 Overview of conducting polymers 11

1.1.1 Introduction 11

1.1.2 Historical back ground of the development of conducting polymers 13

1.1.3 Mechanism of electrical conduction in CPs 15

1.1.4 Current synthesis of conducting polymers 17

1.2 Polypyrrole (PPy) and Electrochemical polymerization of PPy 18

1.2.1 Properties of Polypyrrole 18

1.2.2 Electrochemical synthesis of Polypyrrole 20

1.2.3 Effect of Synthesis conditions on Electrochemical Polymerization 26

1.3 Application of Biosensors 27

1.3.1 General Introduction to DNA sensor 28

1.3.2 Immobilization of probe DNA on polymer based electrode 34

1.4 Aim of the Study 36

Chapter 2: EXPERIMENTS 37

2.1 Electrochemical polymerization of polypyrrole 37

2.1.1 Materials 37

2.1.2 Instrumentation 37

2.1.3 Experiment procedure 40

2.2 DNA immobilization and measurement Setup 42

2.2.1 Chemicals 42

2.2.2 DNA strand immobilization 42

2.2.3 Measurement setup 44

Chapter 3: RESULTS AND DISCUSSIONS 48

3.1 Electrochemical synthesis of PPy nanowires 48

3.1.1 Electroactivities of Polypyrrole 48

3.1.2 Effects of parameters on electrochemical polymerization of polypyrrole 51

3.1.3 Chemical composition and functional groups of obtained PPy nanowires 60

3.2 DNA sensors characteristics 70

3.2.1 Characteristics of DNA sensor is a function of time 70

3.2.2 Hybridization detection using DNA sensor 71

3.2.3 The reproducibility of DNA sensor 73

CONCLUSION 76

RECOMMENDATIONS Error! Bookmark not defined REFERENCES 78

LIST OF ABBREVATION CPs Conducting Polymers

DNA Deoxyribonucleic Acid

PCR Polymerase Chains Reaction

EDC 1-Ethy-3-(3-dimethyl-aminopropyl)-carbodiimide

MIA 1-methyl-imidazole

VB Valence Band

CB Conduction Band

PBS Phosphate Buffer Solution

SEM Scanning Electron Microscopy

FT-IR Fourier Transform Infrared Spectroscopy

SERS Surface Enhanced Raman Spectroscopy

LIST OF TABLE

Table 1.1 The chronology of the development of some important conducting polymers

Table 1.2 Advantages and disadvantages of chemical and electrochemical synthesis of

conducting polymers

Table 1.3 Advantages and Disadvantages of Chemical and Electrochemical synthesis

of PPy

Table 1.4 History of biosensor development

Table 2.1 DNA sequence used in this work

Table 3.1 Current density (mA/cm 2 ) vs added volume of pyrrole monomer (mL)

Table 3.2 Current density (mA/cm 2 ) vs different concentration of gelatin (%wt)

Table 3.3 Current density (mA/cm 2 ) vs Reaction time (second)

Table 3.4 Absoption peaks in FT-IR spectrum

Table 3.5 Comparison between SERS peaks in this work and and those in literature

LIST OF FIGURE

Figure 1.1 Conductivity of conducting polymer compared with other materials

Figure 1.2 Three typical types of conducting polymer

Figure 1.3 Band theory and doping-induced structural transitions of polypyrrole

Figure 1.4 Three steps of polymerization process of Polypyrrole

Figure 1.5 Aromatic and Quinoid structrure of PPy

Figure 1.6 Three electrode setup for electrochemical synthesis composed of working

electrode (WE), counter electrode (CE) and Reference electrode (RE)

Figure 1.7 Cyclic voltammogram of PPy nanowires and cauliflower-like in KCl

solution at scan rate of 25 mV/s

Figure 1.8 Potentiostat curve of the synthesis of PPy on Nikel electrode and ITO

electrode

Figure 1.9 A typical structure unit of gelatin polypeptide

Figure 1.10 The schematic of a biosensor

Figure 1.11 General DNA sensor design based on CPs

Figure 1.12 The principle of DNA sensor

Figure 1.13 The total biosensors market showing the world revenue forecast for

2009–2016

Figure 1.14 Four base types of DNA

Figure 1.15 Hydrogen bonds between the A-T and G-C bases of the two trands of

DNA

Figure 2.1 Schematic of electrochemical synthesis system of polypyrrole

Figure 2.2 Covalent immobilization between PPy films and phosphate DNA on Pt

micro-electrode using EDC, MIA catalysts

Figure 2.3 Differential measurement using Lock-in Amplifier

Figure 2.4 The wave form of the Lock-in Amplifier SR830

Figure 2.5 Equivalent electrical circuit of differential system

Figure 3.1 Cyclic voltammogram of Ppy between -1.0 V and +1.0 V at 250 mV/s scan

rate

Figure 3.2 Potentiostatic polymerization curve for the electrodeposition of

Polypyrrole

Figure 3.3 Potentiostatic curves for the electrodeposition of Polypyrrole from 0.1M

LiClO 4 electrolyte at different conditions

Figure 3.4 The saturated current density of the electrochemical curve vs.pyrrole

monomer concentration.

Figure 3.5 SEM images of Ppy structrures synthesized at different added volume of

pyrrole

Figure 3.6 The current density recorded vs different concentration of gelatin

Figure 3.7 SEM images of Ppy structures potentiostatically synthesized at different of

gelatin concentration

Figure 3.8 The current density recorded vs different sweeping (reaction time)

Figure 3.9 SEM images of Ppy structures potentiostatically synthesized at different

reaction time

Fig 3.10 Morphologies of PPy nanowires prepared at optimized condition

Figure 3.11 FI-IR spectra of obtained Ppy nanowires

Fig 3.12 Path of the stylus over the sample in the measurement of Pt thickness

Figure 3.13 The thickness of Platinum film

Fig 3.14 Distribution of PPy nanowires over Pt surface electrode

Figure 3.15 Surface Enhanced Raman Spectroscope of polypyrrole film deposited on

Platinum surface

Figure 3.16 Response time and Reaction time of the DNA sensor

Figure 3.17 The curve of DNA sequence hybridization, C DNA probe =0.05M, T=300 0 K

Figure 3.18 The reproducibility of DNA sensor

PREFACES

Recently, Polypyrrole (PPy) is one of the most extensively used conducting

polymers in biosensor designs due to its good biocompatibility and polymerization at

neutral pH [30]

The electronic structure of PPy is highly sensitive to change in polymeric chain

environment and other perturbations in the chain conformation caused by, for

example, a biological recognition event such as DNA hybridization [30] The changes

in the delocalized electronic structure can provide a signal for the presence of a target

analyte molecule These advantages of conducting PPy make them suitable for

biosensors and chemical sensors which play important role in public health and

environment [18]

The drawback of DNA sensor based PPy membrane includes limited sensitivity

and reproducibility due to the low conductivity of PPy in film and cauliflower-like

form, presented in previous work (1) In this thesis, we aim at the synthesis of PPy

nanowires using electrochemical technique with the desire of obtaining better

characteristics of DNA sensor for Ecoli bacteria DNA detection

The synthesis of PPy nanowires was obtained by using potentiostat method at

0.75V, in LiClO4 0.1M (PBS, pH =7) electrolyte containing 0.5 mL pyrrole monomer

and 0.08%wt gelatin It should be noted that gelatin is used as a ‘soft template’ to

orientate the growth of PPy nanowires

The PPy nanowires 50 nm of diameter provide large and fine surface Especially,

N-H group of PPy nanowires was orientated upward from the surface which takes

advantage for DNA probe immobilization As the result, the DNA based PPy

nanowires has good characteristics for Ecoli DNA detection, including a short

response time (~10 seconds), small detection limit (0.1 nM) as well as good

reproducibility

(1)

P.D Tam et al / Materials Science and Engineering C 30 (2010) 1145–1150

The dissertation, divided into 4 chapters, reports a proper potentiostat technique

to prepare conducting polypyrrole nanowires on Pt electrode, and then initial

applications of DNA sensor using microelectrode based PPy nanowires

In chapter one, the fundamental of conducting polymers, Polypyrrole and DNA

sensor will be introduced

In chapter 2, experiments for electrochemical polymerization of PPy films and

the application of PPy based electrode for DNA sensor will be described

In chapter 3, achieved results of the thesis will be presented Different

electrochemical parameters were studied to establish the synthesis condition to obtain

PPy nanowires The electrochemical behaviors, morphologies and chemical

composition of polypyrrole nanowires obtained potentiostatically have been analyzed

and discussed Later on, the trial application of DNA sensor and the detection of target

DNA sequence of Ecoli bacteria were studied Some recommendation and perspective

are also given

Chapter 1 INTRODUCTION 1.1 Overview of conducting polymers

1.1.1 Introduction

Polymers are long chain giant organic molecules, ‘poly’ meaning ‘many’ and

‘mer’ meaning ‘part’ (in Greek) Originally, polymers are nonconductor The term

‘conducting polymer’ is used for polymers which can exhibit significant level of

electrical conductivity That property is due to the presence of ‘free electron’ within

the body of the specimen Conducting polymer are usually poly-conjugated structures

which are in the pure state but when treated with an oxidizing or a reducing agent can

be converted into polymer salts with considerably increased electrical conductivity

12 doped polyacetylen AsF5 doped polyacetylen Copper

Conducting polymer can be classified into three distinct groups:

electron-conducting polymer, ion-electron-conducting polymer, redox polymer (Fig.1.2)

Ion-exchange conducting polymer

-CF

O O

3

3 3 6

+ Na +

3 3 6

+

Solution Layer

Polyanilin e

Polyparaphenylene

Polypyrrole

Redox conducting polymer Electron conducting polymers

N H

The chemical stability of a polymer in atmospheric conditions depends on the

value of the redox potential If the reduction potential of a polymer is above the

reduction potential of oxygen (-0.146V) the polymer is naturally stable in air But the

same polymer maybe attacked by atmosphere if its oxidation potential is higher than

that of water (1.23V)

Electron-rich heterocycle based polymers such as polythiophene and

polypyrrole are very stable in the p-doped form and this has made these systems two

of the most studied conducting polymers Their stability is due to their lower polymer

oxidation potentials which follow the order of PAc>PTh>PPy [27]

Moreover, compare with polymers which have similar oxidation potential,

polypyrrole stands out as the most conductive polymer (40S/cm~2.5e-2.cm) It was

believed that polypyrrole could have more limited stability (environmental, thermal,

chemical) than conventional inert polymers due to the presence of dopant and their

dynamic and electroactive nature Therefore, polypyrrole seems to be a good

candidate for researchers now to attempt at the synthesis of conducting polymers, in

particular for biosensing application

1.1.2 Historical back ground of the development of conducting polymers

Polyaniline (PAni), known as ‘aniline black’, is one of the oldest conductive

polymers known It was first prepared by Letheby in 1862 by anodic oxidation of

aniline in sulfuric acid [22], and was used in the printing industry [13] The first

polymerization to form polyacetylene (PAc) as an insoluble and infusible powder was

reported in 1958 by Natta and coworkers [14]

The modern era of conducting polymers began at the end of 1970s when the

discovery that polyacetylene (PAc) could be synthesized to form highly conducting

doped films [16] by Alan Heeger, Alan MacDiarmid and Hideki Shirakawa (2000

Nobel Prize in Chemistry) They stated that polymer plastics can be made to conduct

electricity if alternating single and double bonds link their carbon atoms and electrons

are either removed through oxidation or introduced through reduction [8, 16]

(SN)x synthesized by Burt 1910

Metallic conductivity of (SN)x reported by Waltaka et al 1973

Semiconductivity P.A discovered by Shirakawa et al 1971

Conducting polyppara phenylene sulfide by J.E Forder 1983

Polymer surface modification, Chan et al 1993

PPy, by Tat’yanan V Vernitskaya et al 1997

Nobel prize 2000 for MacDiarmid, Heeger, Shirakawa 2000

PANi by Z.Wei, M.Wan 2002

poly (aniline-co-o-anisidine-co-o-toluidine) by Borole, Kapadi et al 2006

PPy & PTh byThapa et al, USA; P3HT by Park, Korea

Poly (3-hexylthiophene)

2007

Table 1.1.The chronology of the development of some important conducting polymers

[19]

After the first publication, there has been an explosive growth of research into

the whole range of conjugated polymer structures, table 1.1 Since then, many new

conducting polymer structures have been developed over the last three decades with

the desire of obtaining better properties than PAc Although none of them has

exhibited higher conductivity than PAc, these polymers have been useful in designing

new structures that are soluble and stable

1.1.3 Mechanism of electrical conduction in CPs

The conduction properties of conducting polymers have previously been

explained on the basics of the band theory of solids According to this theory of solids,

when a large number of atoms or molecules are brought to form a polymeric chain or a

crystalline solid, an energy band is formed through the interaction of constituent

atomic or molecular orbitals The band of highest energy that is completely filled by

electrons is generally called the valance band The electrons associated with bands are

involved in chemical bonding and are consequently rather localized and are not free to

move through the solid [19]

To explain the conduction mechanism in conducting polymers, a new model

called soliton model was introduced by MacDiarmid et al in 1983 [2] In this model,

charged solitons are a type of charge defects prepared on doping are believed which to

be the conducting species for charge transport It should be noted that this theory

agreed with PAc (because it has a degenerate ground state, two geometric structures

corresponding to the same energy) but not with all other conducting polymers having

non-degenerate ground state

The failure of soliton theory has led to a new theory called polaron and

bipolaron theory According to this concept, the polymer chain is ionized on doping

and this ionization process creates a polaron (radical ion) on the chain At low doping

level, these polarons are carriers of electricity On increasing the doping level, the

concentration of polaron increases and leads to the possibility of interaction with each

other, thus two polarons may get coupled to form a bipolaron doubly charged but

spinless

In the case of Polypyrrole, the conduction mechanism can be explained based

on the formation of polaron and bipolaron, corresponding with lightly doped PPy and

heavily doped PPy, respectively As presented in figure 1.3, doping process has strong

influence on the local Fermi level, results in the formation of polaron and bipolarons

and their energy gaps which are smaller than the gap of undoped state

Figure 1.3 Band theory and doping-induced structural transitions of

polypyrrole (a) Band theory of conjugated polymers (b) Structural changes

associated with polaron and bipolaron formation as a result of oxidative doping in

polypyrrole [27]

In neutral form, PPy is semiconducting with poor conductivity, but upon

oxidation (p-doping) or reduction (n-doping), interband transitions form between VB

and CB, lowering the effective band gap, resulting in the formation of polaron and

bipolaron as charge carriers along the PPy backbone [27] According to the band

theory, the smaller band gap obtained, the higher conductivity measured This leads to

the enhancement of conductivity of conducting PPy In figure 1.3b, dopant used is

negative particle ( , ), hence PPy becomes a p-type semiconductor The

existence of the structural changes associated with polaron and bipolaron, presenting

the level of doping (Fig 1.3a), as the result of oxidative doping in PPy

1.1.4 Current synthesis of conducting polymers

CPs can be synthesized chemically or electrochemically, which each method

has advantages and disadvantages [28] as summarized in table 1.2 Different methods

of chemical synthesis include either condensation polymerization (i.e, step growth

polymerization) or addition polymerization

Chemical

polymerization

 Larger-scale production possible

 Post-covalent modification of bulk CP possible

 More options to modify CP backbone covalently

 Cannot make thin films

 Synthesis is more complicated

Electrochemical synthesis is a common alternative for making CPs, particularly

because this synthesis procedure is relatively straightforward Although this technique

is not new, the first electrochemical preparation of CPs was found in 1968 when

‘pyrrole black’ was form as a precipitate on a platinum electrode by exposing an

aqueous solution of pyrrole and sulfuric acid to an oxidation potential [31] Over the

past forty years, electrochemical method has been used widely for synthesis of

conducting polymers [5,11,19]

1.2 Polypyrrole (PPy) and Electrochemical polymerization of PPy

1.2.1 Properties of Polypyrrole

Pyrrole was known to form a conductive ‘pyrrole black’ [15] via spontaneous

polymerization, and its history can be dated back in 1916 [1] In 1968, it was noted

that pyrrole could be electrochemically polymerized using variety of oxidation agents

to give a black conducting powder It can be synthesized in both aqueous and

non-aqueous solution during electrochemical polymerization

Among all known conducting polymers, polypyrrole (PPy) stands out as an

excellent one because of its good environmental stability, high conductivity and ease

of synthesis It is stable in a wide range of potential, during thousands of

charge-discharge cycles, and under properly selected conditions its response is fast

In contrast to polyaniline, it can operate both in acidic and neutral solutions,

which makes the polypyrrole electrode attractive for use as sensor material in the

bioelectroanalytical chemistry Furthermore, polypyrrole is relatively air stable organic

conducting polymer, which suffers from poor processability The use of new tailor

made reactive statistical copolymers for the synthesis of sterically stabilized pyrrole

colloids is described Moreover, compared to other heterocycles its oxidation potential

is low [27]

For all the reasons, polypyrrole has been an interesting material to study

Polypyrrole can be prepared in various forms depending on the method used and the

preparation conditions A general difficulty of the reproducible polypyrrole

preparation arises from its complexity

The structures and hence the properties of the resulting polypyrrole are strongly

influenced by a number of parameters that are not perfectly controlled

Polypyrrole and a wide range of its derivatives may be prepared by simple

chemical or electrochemical method shown in the table 1.3

Chemical synthesis of PPy Electrochemical synthesis of PPy

Advantages Easy to produce amounts of

PPy in various forms

Convenient carried out

Process is simply controlled (through current or applied potential)

Disadvantages Poor producibility Prepare PPy only in thin film

deposited on the surface of electrode

Table 1.3 Advantages and Disadvantages of Chemical and Electrochemical synthesis

of polypyrrole

Chemical synthesis is a simple and fast process to procedure fine powders of

PPy However, the use of chemical polymerization limits the range of PPy that can be

prepared since a limited number of counterions can be incorporated

In the last few years, the goal of researcher has been to improve physical

properties of PPy, like processibility and mechanical integrity To achieve this goal,

composites and copolymers of PPy with insulating thermoplastic were synthesized In

preparation of conducting composites, the electrochemical method is preferred because

it is easy, clean and selective

1.2.2 Electrochemical synthesis of Polypyrrole

The electrochemical polymerization has been widely used for synthesis of

polypyrrole, as presented in a number of previous studies [5,11,18,30,46]

Although most of their pyrrole units are linked at the α-α (or 2, 5) positions, a

significant number of the units are coupled through the α-β and β-β cross linkages [40]

the less desirable 3,4 or 2,3 coupling contributes to the formation of soluble oligomers

and reduces the conjugation length and lower conductivity

The mechanism of the electrochemical polymerization of pyrrole is believed to

proceed via the radical cation of the monomer [27], shown in figure 1.4

Firstly, the initial oxidation step produces a radical cation which can either react

with another radical cation to produce a dimer or undergo an electrophilic attack with a

neutral monomer The electrochemical polymerization reaction occurs only when the

applied potential is sufficient to oxidize the monomer

At the applied potentials, the coupling of two radicals is more likely because the

number of neutral species at the electrode surface will be essentially zero at these

potentials The charge consumed during polymer formation has linear time

dependence (at least initially) and is independent of pyrrole concentration If there is

no nucleophile in the system which is thought to be capable of reacting with the

radical cations, they will give a dimer cation which readily eliminates 2H+ [27]

Step 1: Initiation Reaction

N H

N H

+.

N H

N H

N H

+ + 2H

Step 3: Termination Reaction

Figure 1.4 Three steps of polymerization process of Polypyrrole

The chain growth is terminated either when the radical cation of the growing

chain becomes too unreactive or, more likely, when the reactive end of the chain

becomes sterically blocked for further reaction Polymer chain bears a charge of unity

of every three to four pyrrole rings The level of oxidation is an intrinsic characteristic

N H

N H

N H

N H

N H

N H

N H

N H

N H

of the polymer and is not sensitive to the nature of the anion However, the anion

influences not only the structural properties and the electroactivities of the films, but

also the mechanical behaviors of the films The products obtained from the

polymerization of PPy can be aromatic and quinoid type, shown in figure 1.5

N

N H

H N

H N

N

N H

Aromatic

Quinoid

Figure 1.5 Aromatic and Quinoid structure of PPy

Nowadays, electrochemical polymerization is performed using three-electrode

configuration (working, counter, and reference electrode) in a solution of monomer,

appropriate solvent, and electrolyte as seen in Fig 1.6

Figure 1.6 Three electrode setup for electrochemical synthesis composed of Working

Electrode (WE), Counter Electrode (CE) and Reference Electrode (RE)

Current is passed through the solution and electro-deposition occurs at the

positively charged working electrode or anode Monomers at the working electrode

surface undergo oxidation to form radical cations that react with other monomers or

radical cations, forming insoluble polymer chains on the electrode surface

Cyclic voltammetry (CV)

Cyclic voltammetry is one of the most useful methods, which provides us a

great deal of useful information about the electrochemical behavior of electroactive

species In this method, the potential of the working electrode to reference electrode is

scanned in the anodic and cathodic directions and the current density flow as a

function of this potential is measured

A cyclic voltammogram helps us to understand the electroactivity and redox

potential of a material, mechanism of the electrochemical reactions, reversibility of

electron transfer, whether the reaction products are futher reduced or oxidized and the

growth rate of the conducting polymers An example of cyclic voltammogram of PPy

obtained in previous work [11] is given in Fig 1.7

Figure 1.7 Cyclic voltammogram of PPy nanowires and cauliflower-like in

KCl solution at scan rate of 25 mV/s (adopted from reference [11])

Constant Potential Electrolysis (Potentiostatic mode)

This method is carried out in a three electrode cell, which ensures effective

potential control and maximize the reproducibility of the polymerization process The

potential of the working electrode with respect to a reference electrode is adjusted to a

desired value and kept constant by a potentiostat This potential may be called the

polymerization potential (Epot) and it is determined by means of cyclic voltammetry

(will be further explained in chapter 3)

Since the potential is constant during the electrolysis, unwanted electroactive

species are eliminated and the initiation proceeds only through monomer Some

potentiostat curves correspond with different electrodes are shown in Fig 1.8

Figure 1.8 Potentiostat curve of the synthesis of PPy on Nikel electrode and

ITO electrode [11]

As shown in figure 1.8, chronoamperometric curve depends on the material of

electrode (Ni, Pt, ITO,ect.) It should be noted that higher current density observed,

higher conductivity of PPy film measured

Gelatin

Recently, a new method has emerged as an alternative way for ‘hard template’

It called ‘soft template’ method, which utilizes some molecules or molecular

assemblies as ‘soft template’, involving surfactant, crystalline phase, lipid tubule, as

well as biomolecules, to orientate the growth of the nanostructures of conducting

polymer [11]

Gelatin (or gelatine) is a translucent, colorless, brittle (when dried), flavorless

solid substance, derived from the collagen inside animals' skin and bones, shown in

Fig 1.9

N

H2C C

H2

CH2

C C

C N C C N C C N C

C

O H

CH2

SH H

O H

CH2

CH2

C

NH2O H

CH2

C O

N C H NH CH

Figure 1.9 A typical structure unit of gelatin polypeptide

(Source: http://www.nanoscalereslett.com/content/6/1/22)

Gelatin contains 84-90% protein, 1-2% mineral salts, 8-15% water It is free from

additives and preservatives

In this work, gelatin is used as a ‘soft template’ with 1D linear molecules which

can self-assemble into 1D structures During the polymerization, the conducting

polymers can grow along the 1D ‘soft template’, leading to the formation of the 1D

polypyrrple nanomaterials

1.2.3 Effect of Synthesis conditions on Electrochemical Polymerization

The nature of the process occurring and final properties of the electrogenerate

polymers are affected by many parameters such as the nature and shape of the

electrodes, solvent, electrolyte, temperature, synthesis potential, cell geometry and

monomer concentration [42]

Shape of the electrode

Since the polymerization proceeds via oxidation and reduction reactions, it is

necessary that the electrode should not be oxidized currently with the aromatic

monomer For the reason, inert electrodes, such as Pt, Au and ITO are mostly used to

prepare conductive films Saturated calomel electrode (SCE), Ag/Ag+ and Ag/AgCl

electrodes can be used as the reference electrodes [42]

For synthesis of PPy, Platinum electrode takes advantage because the oxidation

potential of pyrrole is reduced and current density increased in comparison with Ti, Fe

or Al used It is because the formation of the thinner platinum-oxide which impedes

electron transfer during electropolymerization

Solvent

Solvent should be capable of dissolving monomer and counterion at appropriate

concentrations In addition, it should present a high dielectric constant to ensure the

ionic conductivity of the electrolytic medium and a good electrochemical resistance

against decomposition at potentials required to oxidize the monomer The solvents

with poor nucleophilic character should be used since more nucleophilic solvent are

likely to attack the free radical intermediates Therefore, aprotic solvents such as

acetonitrile may be used during electrochemical polymerization [42]

In this work, we chose double distillated water which can dissolve LiClO4,

gelatin and phosphate buffer However, in the addition of pyrrole monomer and

gelatin, the solution should be slightly heated and deaerated continuously at least 15

minutes with N2 to avoid the oxidation of PPy and achieve the dissolution

Electrolyte

The requirements in selecting the supporting electrolyte are, basically, the

solubility of the salt, its degree of dissociation and the reactivity of both anion and the

cation In addition to these, the counterion should be stable both chemically and

electrochemically; otherwise, breakdown products can interfere in the polymerization

process A typical electrolyte used in this research consists of LiClO4, phosphate

buffer, gelatin and pyrrole monomer

Temperature

Temperature is the other parameter that should be taken into consideration

during electropolymerization It has a substantial influence on the kinetics of

polymerization as well as on the conductivity, redox properties and mechanical

characteristics of the films At high temperatures, lower conducting films are produced

as a result of the side reactions such as solvent discharge and nucleophilic attacks on

polymeric radicals

Due to the low oxidation potential of pyrrole, our experiments were performed

at ambient temperature In addition, the electrolyte is phosphate buffer (pH=7), thus

the neutral condition can be suitable for synthesis PPy for the immobilization of DNA

probe on electrode’s surface

In this work, we do not study effect of all the parameters Three parameters

which will be studied under this work are concentration of pyrrole, gelatin and reaction

time The experiments will be further described in chapter 2

1.3 Application of Biosensors

Among conducting polymers, polypyrrole is the most frequently used in

commercial applications, such as batteries [12], supercapacitors [25], sensors [32],

anhydrous electrorheological fluids [21], microwave shielding and corrosion

protection, etc

Recently, conducting polymer-based DNA sensor have shown applicability in a

number of areas related to human health (infectious diseases, drug discovery, food,

etc.), thus it has been received everlasting interests of scientists all over the world

[30,38]

This thesis focuses on preparation of conducting polypyrrole for DNA sensor

application

1.3.1 General Introduction to DNA sensor

A biosensor is a device for the detection of an analyte that combines a biological

component with a physicochemical detector component [33]

A biosensor consists of 4 parts (illustrated in Fig 1.10):

 The substances (biological material (tissue, micro-organisms, organelles, cell

receptors, enzymes, antibodies, nucleic acids, ect.); a biologically derived

material or biomimic) The sensitive elements can be created by biological

engineering;

 The detection (works in a physicochemical way; optical, electrochemical,

thermometric, piezoelectric or magnetic);

 The transduction: transducer between (associates both components);

 The signal conditioning (amplifier) is used to enhance the signal (output) from

the initial signal of the detection

Operation principle of biosensor

As shown in figure 1.10, the specific interactions between the analyte and the

biorecognition (biological detection) element produce a physico-chemical change,

which is detected by the transducer and measured by peripheral circuits

Figure 1.10 The schematic of biosensor (source: http://basicsofbiosensing.blogspot.com/ )

The fundamental advantage of biosensors over nearly all other sensor devices is

their high selectivity which is benefited the selectivity of this bio-receptor The amount

of electronic signal generated is proportional to the concentration of the analyte,

allowing for both quantitative and qualitative measurements in time

DNA sensor is a special type of biosensor, which used DNA strands as

sensitive biological element, Fig 1.11

Figure 1.11 General DNA sensor design based on CPs [18]

(source: http:// www.europhysics.com, 2002)

DNA biosensors are integrated receptor-transducer devices that use DNA as

biomolecular recognition element to measure specific binding processes with DNA,

usually by electrical, thermal, or optical signal transduction [38]

Figure 1.12 The principle of DNA sensor

As presented in Fig 1.12, the matching between analyte (DNA target) and

recognition layer (DNA probe on surface electrode) provides us a signal transduction

of hybridization, which allow us to record electronic read-out

 History of biosensor development

1916

First report on the immobilisation of proteins: adsorption of invertase on activated charcoal

1922 First glass pH electrode

1956 Invention of the oxygen electrode (Clark)

1962 First description of a biosensor: an amperometric enzyme electrode for

glucose (Clark)

1969 First potentiometric biosensor: urease immobilised on an ammonia

electrode to detect urea

1970 Invention of the Ion-Selective Field-Effect

Transistor (ISFET) (Bergveld)

5/1972 First commercial biosensor: Yellow Springs

Instruments glucose biosensor

1975 First microbe-based biosensor; First immunosensor: ovalbumin on a

platinum wire; Invention of the pO2 / pCO2 optode

1976 First bedside artificial pancreas (Miles)

1980 First fibre optic pH sensor for in vivo blood gases (Peterson)

1982 First fibre optic-based biosensor for glucose

1983 First surface plasmon resonance (SPR) immunosensor

1984 First mediated amperometric biosensor: ferrocene used with glucose

oxidase for the detection of glucose

1987 Launch of the MediSense ExacTech™ blood glucose, Biosensor

1990 Launch of the Pharmacia BIACore SPR-based biosensor system

1992 i-STAT launches hand-held blood analyser

1996 Glucocard launched

1996 Abbott acquires MediSense for $867 million

1998 Launch of LifeScan FastTake blood glucose biosensor

1998 Merger of Roche and Boehringer Mannheim to form, Roche Diagnostics

2001 LifeScan purchases Inverness Medical's glucose testing business for

$1.3 billion

Table 1.4 History of biosensor development (Source: http://nanohub.org )

Biosensor market

The biosensors market is categorized as a growth market, with the number of

applications increasing as each new biosensor is developed

( http://www.sensorsmag.com )

Figure 1.13 The total biosensors market showing the world revenue forecast for

2009–2016 (source: http://www.sensorsmag.com )

As illustrated in figure 1.13, the global revenue for the biosensor market will

continue to exhibit strong growth and will exceed $14 billion mark in the next seven

years The grow rate is estimated up to 11.5% from 2009 to 2016

Biosensors have been developing and having considerable potential in many

applications and commerce

Introduction to DNA

To design efficient DNA-electrochemical biosensors, it is essential to know the

structure and to understand the electrochemical characteristics of DNA molecules

Deoxyribonucleic acid (DNA) is a nucleic acid which carries genetic

instructions for the biological development of all cellular forms of life and many

viruses [33] DNA is sometimes referred to as the molecule of heredity as it is

inherited and used to probate traits During the reproduction, it is replicated and

transmitted to offspring

DNA composed of 4 bases: adenine (A), thymine (T), cytosine (C) and guanine

(G) [23]

Adenin (A) Thymin (T) Guanin (G) Cytosin (C)

Figure 1.14 Four base types of DNA

DNA consists of two antiparallel polynucleotide chains formed by monomeric

nucleotide units Each nucleotide is formed by three types of chemical components: a

phosphate group, a sugar called deoxyribose, and four different nitrogen bases

Figure 1.15 Hydrogen bonds between the A-T and G-C bases of the two strands of

DNA (adopted from reference [18])

The phosphate-deoxyribose sugar polymer represents the DNA backbone

(Fig 1.15, adopted from Ref [18]) The cellular genetic information is coded by the

purine bases, adenine (A) and guanine (G), and the pyrimidine bases, cytosine (C) and

thymine (T), as a function of their consecutive order in the chain The two strands of

nucleotides are twisted into a double helix, held together by hydrogen bonds between

the A-T and G-C bases of each strand

1.3.2 Immobilization of probe DNA on polymer based electrode

To make a biosensor the biological component has to be properly attached to

the transducer This process is called immobilization There are five methods of doing

this: Adsorption, Micro encapsulation, Entrapment, Cross-linking, Covalent bonding

[18]

 Adsorption

Many substances adsorb enzymes on their surfaces Physical adsorption is weak

and involves the formation of Van der Waals bonds Chemical adsorption is stronger

and involves the formation of covalent bonds Adsorbed biomaterial is susceptible to

changes in pH, temperature, ionic strength, and the substrate It should only be used

over a short time-span

 Micro encapsulation

A semi permeable membrane is used to trap the biomaterial on the transducer

This keeps close contact between the biomaterial and the transducer Membrane

materials include cellulose acetate, polycarbonate,and polytetrafluoroethylene

(Teflon) It is stable towards changes in temperature, pH, ionic strength and chemical

composition

 Entrapment

The biomaterial is mixed with a monomer solution, which is then polymerized

to a gel, thus trapping the biomaterial This method creates large barriers which inhibit

the diffusion of the substrate This slows the reaction down and the response time of

 Cross-Linking

The biomaterial is chemically bonded to solid supports or to another supporting

material such as a gel It is useful to stabilize adsorbed biomaterials However it causes

damage to the enzyme, limits the diffusion of the substrate, and there is poor

mechanical strength

 Covalent Bonding:

Some functional groups which are not essential for the catalytic activity of an

enzyme can be covalently bonded to the transducer or membrane Advantage is that

the enzyme will not be released during use Reactions need to be performed with low

temperature, low ionic strength and pH

In order to achieve an increased life-time stability of DNA on electrode, it is

necessary that there should be a strong and an efficient bonding between the DNA

strand and the immobilization material Hence, covalent linking of biomolecules on

transducer is an efficient method of immobilization which might provide low

diffusional resistance and a sensor shows good stability under adverse condition

In this work, we used covalent method to link the phosphate group ( ) of

probe DNA with the amine group (-NH) of PPy for DNA immobilization The further

detail will be described in chapter 2 The catalysts of the reaction are EDC and MIA

EDC is a water-soluble carbodiimide crosslinker that be generally used as a

carboxyl activating agent for the coupling of primary amines to yield amide bonds

Additionally, EDC can also be used to activate phosphate groups of DNA strand

However, the life-time of EDC-activated DNA in water is very short Therefore, MIA

is used to form alternative functional group which helps activated-DNA process

become more stable in aqueous solution, preparing for a reaction between PPy and

DNA probe

1.4 Aim of the Study

Polypyrrole, as discussed in this chapter, has several properties (both

bio-chemical and physical ones) which are suitable for biosensor development Previously,

PPy used for DNA sensor were often in cauliflower form, hence polypyrrole

nanowires are believed to have more uniform and larger surface which might

contribute to the improvement of sensitivity and selectivity of those sensors, The

purpose of this work is to synthesize PPy nanowires using electrochemical technique

for DNA sensor development

Chapter 2 EXPERIMENTS

In electrochemical micro-electrodes based DNA sensor, the output signal is

measured as the change of conductance of conductive membrane [23] Basically, DNA

strand is not conductible, so we need to prepare a conducting polymer film on surface

of electrode for the purpose of improving conductivity and immobilizing DNA target

on the sensor In this work, we chose Polypyrrole to prepare a conductive membrane

on Pt electrode

As mentioned in the first chapter, electrochemical synthesis takes advantages in

preparation of polypyrrole films on Pt electrode Also, PPy films synthesized in neutral

solution might be suitable for the immobilization of DNA strand

This chapter is divided into two main parts, including the electrochemical

polymerization of PPy films and the application of PPy based electrode for DNA

sensor

2.1 Electrochemical polymerization of polypyrrole

2.1.1 Materials

Pyrrole (99.9%) was purchased from Merck&Co., Inc (Germany) All solutions

prepared with double distilled water were H2SO4 solution (0.5M), LiClO4 buffer

(0.1M), phosphate buffer solution (pH=7) Other chemicals were of analytical grade

and were used as received

2.1.2 Instrumentation

 Potentiostat

All controlled-potential experiments were performed with the Autolab

PGSTAT302 The three-electrode system consisted of a platinum working electrode

(effective area 0.01 cm2), a Ag/AgCl reference electrode (saturated KCl) and a counter

electrode made of Platinum, Fig 2.1

Figure 2.1 Schematic of electrochemical synthesis system of polypyrrole

The function of potentiostat is to maintain the potential of the working electrode

(WE) at an adjusted level with respect to a fixed reference electrode (RE) The

potential difference between the WE and RE is equal to input potential that can

controlled externally

The current density driven by the potentiostat (between WE and RE) can be

determined by measuring the voltage drop across a small resistance R connected to the

counter electrode in series

In a three electrode potentiostatic system, the major current density passes

through the counter electrode (CE) and WE The current density amplifier supplies

current density to the cell (between WE and CE), regardless of the solution resistance

By this way, the purpose of maintaining potential control between the two electrodes

has been accomplished

 Electrolysis cell

Constant potential electrolysis (CPE) was carried out in a 100 mL – beaker with

three electrode system namely working electrode (WE), counter electrode (CE) and

reference electrode (RE)

The working electrode was Platinum microelectrode has comb configuration

with effective area of 0.01 cm2

The counter electrode was a disk of Platinum which is bigger than WE, and a

Ag/AgCl (10-2M) was utilized as the reference electrode (saturated KCl)

Characterization of polypyrroles

The obtained PPy films were characterized and analyzed by a series of

technique including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform

Infrared Spectroscopy) and SERS (Surface Enhanced Raman Spectroscopy)

 Scanning electron microscopy (SEM)

SEM is a surface analytical technique which is employed to study the

morphology of conducting polymer film surfaces and provides valuable information

on the structure of the monomer, the nature of dopant and the thickness of the film

SEM of polymer films were performed using Scanning Electronic Microscope S4800

from Hitachi, National Institute of Hygiene and Epidemiology of Viet Nam

 Fourier Transform Infrared spectroscopy (FT-IR)

FT-IR is a useful method for the characterization of monomers and conducting

polymers because it does not require polymers to be soluble It is used for the detection

of functional groups In this work, the spectrometer used in order to obtain the spectra

was Thermo Nicolet 6700 FT-IR, Hung Yen University of Technology and Education

 Surface Enhanced Raman Spectroscopy (SERS )

Since the discovery of Surface-Enhanced Raman Scattering (SERS) in 1974

[26], SERS has played an important role in studies of molecules adsorbed onto metal

surfaces The enhancement factor can be as much as 1010 to 1011 The molecules

interact with the metal surface and this increase the intensity of some of the Raman

peaks SERS has proven useful in determining the orientation of adsorbed molecules

relative to the metal surface

In some researches, SERS spectra of polythiophene and polypyrrole were able

to show that pyrrole (in some instances) adsorb with aromatic ring parallel to the silver

electrode (although this depend upon the method used to prepare the roughened

silver)