dna polymer

Conducting polymers for electrochemical DNA sensing Hui Penga,*, Lijuan Zhanga, Christian Soellera, Jadranka Travas-Sejdica,b,** a Polymer Electronic Research Centre, The University of A

Conducting polymers for electrochemical DNA sensing

Hui Penga,*, Lijuan Zhanga, Christian Soellera, Jadranka Travas-Sejdica,b,**

a Polymer Electronic Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand

b MacDiarmid Institute for Advanced Materials and Nanotechnology, New Zealand

a r t i c l e i n f o

Article history:

Received 30 September 2008

Accepted 24 December 2008

Available online xxx

Keywords:

Conducting polymers

Electrochemical DNA sensor

Electropolymerization

a b s t r a c t

Conducting polymers (CPs) are a class of polymeric materials that have attracted considerable interest because of their unique electronic, chemical and biochemical properties, making them suitable for numerous applications such as energy storage, memory devices, chemical sensors, and in electro-catalysis Conducting polymer-based electrochemical DNA sensors have shown applicability in a number

of areas related to human health such as diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology due to their simplicity and high sensitivity This review paper summarizes the advances in electrochemical DNA sensing based on conducting polymers as active substrates The various conducting polymers used for DNA detection, along with different DNA immo-bilization and detection methodologies are presented Current trends in this field and newly developed applications due to advances in nanotechnology are also discussed

Ó 2008 Elsevier Ltd All rights reserved

1 Introduction

DNA analysis plays an ever-increasing role in a number of areas

related to human health such as diagnosis of infectious diseases,

genetic mutations, drug discovery, forensics and food technology

Conventional methods for the analysis of specific gene sequences

are based on either direct sequencing or DNA hybridization The

sequencing technology was invented by Maxam and Gilbert[1]and

Sanger et al.[2]in the 1970s In the same period, solid-supported

hybridization became a widespread method for DNA analysis using

membrane-based blots [3,4] However, these approaches have

some disadvantages, such as the inability to use a large number of

DNA samples, low selectivity between closely related sequences

and they are often time consuming In the early 90s, gene array

technologies which relied on the anchoring of multiple specific

probe DNA fragments or oligonucleotides (ODNs) onto solid

surfaces and detection of fluorescently or radioactively tagged

analyte oligonucleotides appeared as promising tools for the

simultaneous analysis of multiple DNA sequences [5–7] These

array technologies have had a huge impact on genomics and

pro-teomics applications, although they have shortcomings arising

from, for example, limited tagging efficiency, hazardous waste

disposal and complex multi-step analysis In order to seek faster, sensitive and label-free DNA detection, a number of approaches have been suggested based on optical [8–11], acoustic[12] and electrochemical[13–15]techniques

Electrochemical DNA sensors are regarded as particularly suit-able for direct and fast biosensing since they can convert the hybridization event into a direct electrical signal [16–18] This means that there is no need for complex signal transduction equipment and the detection can be accomplished with an inex-pensive electrochemical analyzer Electrochemical DNA sensing approaches include the intrinsic electroactivity of DNA [19–22], electrochemistry of DNA-specific redox reporters[23,24], electro-chemistry of nanoparticles[25–27]and conducting polymers (CPs)

[18,28] Conducting polymers (CPs) are polyconjugated polymers with electronic properties resembling those of metals, while retaining properties of conventional organic polymers Since the observation

of the remarkably high electrical conductivity of a halogen-treated polyacetylene[29], a number of other conjugated polymers have been transformed from an insulating into a highly conductive state The most widely investigated conducting polymers include poly-aniline, poly(phenylenevinylene), polypyrrole and polythiophene (Fig 1) The award of the Nobel Prize in Chemistry in 2000 to H Shirakawa, A MacDiarmid and A Heeger for their pioneering work

on conducting polymers widely recognized the importance of these materials and has prompted even more vigorous research in the field Compared to saturated polymers, CPs have a unique elec-tronic structure which is responsible for their electrical conduc-tivity, low ionization potentials and high electron affinity For CPs in

* Corresponding author.

** Corresponding author MacDiarmid Institute for Advanced Materials and

Nanotechnology, New Zealand.

E-mail addresses: h.peng@auckland.ac.nz (Hui Peng), j.travs-sejdic@auckland.

ac.nz (J Travas-Sejdic).

Contents lists available atScienceDirect

Biomaterials

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / b i o m a t e r i a l s

0142-9612/$ – see front matter Ó 2008 Elsevier Ltd All rights reserved.

doi:10.1016/j.biomaterials.2008.12.065

the ground state (insulating or semiconducting state),p-bonds (p–

p*) are partially localized due to a phenomenon called the Peierls

distortion[30] During the doping process, the excitation across the

p–p* band gap creates self-localized excitations of conjugated

polymers with localized electronic states in the gap region [30]

These self-localized excitations are called polarons, bipolarons and

solitons and underlay electrical conduction in CPs The unique

properties of CPs have led to a variety of applications for these

materials, such as light emitting diodes (LEDs)[31], electrochromic

materials[32], anti-static coatings[33], solar cells[34], batteries

[35], anti-corrosion coatings[36], chemical sensors and biosensors

[37]and drug release systems[38–40]

Conducting polymers can be synthesized chemically[41,42]and

electrochemically [43,44] In terms of biological applications,

electrochemical polymerization is widely used because of several

advantages: (i) it is performed at ambient temperatures and

microelectrodes or electrodes with a large surface area can be used;

(ii) the polymer film formed is confined to the electrode and its

shape can thus be controlled by electrode design, while the

thick-ness can be controlled in the nanometer to micrometer range; (iii)

the properties of the CP film can be widely modulated by varying

electrochemical polymerization conditions Electrochemical

poly-merization can be carried out potentiostatically, amperometrically

or with potential scanning and the whole process may only take

a few seconds [45] During polymerization, the monomers are

oxidized to form radical cations, followed by coupling reactions to

form oligomers that eventually lead to deposition of the polymers

on the electrode surface More detailed descriptions of

electro-chemical polymerization can be found elsewhere[41,46]

The electronic structure of CPs is highly sensitive to changes in

the polymeric chain environment and other perturbations in the

chain conformation caused by, for example, a biological recognition

event such as DNA hybridization The changes in the delocalized

electronic structure or in other CP properties are manifested in

altered optical and electrical properties, and, when measured, can

provide a signal for the presence of a target analyte molecule[47]

These advantages of CPs make them suitable materials for chemical

sensors and biosensors An excellent review on chemical sensors

based on CPs by Swager and collaborators[47]outlines numerous

synthetic approaches towards the specific recognition probes

attached to a conjugated polymer backbone More recently, Bai et

al reviewed the application of CPs as gas sensors[48]

This review paper focuses on the applications of conducting

polymers specifically in DNA sensing, with a special attention paid

to current trends and applications developed recently in the field

due to advances in nanotechnology

2 Immobilization of DNA probes

A typical configuration for DNA sensors based on CPs is shown in

Fig 2 Single-stranded DNA probes are immobilized on or within

a conducting polymer layer The target DNA is captured by base-pairing to generate a recognition signal, which is recorded through

an electrode (gold, platinum, glassy carbon, etc.) Because the recognition event takes place at the CP/electrolyte interface and the recognition signal generated reaches the transducer through the CP layer, the properties of the CP and the orientation of the immobi-lized DNA probes on the CP are crucial to the sensor performance The procedure of DNA probe immobilization should retain the probe’s affinity for complementary target DNA Ideally, the orien-tation of probes should be predictable and readily accessible to the analyte DNA[49] Generally, immobilization methods fall into the classes of electrochemical entrapment, covalent immobilization or affinity interactions

The electrochemical entrapment method originates from the pioneering work on enzyme sensors by Umana and Waller[50] It involves the electrochemical oxidation of a suitable monomer to the corresponding conducting polymer from a solution that contains oligonucleotide (ODN) probes Wang et al first illustrated that ODNs can act as the sole dopant during the growth of

n

N

H

N H

n

n polyacetylene

poly(aniline)

poly(phenylene vinylene) polypyrrole polythiophene

Fig 1 Structures of some of the most common conducting polymers.

target DNA

immobilized DNA probe conducting polymer layer transducer

signal

Fig 2 General DNA sensor design based on CPs.

polypyrrole films while maintaining their hybridization activity

[51,52] The significant advantage of this immobilization method is

its simplicity Potential drawbacks include the possibility of

damaging ODN probes due to the high potentials employed during

polymerization, and poor target accessibility to the incorporated

probe in the bulk of the resulting film[49]

Covalent attachment can overcome the disadvantages of the

electrochemical entrapment method and improve probe

accessi-bility by target DNA[53] Generally, ODN probes are functionalized

with –NH2, –COOH, etc., and are then covalently attached to either

a functionalized monomer or a functionalized polymer The

reac-tions used for the covalent immobilization are shown inFig 3

Livache et al developed a process that utilizes pyrrole monomer

bearing an ODN to copolymerize with pyrrole (Fig 3a) [54,55]

allowing the immobilization of multiple probes on electrode arrays

Later the same group used a similar process to prepare an ODN

array consisting of a matrix of 48 addressable 50-mm

microelectrodes that was applied to detect hepatitis C virus in blood samples[56] In another approach for covalent ODN attachment

a film is first electropolymerized from a solution containing func-tionalized monomers (either exclusively or in a mixture with non-functionalized monomers), followed by covalent attachment of

a 50-end modified ODN onto CP functional groups In this method, the conducting polymer films can be prepared under conditions that are potentially incompatible with the maintenance of ODNs, such as organic solvents and high polymerization potentials, while the subsequent attachment of the ODN probes is performed under mild conditions that ensure ODN integrity Garnier et al prepared

a functionalized polypyrrole, poly(3-acetic acid pyrrole-co-3-N-hydroxyphthalimide pyrrole), which bears an easy leaving group, N-hydroxyphthalimide (Fig 3b)[28,57] In a further step, an amino-substituted ODN was then grafted onto this precursor copolymer

by direct chemical substitution of N-hydroxyphthalimide The authors concluded that 3-substitution is more favorable than

N

H 3 C

O NH(CH 2 ) 6 NHOC(CH 2 ) 6 CONH(CH) 2 N

O

HO

P O

O

O

ODN +

N

H2 ) 6

) 6

) 2

N

N

H3C

O

O

HO

P O

O O

DN

N

N

Electropolymerizaiton

a

N N

ODN Electropolymerization

b

OH

O

O N O

O

O

N N

OH O

O N O

O

O

N N

OH O

O H O

N

O

OH

O OH

N +

Electropolymerization ODN probe

H2N

O HN

N

c

S S

+

O

S N O O

S S

O

S N O O

S S

O

S Cl O O

S S

O

S NH O O

H2N

ODN Electropolymerization 1 Cathodic cleavage

2 Chlorination

d

Fig 3 Reactions for covalent immobilization of ODN probes a, b, c and d are from Refs [18,28,54] and [61] , respectively.

N-substitution with regard to maintaining the high intrinsic

conductivity of the polymer Peng et al reported an acid

func-tionalized polypyrrole, poly[pyrrole-co-4-(3-pyrrolyl) butanoic

acid], where the amino-functionalized ODN was covalently

attached to the polymer film using

1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as a catalyst (Fig 3c)[18] Thompson

et al proposed a different approach, in which a single-stranded

ODN probe was linked to the conducting polymer by forming

a bidentate complex between Mg2þand an alkyl phosphonic acid

group on the polymer and the phosphate group of the ODN[58]

Recently Gautier et al developed a process that involves

electro-chemical copolymerization of 3-methylthiophene and

3-(oxy-alkyl)-thiophene bearing an arylsulfonamide group The

sulfonamide terminal functional group of the copolymer film

was electrochemically cleaved and chemically modified with

N-chlorosuccinimide (NCS) which led to a sulfonyl chloride

func-tionality as a prerequisite for amino-ended single-stranded DNA

probe immobilization (Fig 3d) [59–61] An advantage of this

approach is the ability to electrochemically cleave immobilized

ODN probes, which leads to the potential application for the

immobilization of addressable multiple ODN probes on an

elec-trode array Due to this advantage, the research group developed an

electrochemically controlled DNA delivery system by using similar

procedure[62]

The immobilization of ODN probes on the CP film can also be

achieved via an affinity interaction The most common approach

involves the avidin–biotin interaction which is extremely specific

and the strongest known non-covalent biological bond (association

constant Ka¼ 1015M) This approach can be highly versatile due to

the ability to anchor different biomolecules on the same support

and the sensor can be regenerated by treatment with a detergent

solution that breaks the avidin-biotin bridge but does not affect the

support matrix Cosnier and Lepellec first prepared

poly(pyrrole-biotin) films for grafting glucose oxidase[63] Later Dupont-Filliard

et al exploited this principle to reversibly immobilize ODN probes

[64] Briefly a biotinylated polypyrrole film was synthesized,

fol-lowed by immobilization of avidin units by biotin-avidin

interac-tion, and the film was exposed to biotinylated ODN probes, as

shown inFig 4 The reversibility was achieved by a treatment in

aqueous solution of sodium dodecylsulfate to cleave the avidin– biotin connection[64,65]

3 Transduction mechanisms The transduction process converts the recognition event into

a measurable signal According to the quantity measured, the transducing mechanisms can be classified as electrochemical, optical, mass or thermal Among these, electrochemical trans-duction is well suited to DNA sensing where a biorecognition event results in a direct electrical signal and the whole sensing system can

be very effectively miniaturized Indeed, portable systems for diagnosis and on-site environmental monitoring are now being developed[66] For electrochemical DNA sensors based on CPs, the polymer is not only used as an immobilization matrix but also plays

an active role in transduction CPs can be reversibly doped and dedoped using electrochemical techniques, with doping and dedoping resulting in significant changes in electrical and spec-troscopic properties These changes can be modulated by probe– analyte interactions[67,68], and then become an analytical signal for analyte detection that can be quantified as a change in current at

a fixed applied potential (amperometry), a change in conductivity (conductometry), impedance (impedimetry) or potential (potentiometry)

3.1 Amperometric detection and cyclic voltammetry Amperometry is the most common approach used generally with biosensors based on CPs due to the simplicity of the method and fast response In this approach, the performance of biosensors

is governed by the efficacy of the electron transfer between the biomolecules, such as oxidoreductase, and the underlying electrode surface, involving the CP layer In the case of DNA sensors, the efficacy of modulating the electrical conductivity and the redox properties of the CP by hybridization plays a key role PPy/probe-modified electrodes, made by Wang et al.[51,69] using oligonu-cleotide probes as sole counter anions, displayed transient anodic peaks upon adding a complementary target, and opposite (cathodic) signals upon spiking of the solution with

non-Fig 4 Schematic of a sensor design based on electrocopolymerization of biotinylated pyrrole and the use of biotin–avidin interactions Reprinted with permission from Ref [64]

with permission Copyright Elsevier 2008.

complementary strands The direction of signals (relative to the

base line) was dependent on the sequence of probe dopants and of

the added target oligomers The authors suggested that such

a response reflected the change in the conductivity of the host PPy

network caused by an increase in charge density in the case of

a complementary target, or induced by electrostatic repulsion in

the case of non-complementary sequences

Cyclic voltammetry is widely used with CP-based DNA sensors as

the readout method, because the doping and dedoping processes

monitored during potential scanning can be efficiently modulated

by the hybridization event Garnier et al.[28,70,71]illustrated this by

using polypyrrole grafted with single-stranded ODNs that was

electropolymerized onto a Pt electrode The cyclic voltammograms

showed a significant decrease in the oxidation current and a positive

shift in oxidation potential of the polymer film after hybridization

with complementary ODN samples (Fig 5) Such a change has been

ascribed to the formation of bulky and rigid double-stranded DNA

upon hybridization, thus increasing the energy required to planarize

the polymer upon oxidation The detection sensitivity increased

with increasing length of the recognition base sequence, while the

oxidation potential in cyclic voltammograms was shifted to even

higher potentials In similar studies, it was found that better

sensi-tivities are achieved by using thinner sensor films due to the larger

surface to volume ratio[18,71] Furthermore, modulation of

elec-trical properties of the conjugated backbone of CPs by single bases

has been confirmed by Emge and Bauerle[72,73] First, they

func-tionalized bithiophene monomers with pyrimidine and triazine

bases After polymerization, the addition of small quantities of

complementary bases strongly affected the electrochemical

prop-erties of the obtained polymer, also resulting in the decrease of the

oxidation currents and positive potential shift that described above

3.2 Conductometric detection

Conductometry measures the conductivity change of CPs arising

electrochemically synthesized poly(3,4-ethylenedioxythiphene) microtubules in the presence of ssDNA using a polycarbonate membrane as a template, and developed a label-free conducto-metric DNA sensor based on these microtubules [74] The conductivity measurements were done at a gate potential of þ0.8 V (vs SCE) where the response was at its highest The authors investigated the effect of ssDNA length on the sensor response The results showed that the sensor response, as well as the linear range, increased with an increase in the ssDNA length For the sensor based on ssDNA with 20 bases, the linear range was 8.0  108to 1.0  105g mL1 and the detection limit was 8.0  108g mL1 Ramanathan et al prepared polypyrrole nanowires in the presence

of avidin-conjugated ZnSe/CdSe quantum dots by electrodeposition within a channel between two electrodes on the surface of a silicon wafer[75] After addition of biotin end-modified DNA, the resis-tance of the nanowires increased significantly

3.3 Impedimetric detection Electrochemical impedance spectroscopy (EIS) has become

a powerful tool in the study of corrosion, semiconductors, batteries and electro-organic synthesis because it provides kinetic and mechanistic information In EIS experiments, a sinusoidally varying and interrogating voltage (typically in the range of 5–10 mV peak-to-peak) relative to a suitable reference electrode is applied to the working electrode and the resulting current response is measured The real (Zre) and imaginary (Zim) components (or magnitude çZj and phaseq) of the impedance are calculated as the ratio between the system voltage phasor and the current phasor, which are generated by a frequency response analyzer during the experiment

[16] Compared to dc techniques, EIS offers the following advan-tages: (i) a very small excitation amplitude is used which causes only minimal perturbation of the electrochemical test system, reducing errors introduced by the measurement technique; (ii) EIS provides valuable mechanistic information, because electro-chemical impedance experiments obtain data on both electrode capacitance and charge-transfer kinetics Furthermore, a purely electronic model consisting of a specific combination of resistors and capacitors can be used to represent the electrochemical system, and then the electrochemical system can be characterized by using established AC circuit theory in terms of equivalent circuits The immobilization of DNA probes and the hybridization event not only induce changes in the intrinsic properties of the CP film but also bring about changes in the various interfacial film prop-erties, such as the capacitance and interfacial electron transfer resistance [17] EIS can detect these changes, which can be exploited in the development of truly label-less biosensing Indeed, EIS has been successfully employed in the label-free detection of DNA hybridization based on conducting polymers, although a full mechanistic understanding of the above-mentioned contributions

to the overall EIS spectra change for a CP films has still to be developed Peng et al showed that there is a considerable differ-ence in the AC impedance spectra of a functionalized polypyrrole containing covalently grafted ODN probes before and after hybridization with different concentrations of complementary ODNs obtained in the presence of Fe(CNrad)64/ Fe(CNrad)63 at open circuit potential [18] The spectra were modeled using

a modified Randles equivalent circuit, but with a constant phase element (CPE) used instead of a double layer capacitance The fitting results showed that the charge-transfer resistance increased with an increase in the concentration of complementary ODNs The values of the heterogeneous standard charge-transfer rate constant (ka), which represents the kinetic facility of a redox couple, decreased with an increase in complementary ODN concentration, suggesting that the ODN duplexes form a barrier to ion movement

Fig 5 Cyclic voltammograms of poly(3-acetic acid pyrrole-co-3-ODN-acetamido

pyrrole) deposited on a platinum electrode, after incubation in a buffered aqueous

solution of (a) non-complementary ODN (b), increased concentration of

complemen-tary ODN (c–e, 66, 165 and 500 nmol, respectively) The incubation solution was 5 mL.

Reprinted with permission from Ref [28] Copyright Elsevier 2008.

and the redox couple (Fe(CNrad)64/ Fe(CNrad)63) was less

acces-sible to the electrode due to electrostatic repulsion Tlili et al

reported detection of DNA hybridization using non-Faradic

elec-trochemical impedance spectroscopy[76] The DNA probes were

covalently attached to the precursor copolymer, poly(3-acetic acid

pyrrole, 3-N-hydroxyphthalimide pyrrole) and the impedance

measurements were performed without any redox species at a DC

potential of 1.4 V The authors chose this potential in order to

minimize the Warburg impedance and to emphasize the

contri-bution of the impedance at the PPy-DNA/electrolyte interface At

this potential, polypyrrole was in non-doped and semiconducting

state; therefore, no parasitic electrochemical reaction occurred

during the measurement The resulting impedance spectra were

fitted using a modified Randles equivalent circuit with a CPE in

order to reflect the non-homogeneity of the layer The results

showed that the charge-transfer resistance decreased upon

graft-ing of DNA probes and increased upon hybridization with

complementary DNA The authors explained these observations as

being due to the effect of the negative charge of ss-DNA and ds-DNA

and to their conformational structures In the case of grafting of

ss-DNA onto functionalized polypyrrole, which could be considered as

a p-type semiconductor under the experimental conditions, the

grafting of negatively charged ss-DNA led to an increase in the

majority carrier density and a decrease in the resistance of space

charge region On the other hand, the ss-DNA is in a flexible random

structure and can penetrate into the polymer pores to increase the

ionic concentration in the polymer film After hybridization, the

ds-DNA is in a helical formation, resulting in significant stiffness of the

functionalized polypyrrole that leads to a decrease in intrinsic

conjugation of the polymer backbone and causes an increase in the

charge-transfer resistance

Peng et al suggested a DNA detection system in which DNA

sample fragments were entrapped in the PPy film and CdS

nano-particle labeled ODN probes were used to amplify the impedance

change[77] The equivalent circuit model consisted of a solution

resistance (Rs), an element of resistance (Re) in parallel with

a capacitance (Ce), representing the Au electrode, and an element of

interfacial charge-transfer resistance (Rct) in series with a Warburg

impedance (W) A constant phase element (CPE) (in parallel with W

and Rct) acts as a non-ideal capacitor The experimental data was

well fitted by this model and the fitting results showed an increase

in charge transfer resistance upon binding of complementary CdS–

ODN nanoparticle probes Besides taking the change of

charge-transfer resistance as the sensor response, the authors also

suggested that the change in impedance at a fixed frequency could

be used as the sensor response

In another study a terthiophene polymer bearing a carboxyl

group was polymerized onto a glassy carbon electrode and used for

the preparation of a DNA sensor[78] The impedance spectra were

recorded before and after hybridization at open circuit potential

without any redox species A decrease in total impedance was

obtained after hybridization, and the highest differences in

admittance (¼1/jimpedancej) were observed at w1 kHz The

mechanism for the decrease in the impedance was not investigated

in detail, which the authors attributed to the higher conductivity of

double stranded ODN compared with ss-ODN Peng et al reported

the synthesis of a terthiophene bearing an unsaturated side chain

and used the resulting polymer for DNA hybridization detection

[79] The impedance spectra were measured without a redox probe

at 800 mV, a potential at which the polymer is in an oxidized state

The hybridization caused a decrease in the impedance, similar to

Ref.[78] Peng et al further studied the mechanism using

electro-chemical quartz crystal microbalance (EQCM) and the authors

suggested that the dopants play an important role in the

imped-ance change [80] When CF3SO3was used as the dopant (which

provided the best sensor response), EQCM results illustrated that the dominant ion movement during the polymer redox processes is cation movement Thus, the hybridization, which caused an increase in the negative charge due to the formation of duplex DNA, facilitated cation movement during the doping process, resulting in

an increase in the conductance of the polymer film

Gautier et al prepared a functionalized polythiophene matrix for label-free DNA hybridization detection by copolymerizing an arylsulfonamide modified thiophene with 3-methylthiophene

[61,81] The impedance spectra were recorded without any redox species at þ1.1 V The hybridization caused a decrease in the impedance (Fig 6A) An equivalent circuit was suggested to describe this system, as shown inFig 6B The resistive component

Rewas attributed to the sum of the electrolyte resistance and the resistance of the electrode material The first parallel element circuit (C1, R1) and the second one (Q2(R2Q1f)) are responsible for the semicircles observed in experimental data in the high frequency and low frequency domains, respectively The fitting results showed that the resistance R1decreases while the capaci-tance C1did not significantly change upon hybridization For the second circuit element (Q2(R2Q1f)), R2 also decreased while the pseudo-capacitance C02increased slightly The authors suggested that the decreases of the resistive components under hybridization were caused by the formation of the double helix structure which liberates the surface from the random coil conformation of the ss-DNA and restores a partial anionic exchange at the interface between film and electrolyte The increase in the density of nega-tive phosphate groups at the surface was responsible for the increase of the pseudo-capacitance C02

Fig 6 A: Nyquist diagrams recorded at þ1.1 V vs SCE from 100 kHz to 0.1 Hz with perturbation amplitude of 10 mV, onto a DNA probe modified-copolymer-coated Pt quartz crystal The working electrode was dipped in a deaerated TE/0.6 M NaCl buffer solution (triangles), next in the same solution after exposure to the non-comple-mentary DNA sequence (squares) and finally after exposure to the fully complenon-comple-mentary DNA target (circles) The filled symbols correspond to the experimental data and the empty to the calculated data B: Equivalent circuit Reprinted with permission from Ref [61] Copyright Elsevier 2008.

More recently, the same group compared non-Faradaic

imped-ance spectra with Faradaic impedimped-ance spectra for the same system

to reveal different changes in the impedance modulus [82]

Generally, the Faradaic impedance measurement described the

kinetics of electron transfer processes while the non-Faradaic

impedance was related to alterations in the capacitance and

molecular layer organization [16] The authors found that the

impedance features obtained by the Faradaic approach were

dramatically different from those obtained using the non-Faradaic

approach For the non-Faradaic impedance, the hybridization

caused a decrease in the semicircle diameter in Nyquist plots, while

an increase in the semicircle diameter was observed in Faradaic

measurements due to electrostatic repulsion between the negative

charges of the redox probes and negatively charged DNA

Further-more, the effect of the length of target DNA has been investigated

Target ss-DNA with 675 bases that was much longer than the probe

(42 bases) caused an increase in the impedance modulus, which

was diametrically opposite to the change in non-Faradaic

imped-ance observed upon hybridization with a 37-base DNA target The

authors ascribed to differences in the organization of the DNA

modified layer after hybridization When the probe and the target

have the same length, the hybridization resulted in an opening of

the interface to mobile ions in solution When the target was much

longer than the probe, the double helix extended into the solution

by way of a flexible single-stranded DNA sequence, which

pre-vented access of the anions and caused an increase in the

impedance

3.4 Photocurrent spectroscopic detection

Photoelectrochemical techniques have been employed

increas-ingly for investigating thin photoconducting films and corrosion

layers on metals and alloys[83,84], because of the simplicity of the

experimental setup, the possibility of monitoring in situ surface

changes with time, and the ability to investigate very thin films

Recently this technique has been used for the detection of DNA

hybridization[85–87]and damage[88,89]

Conducting polymers can exhibit a good photocurrent response,

so this technique has great potential for label-free DNA

hybridiza-tion detechybridiza-tion However, DNA sensing based on the photocurrent

properties of conducting polymers has not been fully exploited The

initial work on photocurrent spectroscopy as a transduction tool for

direct detection of DNA hybridization based on CPs was undertaken

by Lassalle et al.[90–92] The photocurrent response under white

light illumination was analyzed for a polypyrrole copolymer with

one monomer unit modified by grafted ODN, and the copolymer

exposed to a blank buffer solution, and buffer solutions containing

either complementary or non-complementary ODNs (Fig 7) When

exposed to the blank buffer, and after interaction with

non-complementary ODN, the pyrrole-ODN showed a similar

photo-current response, while the photophoto-current was much lower

following hybridization The reason for the decrease of

photocur-rent was not clear It was suggested that it may originate from

a different physiochemical behavior for the polymer film caused by

formation of a DNA double helix The band gap energies for direct

electronic transition in these three cases were estimated, with

values of 2.9, 2.85, and 3.1 eV for the polymer, the polymer after

interaction with non-complementary ODN, and for the hybridized

film, respectively These values were higher than those obtained for

polypyrrole (e.g 2.2–2.6 eV), indicating a less photosensitive film

due to the presence of oligonucleotides The photocurrent

evolu-tion during hybridizaevolu-tion revealed that the kinetics of the process

could be followed However, these sensing films were not

completely characterized in terms of sensitivity, reproducibility and

the linearity of a calibration curve

4 DNA sensors based on different classes of CPs 4.1 DNA sensors based on polypyrrole and its derivatives Polypyrrole is one of the most extensively used conducting polymers in biosensor designs due to its good biocompatibility and polymerization at neutral pH[93] Wang et al illustrated that short ss-ODN probes could be entrapped in a polypyrrole film as the dopant during film growth and still maintain an affinity for target ODNs [51,52] Alocilja et al reported a DNA sensor for rapid detection of Escherichia coli prepared using same methodology

[94] The recognition element was a 25-base pair oligonucleotide specific for E coli derived from the uidA gene that codes for the enzymeb-Dglucuronidase A DNA concentration of 1m mL1was detected by cyclic voltammetry and the analysis was complete in

15 min A pulsed amperometric detection of target DNA in PCR-amplified amplicons with platinum electrodes modified by single-stranded DNA (20 bases) entrapped within polypyrrole has also been reported [95] The detection time was 30–35 min and the sensor response to a complementary target was higher than for

a non-complementary target by a factor of at least 6–8 Komarova

et al prepared ODN-doped PPy sensor films to detect the pathogen Variola major by means of chronoamperometry[96] It was estab-lished that thinner films with smaller or more highly concentrated dopant ions produced stronger amperometric signals Blocking of the film surface with fragmented half thymus DNA resulted in complete disappearance of the non-specific signal when ultra-thin (Langmuir–Blodgett) films were tested, while the specific signal from complementary ODN remained unaffected Additionally, lowering the potential during the hybridization reduced the non-specific signal Under optimal conditions a detection limit of 1.6 fmol of target ODN in 0.1 mL (16 pM) was achieved For this type

of DNA sensors, the obvious advantages are simplicity and fast detection, however, steric hindrance and poor accessibility of the probe entrapped within the film to the analyte result in poor hybridization efficiency that greatly limits sensitivity and selectivity

In order to overcome these disadvantages, a variety of func-tionalized pyrrole monomers have been developed and used for DNA detection, as shown inFig 8 Livache et al developed a pyrrole monomer bearing an ODN (Fig 8, monomer 1) and copolymerized this with pyrrole to realize addressable multiple probe immobili-zation [54,55] The hybridization was detected by photocurrent

Fig 7 Normalized photocurrent spectra of copolymer film (:), of copolymer film in presence of a non-complementary oligonucleotide (A) and copolymer film in pres-ence of a complementary oligonucleotide (-) Reprinted with permission from Ref.

[92] Copyright Elsevier 2008.

spectroscopy[92] The same group also prepared a biotinylated

pyrrole monomer (Fig 8, monomer 2) [64] The resulting

bio-tinylated polypyrrole film could be used for reversible biobio-tinylated

ODN probe immobilization using the strong biotin/avidin

interac-tion Thompson et al prepared a pyrrole monomer modified with

a phosphonic acid group (Fig 8, monomer 3)[58] The DNA probe

was immobilized onto the resulting polymer film with the help of

magnesium cations that served as a bridge between the

phos-phonic acid group of the grafted polymer and the phosphate group

of the oligonucleotide probe This type of linkage makes the

oligonucleotide offset from the surface of the polymer, giving it

some freedom of movement and easing the effect of steric

hindrance on the hybridization event The hybridization was

detected using cyclic voltammetry, while the sensitivity was not

investigated The same group developed this system further at

a microelectrode[97]and applied the sensor to hepatitis C virus

detection[98]

Ionescu et al used poly(pyrrole-NHS) (Fig 8, monomer 4) to

covalently anchor an amino-21-mer oligonucleotide probe for

detecting a short cDNA sequence from the West Nile Virus (WNV)

by an amperometric method[99] After incubation with a target model of the WNV cDNA, the modified electrode was further incubated in a complementary biotinylated 15-mer WNV cDNA solution followed by specific attachment of a biotinylated glucose oxidase via an avidin bridge The hybridization event was then monitored at 0.6 V vs Ag/AgCl by amperometric detection of H2O2, generated by the enzyme marker in the presence of glucose Since the product of the enzyme-catalyzed reaction was detected, the sensitivity of the sensor was related to the permeability of the (pyrrole-NHS) film With the aim of increasing permeability, the polymer film was overoxidized until its conductivity significantly decreased A relatively short hybridization period (2 h) allowed the convenient quantification of the WNV DNA target in the range of

1010–1015g mL1and a detection limit of 1 fg mL1 Because 3-substitution of pyrrole is more favorable than N-substitution, with regard to maintaining the high intrinsic conductivity of the polymer, Garnier et al prepared a functional-ized pyrrole monomer bearing an easy leaving group, N-hydroxy-phthalimide (Fig 8, monomer 5) [28,57] An amino-substituted ODN was grafted onto the precursor copolymer, poly(3-acetic acid

N

NH N

O O

HO

O

O

ODN

N

P O

Mg2+O P

O HO O ODN

N H

O O

N

N H

O OH

N H

HO O

N H

O OH

N (CH2)13

8

NH C (CH2)4

NH HN

O

O

N

H

9

HN

O

N O

O O

N

4

O O

N

S S

Fig 8 Structures of functionalized pyrrole monomers for DNA sensors.

pyrrole-co-3-N-hydroxyphthalimide pyrrole), by a direct chemical

substitution of the easy leaving group, N-hydroxyphthalimide (the

sensor response to the target ODN is shown inFig 5) The detection

limit was found to be 2 nM A pyrrole monomer bearing a relatively

long butanoic acid side chain (Fig 8, monomer 7) reported by Peng

et al., was expected to position the ODN probe away from the

copolymer backbone and allows easy hybridization with

comple-mentary ODN sequences [18] In both of the above cases, the

hybridization was detected by cyclic voltammetry and the

sensi-tivities were related to the redox properties of polymers In order to

increase the sensitivity of DNA detection, a

ferrocenyl-functional-ized pyrrole (Fig 8, monomer 6) was prepared [100] and ODN

probes were subsequently grafted onto the obtained

ferrocenyl-functionalized polymer film In this arrangement the ferrocenyl

group on the polymer was used as a probe for DNA detection due to

its high sensitivity to changes in the electronic and steric

envi-ronments and narrow and reversible redox signature Upon

hybridization, a shift in the oxidation wave of the ferrocenyl groups

to more positive potentials and a decrease in the oxidation current

were observed The results were explained by a decrease in the

permeability of the polymer film to dopant ions and changes in the

polymer backbone conformation The estimated detection limit

was 2 pMof the target ODN molecule In that study, the

electro-polymerization was carried out first in an organic solvent, followed

by grafting of DNA probes in an aqueous medium This strategy

would be unsuitable for multiprobe addressing on a chip Bouchet

et al developed a technique allowing one-step electro-addressing

of probes on a microarray and sensitive and label-less

multi-detection of DNA targets in solution[101] Firstly, a

electropolymerized in an aqueous medium was synthesized The

electrochemical copolymerization of this monomer with monomer

2, bearing different sequences of ODN, and monomer 3 was then

carried out in a buffered solution using a miniaturized graphite

electrode network, as shown in Fig 9 The hybridization was

detected by changes in the ferrocene oxidation current Good

selectivity between Human Immunodeficiency Virus and Hepatitis

B Virus targets was achieved and the detection limit reached

100 pM

More recently, Peng et al investigated the effect of the ‘linker’

group (a functionalized side chain that links the polymer backbone

and the bioprobe) on the resulting sensor properties [102,103]

Pyrrole monomers with unsaturated side chains were synthesized

(Fig 8, monomers 8 and 9) The motivation for using a conducting

polymer with an unsaturated carbon side chain was based on the

idea that extension of the main chain conjugation into the side

chain may improve polymer susceptibility to changes caused by

DNA hybridization The results demonstrated that a longer side

chain improves the sensor response It was also shown that

copolymers with unsaturated side-chain functionalization have

superior properties for use in biosensor applications compared to

those with a saturated side chain The resulting sensor had good

selectivity and a detection limit of 0.5 nM

Recent advances in nanotechnology have opened up new

possibilities for DNA sensor design The sensitivity and other

attributes of sensor can be improved by using nanomaterials which

have superior physical and chemical properties over their bulk

counterparts, because of effects such as quantum confinement,

a mini-size effect, surface effects and macro-quantum tunnelling

effects Until now, conducting polymer nanomaterials with

different morphologies such as nanoparticles, nanowires and

nanotubes have been prepared by chemical or electrochemical

methods (see a recent review paper[104]) Ramanathan et al.[75]

reported the preparation of biologically functionalized polypyrrole

nanowires by electropolymerization from an aqueous solution of

pyrrole monomer in the presence of a model biomolecule

(avidin-or streptavidin-conjugated ZnSe/CdSe quantum dots) and within

a 100 or 200 nm wide and 3mm long channels between gold electrodes (Fig 10) After addition of biotin–DNA, the avidin– and streptavidin–polypyrrole nanowires generated a rapid change in resistance to 1 nM of biotin–DNA The authors suggested the possibility of single-molecule detection by adjusting the nanowire’s conductivity to a value closer to the lower end of the semi-conductor This work illustrated the concept of biological modifi-cation of PPy nanowires, and the detection of complementary DNA targets based on this principle is currently underway in author’s laboratory Cai et al prepared polypyrrole and multi-walled carbon nanotubes functionalized with carboxylic group (MWNTs–COOH) nanocomposites for indicator free DNA hybridization detection

[105] Firstly, MWNTs–COOH were attached to the glassy carbon electrode and ODN probes were doped within PPy films electro-polymerized on top of this, and the ODN probes served as the sole counter anion during the growth of the film After hybridization,

a decrease in impedance was observed, attributed to a decrease in impedance owing to the higher conductivity of double stranded ODNs compared with the ssODN The process of optimization of hybridization conditions revealed that the sensitivity of the sensor increased dramatically with an increase in the amount of multi-walled carbon nanotubes (MWNT) used The response (difference

in logarithmic impedance values) obtained was five times larger when the optimum amount of MWNTs was used (as compared to the sensor without MWNTs) The estimated detection limit for this simple method was 10 nM, which was further improved to 0.05 nM

by the formation of metallized double-stranded DNA[106] Fu et al used Au–Ag nanocomposites that were adsorbed onto the PPy film

by electrostatic interactions and mercapto ODN probes were self-assembled onto the surface of the modified electrode [107] This sensor was applied to the detection of human immunodeficiency

Fig 9 Monomers involved in the co-electropolymerization onto microelectrodes Reprinted with permission from Ref [101] Copyright Elsevier 2008.

virus (HIV) sequences by electrochemical impedance spectroscopy

and the detection limit reached 0.5 nMof target ODN

Table 1summarizes characteristics of the electrochemical DNA

sensors based on polypyrrole and its derivatives

4.2 DNA sensors based on polyaniline and its derivatives

Compared to polypyrrole, polyaniline is less widely employed in

biosensor designs, due to the polymerization commonly requiring

an acidic environment not suitable for biomolecules However,

polyaniline undergoes two redox processes, mechanically resilient

and environmentally stable, and can be functionalized, making it an

attractive material for DNA sensing applications Gu et al reported

the immobilization of DNA onto a thin layer of polyaniline/poly

(acrylic acid) (PANI/PAA) composite polymer film that was

elec-trodeposited on boron-doped diamond (BDD) thin films[108] The

carboxylic acid residues in the polymer film acted as binding sites

for DNA attachment, whilst the conductive polymer matrix

enhanced electron-transfer between DNA and the diamond surface

The direct oxidation of guanine and adenine in the double helix

DNA was used to detect the hybridization event The advantage of

such a design was a minimal non-specific DNA adsorption, which

was confirmed by fluorescence microscopic and cyclic

voltam-metric measurements This PANI/PAA modified BOD electrode was

further characterized by electrochemical impedance spectroscopy

[109] The impedance measurements showed changes in the

impedance modulus as well as electron-transfer resistance upon

probe DNA immobilization and after hybridization with target DNA

Good selectivity between the complementary DNA targets and the

one-base mismatch targets was demonstrated, as well as sensor

reusability The detection limit was 20 nMmeasured at 1000 Hz

Davis et al investigated the differences between polyethylenimine,

polyaniline and polydiaminobenzene modified screen-printed

carbon electrodes containing single-stranded DNA by an AC

impedance approach [110] Complementary DNA hybridization

gave rise to a lowering of the capacitance of the electrode/polymer film in solution

More recently, Arora et al reported an ultrasensitive DNA hybridization biosensor based on polyaniline electrochemically deposited onto a Pt disc electrode, as shown inFig 11 [111] Acti-vated avidin was covalently attached to the PANI film, followed by the immobilization of a 50-biotin end-labeled oligonucleotide probe via the biotin/avidin interaction The hybridization was detected using both direct electrochemical oxidation of guanine and a redox electroactive indicator methylene blue Compared to a direct elec-trochemical oxidation of guanine, hybridization detection using methylene blue resulted in an enhanced detection limit by about

100 times and reached 2 fM This sensor has been utilized for direct detection of E coli by immobilizing a 50-biotin-labeled E coli probe, and using differential pulse voltammetry in the presence of methylene blue as a DNA hybridization indicator[112] The detec-tion limits for complementary target probe, E coli genomic DNA and E coli were 0.009 ngmL1, 0.01 ngmL1and 11 E coli cells mL1

without PCR amplification and it can be used 5–7 times at temperatures of 30–45C

PANI nanocomposites have also been used in DNA sensing Wu

et al synthesized a polyaniline intercalated graphite oxide nano-composite (PAI/GO) [113] Square wave voltammetric measure-ments showed that single-strand DNA and double-strand DNA alter the redox characteristics of PAI/GO at the PAI/GO modified carbon paste electrode The PAI/GO-modified electrode immobilized with ssDNA by physical adsorption can be utilized to monitor hybrid-ization of complementary ssDNA which resulted in a new peak at

0.27 V A nanocomposite of polyaniline and mercaptosuccinic-acid-capped gold nanoparticles (MSAGNP) was also prepared by the layer-by-layer methodology[114] The MSAGNP inside multilayer films can effectively shift the electroactivity of polyaniline to

a neutral pH which greatly facilitates its biological applications After immobilization of DNA probes onto the gold nanoparticles, DNA hybridization was detected either by electrochemical methods (cyclic voltammetry and AC impedance) or by surface plasmon

Fig 10 (A) SEM image of an Aqd-embedded polypyrrole nanowire (200 nm wide) The EDX analysis of polypyrrole nanowire with embedded Aqd (B) and without embedded Aqd (C) Reprinted with permission from Ref [75] Copyright American Chemical Society 2008.