Luận văn electrochemical synthesis of polypyrrole nanowires and application of biosensor

Three electrade setup for electrochemical synthesis composed of working electrode WE, counter electrode CH} and Reference electrode RE Figure 1.7.. Master Thesis PREFACES Recently, Poly

MINISTRY OF EDECATION AND TRAINING

TIANO! TINTVERSITY OF SCIENCE AND TECHINOLOGY

INTERNATIONAL TRAINING INSTITUTE FOR MATERIALS SCIENCE

BUI DAI NHAN

ELECTROCHEMICAL SYNTHESIS OF POLYPYRROLE NANOWIRES AND APPLICATION

Master Thesis

ACKNOWLEDGMENTS

T would like to express ty appreciation to my supervisor, Dr Mai Anh Tuần for his guidance paticuce, advive and support during the course al International Training Institute for Materials Sence (ITIMS)

T would like to express iy sincere gratitude lo 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 facilrties for my master thesis

My very special thanks goes to my co-supervisor M.Sc Luu Manh Quynh, Institute off Materials Science, Hanoi University of Science, for his endless guidance

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

I wish fo thank to my fiend Tran Thi Trang for her friendship and cooperation, thank to Eng, Phuong Trung Dung who has helped me in doing measurements

1 am indebted to the teaching ITIMS for their motivation and support, particularly the fiiendly and helpful manner of ITIMS statis will remain in my mind, especially the members of Biosensor group in ITIMS for sharing fiiendly research environment

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

Above all, I am gratetil to my beloved family, especially my tather who always be with me wath endless encouragement, inspiration and love

ITMS, Hanci, November 2011

Master Thesis

I hereby declare that all the result in this document has been obtained and presented in accordance with academuc rules and ethical conduct, | 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

1.1.4, Current synthesis of conducting polymers

1.2 Polypyrrole (PPy) and Electrochemical polymerization of PPy

1.2.1 Properties of Polypyrrole

1.2.2, Electrochemical synthosis of Polypyttole «usecase

1.2.3 Effect of Synthesis conditions on Electrochemical Polymerization

1.3 Application of Biosensors, .ccesesseseuesstininieaenatninaniateee

1.3.1, General Introduction ta DNA sensor

1.3.2, Immobilization of probe DNA on polymer based electrode essences

1.4 Aim of the Study

3.1 Electrochemical synthesis of PPy nanowres 48

3.1.2, Efftcts off parameters on electrochemical polymerization of polypymole Š1 3.1.3 Chemical composition and functional groups of obtained PPy nanowires «0

3.3.1 Charaeteristios of DNA sensor is a function of time 70 3.2.2, Hybridization detection nsing DNAA scBsor no 71 3.3.3 The reprodueibillty o£ DNA sensoz 73

RECOMMENDATIONS Error! Bookmark not defined

Master Thesis LIST OF ABBREVATION

EDC 1-Ethy-3-(3-dimethyi-ammopropyl)-carbodiimide

TT-IR Eouier Transform Infrared Spectroscopy

SERS Surface Enhanced Raman Spectroscopy

Master Thesis 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 Advamages and Disadvantages of Chemical and Llectrachemical synthesis

of PPy,

Table 1.4 History of biosensor development

Table 2.1 DNA sequence used mn this work

Table 3.1 Current density (mA/em*) vs added volume of pyrrole monomer (ml)

Table 3.2 Current density (mA‘en?) vs different concentration of gelatin (%4y1)

Table 3.3 Current density (wA/em) 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.

Master Thesis LIST OF FIGURE

Figure 1.1 Conductivity of conducting polymer compared with otter materials

Figure 1.2 Three typical types of conducting polymer

Figure 1.3 Bund theory and deping-induced structural wansttions of polypyrrole

Figure 14 Three steps of polymerization process of Polypyrrole

Figure 1.5 Aromatic and Quinoid structrure of PPy

Figure 1.6 Three electrade setup for electrochemical synthesis composed of working electrode (WE), counter electrode (CH} and Reference electrode (RE)

Figure 1.7 Cyclic voltammogram of PPy nanowires and cauliflewer-like in KC}

solution at scan rate of 25 mV/s

Figure 1.5, Potentiostat curve of the synthesis of PPy on Nikel electrode and ITO electrode

Figure 1.9 A wpical 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 bypes of DNA

Figure 1.15 Hydrogen honds hetween the A-T and O-C bases of the two trands of BNA

Figure 2.1 Schematic of electrochemical synthesis system of polypyrrate

Figure 2.2 Cavalent immobilization hetween PPy fils and phosphate DNA on Pt micro-elecirade using EDC, MIA catalysts

Figure 2.3, Differential measurement using Lock-in Amplifier

Figure 2.4, The wave form of the Luck-in Anpplifier SR830

Figure 2.5 Equivalent electrical circuit of differential system

7

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 FT-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 Phatimun 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, Conayame 0.054, T 300°K, Figure 3.18 The reproducibility of DN: sensor

Master Thesis

PREFACES

Recently, Polypyrole (Py) is one of the most extensively used conducting polymers in biosensor designs due to its good biocompatibility and polymerization at uculral 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 weribrane inchudos limited scnsitivity aud reproducibilily dus to the low conductivity of PPy in Gln and caulilowerlike fom, presented in previous work '® In this thesis, we aim at the synthesis of PPy nanowires using electrochemical technique with the desire of obtaining better charactetistics of DNA sensor for Ecoli bacteria DNA detection,

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

0.75V, in LiC]040.1M (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-IT group of PPy nanowires was origntated upward from the surfice which takes

advantage for DNA mobe immobilivation, As the result, the DNA based PPy

manawires has good charsetcristics for Feoh DNA detection, including a short

response time ¢-10 seconds), smal] detection limit (0.1 nM) as well as good reproducibility

PD, Tam etal, / Materials Science and Engineering C 30 (3016) 1141-1150

9

Master Thesis

‘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 fo obtain PPy nanowires The electrochemical behaviors, morphologies and chemical

composition of polypyirols nanowires obtained potcntiostatically have been analyzed

Master Thesis Chapter 1 INTRODUCTION

1.1 Overview of conducting polymers

1.1.1 Intruduction

Polymers are Jong chain gisnl organic molecules, ‘poly’ meaning Gnany’ andl

‘ner’ meaning ‘part? Gin Greek) Orighmally, polymers are noneonductor, The tenn

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

electrical conductivity That property 1s 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 oxidizmg or a reducing agent can

be converted into polymer salts with considerably increased electrical conductivity (Fig 1.1)

Master Thesis

Conducting polymer can be classified into three distinct groups; electron- conducting polymer, ion-conducting polymer, redox polymer (Fig.1.2)

ov Protonated Polyvynilpyridine Layer Solution

Jon-exchange conducting polymer

Electron conducting polymers

Figure 1.2 Three typical types of conducting polymers

12

Master Thesis

‘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 thal of water (1.23)

Electron-rich heterocycle based polymers such as polythiophene and polypyrole are very stable iu 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 (A0S/cm~2.5e "Q.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, polypyrrale seems fo he a good candidate for rosomchors now Lo atlempl al the synthesis of conducting polymers, in

particular for biosensing application

1.1.2 Historical back ground of the development of conducting polymers

Polyanitine (PAni), known us ‘aniline black’, is one of the oldest cormluetive

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

reparted in 1958 by Natta and coworkers [14]

‘The modem 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 (at polymer plasties can be made to condnel cloctricity if alternating single und double bunds tink duit carbon atoms and cloctrons

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

13

Master Thes

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

Semiconductivity P.A discovered by Shirakawa et al 1971

Conducting polyppara phenylene sulfide by J.-E Forder 1983

Poly (3-hexylthiophene)

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

Table 1.1.The chronology of the development of some important conducting polymers [19]

14

Master Thesis

After the first publication, there has been an explosive growth of research into the whole range of conjugated palymer 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 conductivily than PAc, these polymers have been useful in designing

new stucbures thal 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 #e band theary of solids According to this theory of solids, whan a lage number of aloms or molecules are brought to form a polymeric chain or a crystalline solid, am cnergy band is formed through the iavteraction 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 conducling species for charge transport, Ti should be nioled thal this theory agreed with PAc (because it has a degenerate ground stale, two geometric structures corresponding to the same energy) but not with all other conducting polymers having non-degenerate ground state

The fhilure 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 gel coupled to form a bipolaron doubly charged bul

spinless,

15

Master Thesis

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 stale

a) Conduction

Band

‘Valonee

Band

Neutral Lightly Doped Hemvily Daped

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 conductivily tueasured This leads to

the znhaneomơnt o[ conductivity of conducting PPy tn figure 1.3b, dopant used is

negative particle X (CE C10, ), hence PPy becomes a p-type semiconductor The

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

the luvel of doping (Fig 1.32), as the result of oxidative doping in PPy,

1.1.4 Current synthesis of conducting poly mers

CPs ean be synthesized chemically or electrochemically, which each method

Different methods

has advantages and disadvantages [28] as summarized in table

of chemical synthesis indude cither condersalion polymerization (is, step growth

polymerization) or addition polymerization

« Langerscaleproducionposibie Camol make thin films

© Poct-covalent modification of * SYthesis is more completed

Chemical

More options to modify CP backbone covalently

«Thin itn synthesis possible 4 Diffienlt to remove film from

Electrochemical «Fase of synthesis electrode surface

polymerization atapment ofmoleculesin CP * Post covalent modification of bulk

© Doping is simultancous Pie dificult

Table 1.2 Advantages and disadvantages of chemical andelectrachemical synthesis of

conducting polymers

1

Master Thesis

Electrochemical synthesis is a common altemative 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

to give a black conducting powder TL can be synlhusived in both aqueous and non aqueous satution during lectrocherical polymerization

Among alt 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, duing 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 m the

bioelectroanalytical chemistry Furthermore, polypyrtole 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 pyrole

colloids is described Moreover, cornpared 1o other heterocycles ils oxidation potential

is low |27]

For all the reasons polypyrrole has been on interesting material wo study

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

18

Chemical synthesis of PPy Electrochemical synthesis of PPy

Advantages Easy to produce amounts of Convenient caried out

PPy in various forms Process is simply controlled

(through current or applied

potential)

Disadvantages Poor producibility Prepare PPy only in thn film

deposited on the surface of electrode

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 electruchertical method is preferred because

iLis casy, clean and selective

19

Master Thesis 1.2.2 Electrochemical synthesis of Polypyrrole

ical polymerization has been widely used for syull

polypyzole, as presented in a mamber of previous studies [5,11.18,30,46]

Although most of their pyrrole units are linked at the o-a (or 2, 5) positions, a signiticant number of the units are coupled through the af and B-f cross Linkages [40] the less desirable 3,4 or 2,3 coupling contubutes to the formation of sohuble 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 cither react

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

neutral monomer The electrochemical polymerization reaction accurs only when the

nt ta oxidize the monomer

applied patential is

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

tumber of neutral species af the electrode surface will be essentially zero al these potentials, The charge consumed during polymer foomation has linear time dependence (at least initially) and is independent of pyrrole concentration If there is

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

radical cations, they will give a dimer cation which readily eliminates 211* [27]

20

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

21

Master Thesis

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 bs aromatic and quinoid type, shown in figure 1.5

Nowadays, clsotrochondical polymerization is porformed using dhrce-cleetrods configuration (working, counler, and rofercnus cluetrodc) in a solution of monomer,

appropriate solvent, and electrolyte as seen in Fig 1.6

Figure 1.6, Three electrade sensp for electrochemical synthesis composed of Working

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

22

stor The:

Curent 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 ot radical cations, forming insoluble polymer chains on the electrode surface

Cyelic 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

Tn this method, the potential af the working clovtrade to referen

seamed in the wiodie and cathodic directions and the current density Dow as a function of this potential is measured,

A cyclic voltammogram helps us to understand the clectroavtivity and redox potential of a material, mechanism of the clectrochemical reactions, reversibility of electron transter, 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

stor The:

Constant Potential Electrolysis (Potentiostatic mode)

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

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 (L.,.) and it is determined by means of cyclic voltammetry (will be farther explained in chapter 3)

24

Recently, a new method has emerged as an altemative 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 Iarslucert, colorless, brittle (when dried), flavorless

Figure 1.9 A typical structure unit af gelatin polypeptide

(Source: http: ‘eww nanoscalerestett com/contenti6/1/23)

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

additives and preservatives

In this work, gelatin is used as a ‘sof template” with 11D linear molecnles whieh can sclf-asscmble into 1D structures During the polymerization, the conducting polymers can grow along the 1D ‘soft tamplate’, leading to the formation of the 1D polypyzzple nanomatenals

25

Shape of the electrode

Since the polymerization proceeds via oxidation and reduction reactions, it is necessary that the electrode should not be oxidized curently 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/Agt] electrodes can be used as the reference electrodes [12]

For synthesis of PPy, Platinum elechode takes advantage because the oxidation polontial of pyrrole is reduced and current density inercused in comparison with Ti, Fe

or Al used, It is because the formation of the thinner platimum-oxide which impedes electron transfér during electropolymerization

acelonitnle tnay be used during electrochernical polymerization [42]

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

gelatin and phosphate buffer However, in the addition of pynole monomer and gelatin, the solution should be slightly heated and deaerated continuously at least 15

minutes with N; to avoid the oxidation of PPy and achieve the dissolution

26

Master Thesis

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 LiClO,, phosphate buffer, gelatin and pyrrole monomer

‘Temperature

Temperature is the olher parameter that should be taken inlo consideration ding cechopalymerization It has a substantial infucnoc on the kinetics of polymerization as wel as ơn the cơnductivily, redox propertics ad mecharival characteristics of the films, Ai hìgh tamperaltros, 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 ou elactrode’s surface

In this work, we do nol study cffect of alt the parameters Three parameters which wilt be studied under this work are concentration of pyrrole, gelatin and reaction

time The experiments will be fixther described in chapter 2

1.3 Application of Biosensors,

Among conducting polymers, polypyrele is the most frequemtly used in commercial applications, such as batteries [12], supercapacitors [25] sensors [32], anhydrous clectrurheological Quids [21], microwave shielding and corrosion protection, ete

Reet

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

uumber of arcas rclatcd to human health (infectious discases, drug discovery, food,

27

1.3.1 General Intreduction 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

1eceplors, enzymes, antibodies, nucleic acids, ect); a biologically derived

material or bionimie) The sensilive cements can be created by biological

engineering;

+ The detection (works in a physicochemical way, optical, electrochemical,

thenmotmuetric, pievockevlric or magnelic),

+ ‘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

28

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

®——— immobilized DNA probe

conducting polymer layer transducer

signal

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

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

29

Master Thesis

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

1969 First potentiometric biosensor: urease immobilised on an ammonia

electrode to detect urea

1970 | Invention of the Ion-Selective Field-Effect

‘Transistor (ISFET) (Bergveld)

30

Bui Dai Nhan | 2011

Master Thesis

5/1972 | First commercial biosensor: Yellow Springs

Instruments ghicose biosensor

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

platinum wire; Invention of the pO2 / pCO2 optode

1976 _ | Furst 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 ghucose

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

$13 billion

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

31

Master Thesis

Biosensor market

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

(hp: (Athtt sehisOt:snqg 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

transmitted to offspring

32

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,

3° extremity = sugar 5" extremity = phosphate

Figure 1.15 Hydrogen bonds beeen the A-T and G-C bases of the bwo strands of

DNA (adopted from reference [18])

33

Master Thesis

The phosphate-deoxyribose sugar polymer represents the DNA backbone (Fig 1.15, adopted fom 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 (1), as a function of their consecutive order in the chain The two strands of nucleotides are twisted inlo a double helix, held logether by hydrogen bonds between

ihe A-T and G-C buses of cach 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, Frtrapment, Crosslinking, Covalent bonding

ps]

+ Adsorption

Many substances adsorb enzymes on their surfaces Physical adsenptitm 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 fume-span

> Micro encapsulation

A semi permeable membrane is used 1o trap the biomaterial on the transducer

‘This keeps close contact between the biomaterial and the transducer Membrane materials inchide cellulose acetate, polycarbonate,and _polytetrafluoroethylene (Teflon) It is stable towards changes in temperature, pll, ionie strength and chemical composition

> Entrapment

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

to a gol, thus bapping the biomatorial This method crcates Inge bartiors which inhibit

ihe diffusion of the substrate This slows the reaetion down and the response fine of

the sensor

34

In ordsr 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 Henee, covalent linkmg of biomolzeules on transducer is an efficient method of immobilzation 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 (PO3-) of probe DNA with the amine group (-NH) of PPy for DNA immobilization The tirther detail will be desozibed 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 Towever, the life-time of BDC-activated DNA in water és very short Therefore, MIA

is used to form alternative functional group which helps activated-DNA process bevome more stable in aqueous sokuGon, preparing for a reaction between PPy and

DNA probe

35

36

Master Thesis 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 Polypyrrele to prepare a conduetive metbrane

on PL electrode

As mentioned in the first chapter, electrochemical synthesis takes advantages in preparation of polypyrrole films on Pl slectrode 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

and were used as received

2.1.2 Instrumentation

“ Potentiostat

All controlled-potential experimenis were performed with’ the Aulolah

PGSTAT302 The three-clectrode s

(cfleetive ava 0.0L cur’), a Ag/AgCl reference cleclrode (saturated KCD and a counter electrode made of Platinurn, Fig 2.1

stom consisted of a platinum working cloetrodu

37

Higure 2.1 Schematic of electrochemical suuthesis system of polypyrrole

‘The fumetion of potentiostat ts to maintain the potential of the working electrode

(RE) The

potential diffzrence between the WE and RE is cqual to input polential thal can controlled extemally

GWE) at an adjusted luvel with respect to a fixed reference clucire

‘The current density driven by the potentiostal (belwoun 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 cument 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

+ Electrotysis cell

Constant potential electrolysis (CPE) was camied out ina 100 mL beaker with three electrode system namely working electrode (WE), counter electrode (CE) and reference electrode (RE)

"The working electrode was Platinum microslectrade has comb configuration

with effective area of U.01 cm”

‘The connter electrode was a disk of Platinum which is bigger than WE, and a Ag/AgCl (107M) was utilized as the reference electrode (saturated KCD)

38

Master Thesis

Characterization of polypyrroles

‘The obtained PPy tilms 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 2 surface analytical tochmique which is cmployed to study the urphelogy of conducting polymer film surfaces and provides valuable information

ơn the structure of the monomer, the nature of dopant and the thickness of the film SEM of polymer films were perfoomed using Scanning Electronic Microscope $4800 trom Hitachi, National Institute of Hygiene and Epidenuology of Viet Nam

= Fourier Transform Infrared spectroscopy (FT-IR)

FT-IR is @ useful method for the charaulzrization of monomers and conducting polyrrers heususe if doss not require polytnors to he soluble, Tis used for the detevtion

of functional groups In this work, the spectrometer used in order te 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-Lnhanced Raman Scattering (SERS) in 1974

[26], SERS has played an important role im studies of molecules adsorbed onto metal surfaces The enhancement faclor can be as much as 10” to 10" The molecules interact with the metal surface and this increase the intensity of some of the Raman peaks SERS has proven usefill 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)

39