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Polypyrrole-polyvinyl sulphonate film baseddisposable nucleic acid biosensor

Nirmal Prabhakar a,b, Kavita Arora a, Surinder P. Singh a, Manoj K. Pandey a,Harpal Singh b, Bansi D. Malhotra a,∗

a Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012, Indiab Centre for Biomedical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India

Received 9 November 2006; received in revised form 23 January 2007; accepted 27 January 2007Available online 21 February 2007

bstract

Double stranded calf thymus deoxyribonucleic acid entrapped polypyrrole-polyvinyl sulphonate (dsCT-DNA-PPy-PVS) films fabricated ontondium-tin-oxide (ITO) coated glass plates have been used to detect organophosphates such as chlorpyrifos and malathion. These disposable dsCT-NA-PPy-PVS/ITO bioelectrodes have been characterized using cyclic voltammetry, Fourier-transform-infra-red (FTIR) spectroscopy and atomic

orce microscopy (AFM), respectively. These biosensing electrodes have a response time of 30 s, are stable for about 5 months when stored in desic-ated conditions at 25 ◦C and can be used to amperometrically detect chlorpyrifos (0.0016–0.025 ppm) and malathion (0.17–5.0), respectively. Thedditive effect of these pesticides on the amperometric response of the disposable dsCT-DNA-PPy-PVS/ITO bioelectrodes has also been investigated.

2007 Elsevier B.V. All rights reserved.

vinyl

pbp(1bcs[oom[gcb

eywords: Nucleic acid biosensor; Deoxyribose nucleic acid; Polypyrrole-poly

. Introduction

There is an urgent need for sensitive and faster detection ofrganophosphates (OPs) as these are widely used as compo-ents of various insecticides formulations for agricultural andomestic purposes. OPs are unique class of contaminants andhemical warfare (CW) agents that usually show low environ-ental persistence, but many of them are extremely hazardous to

uman health and ecosystem [1,2]. Wide range of organophos-hates such as carbofurans, dichlorous, paraoxan, malaoxaon,arathion, chlorpyrifos and malathion, etc. have been stud-ed for neurotoxicity. These OPs are powerful inhibitors ofarious nerve agents such as esterase enzymes, acetyl/butyryl-holinesterases, etc. since these have been reported to causeetabolic and organ malfunctioning [3–5]. Besides this, OPs

re known to cause DNA damage and accelerate development

f cancer [4,5].

Many enzyme (acetylcholine esterase and butylcholinesterase) based biosensors for the detection of the organophos-

∗ Corresponding author. Tel.: +91 1125734273; fax: +91 112572698.E-mail address: bansi.malhotra@gmail.com (B.D. Malhotra).

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003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2007.01.084

sulphonate; Electrochemical entrapment; Chlorpyrifos; Malathion

hates have been developed [6–10]. A continuous flow systemased enzymatic biosensor could be used to detect the chlor-yrifos oxon from 0.72 × 10−6 M (240 ppb) to 73 × 10−9 M24 ppb) when the incubation time was increased from 1 to6 min [6]. A potentiometric biosensor based on acetyl andutyl choline esterase cross-linked polyaniline onto glassyarbon electrode has been used to detect various pesticidesuch as Caumaphos, Trichlorfon, Methiocarb, Aldicarb, etc.7]. An amperometric biosensor has been used to detectrganophosphates such as paraoxon, methylparthion usingrganophosphorus hydrolase immobilized onto carbon nanotubeodified transducer up to 0.15 �M and 0.8 �M, respectively

8]. Besides this, amperometric whole cell biosensor usingenetically engineered Moraxella sp. and Pseudomonas putidaontaining surface expressed organophosphorus hydrolases haseen utilized to detect organophosphates [9]. Various techniquesncluding enzymatic biosensors and whole-cell biosensorsave longer response times (8–30 min), low stability of about–5 weeks and low sensitivity [10]. Therefore, estimation of

rganophosphate (OP) neurotoxins with desired sensitivity innvironmental samples is presently a challenge.

DNA biosensors based on guanine oxidation have recentlyeen proposed for detection of pesticides [11]. These DNA

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ensors utilize the interaction of DNA molecule with variousompounds either by monitoring changes in the DNA redoxroperties (i.e. oxidation of guanine) or with an electro-activenalyte intercalated on a DNA layer [11–13]. Electrochemi-al techniques such as cyclic voltammetry [14], square waveoltammetry [15], differential pulse voltammetry [16], chrono-otentiometry [17] have been used to study the interactionf various compounds with DNA immobilized onto respec-ive electrodes. There are reports about the use of immobilizedouble stranded-calf thymus (dsCT-DNA) onto screen-printedarbon electrodes to detect the presence of various toxicants16,15]. The interaction of mitoxantrone with double strandednd single stranded-calf thymus DNA has been studied usingifferential pulse voltammetry and cyclic voltammetry at a car-on paste electrode [18]. However, the inherent stability ofiomolecules in a desired environment including DNA presentlyolds the key towards the pesticide detection

Conducting polymers are known to provide enhancedensitivity to electrochemical biosensors [19]. Besides this,onducting polymers provide considerable flexibility for themmobilization of biomolecules by providing pendant func-ional groups and direct electrochemical synthesis onton electrode surface, while simultaneously trapping theiomolecule [20]. Compared to sol–gels, self-assembledonolayers (SAMs) and carbon-based materials, conducting

olymers can efficiently transfer electric charge resulting due tobiochemical reaction. We report the electrochemical entrap-ent of the double stranded calf thymus (dsCT) DNA onto

olypyrrole-polyvinyl sulfonate (PPy-PVS)/indium-tin-oxideITO) bioelectrode for investigating the interaction of chlorpyri-os and malathion.

. Materials and methods

.1. Chemicals and reagents

Pyrrole (Py), PVS (polyvinyl sulphonic acid), dou-le stranded Calf thymus DNA (dsCT-DNA), malathion,hlorpyrifos, potassium monohydrogen phosphate and potas-ium dihydrogen phosphate and EDTA were procured fromigma–Aldrich, Milwankee, USA. Indium-tin-oxide (ITO)oated glass plates were obtained from Balzers UK. All thehemicals and reagents used in the present studies were ofolecular biology (MB) grade. These reagents were prepared

n de-ionized water (Milli Q 10 TS) and the solutions and glass-ares were autoclaved prior to being used.

.2. Electrochemical preparation of disposablesCT-DNA-PPy-PVS/ITO bioelectrodes

dsCT-DNA entrapped PPy-PVS films were polymerizedy chronopotiometrically in a three-electrodes electrochemi-al cell having Ag/AgCl as reference, platinum as a counter

nd ITO glass plate (1 × 2 cm2) as a working electrode usinghe Potentiostat/Galvanostat (Model 273A, Princeton Appliedesearch). Monomer solution containing 0.1 M Py, 0.1 M PVSnd 10 �g mL−1 dsCT-DNA (suspended in Tris (10 mM)–EDTA

(pbr

1 mM) pH 8.0) was subject to constant current (200 �A) forbout 15 min to obtain dsCT-DNA-PPy-PVS films onto ITOlass plates at working area of about 1 cm2. Nitrogen wasubbled in monomer solution prior to polymerization. Cyclicoltammograms of PPy-PVS films (thickness and conductivity10 �m and 120 S cm−1, respectively) coated onto ITO glass

lates were used to compare the cyclic voltammograms of dis-osable dsCT-DNA-PPy-PVS/ITO bioelectrodes.

The amount of dsCT-DNA entrapped in a PPy-PVS filmas quantified by measuring the optical absorbance (Shimadzu,60A) of DNA at 260 nm in the aqueous solution contain-ng monomer Py (0.1 M) and PVS (0.1 M) before and afterhe polymerization. The amount of the dsCT-DNA in thesCT-DNA-PPy-PVS bioelectrode has been found to be about9 �g cm−2 of bioelectrode.

A series of dsCT-DNA-PPy-PVS/ITO bioelectrodes wererepared and were kept under desiccated conditions at 25 ◦Chen not in use. The performance of dsCT-DNA-PPy-PVS/ITOioelectrodes were analyzed using cyclic voltammetry asfunction of time. The prepared dsCT-DNA-PPy-PVS/ITO

ioelectrodes were treated with analytes (chlorpyrifos andalathion) of desired concentration for 30 s. These treated elec-

rodes were rinsed with autoclaved deionized water prior to CVeasurements. We found that 30 s were sufficient for obtaining

he maximum reduction in guanine oxidation current for eachf the toxicant concentration (chlorpyrifos/malathion).

.3. Characterization

Both PPy-PVS/ITO and dsCT-DNA-PPy-PVS/ITO bioelec-rodes have been optimized for different scan rates (10, 20, 30nd 40 mV s−1) using cyclic voltametry in phosphate buffer0.05 M, pH 7). These PPy-PVS/ITO and dsCT-DNA-PPy-VS/ITO bioelectrodes have been characterized using Fourier

ransform infrared (FTIR) spectroscopic (Perkin-Elmer Spec-rum, BX) and atomic force microscopic (AFM) (NanoscopeII A) studies. The response of these disposable dsCT-DNA-Py-PVS/ITO bioelectrodes has been studied using cyclicoltammetry for the detection of chlorpyrifos and malathion,espectively. The percentage reduction in the oxidation peakrea is calculated as the effect of interaction between the dis-osable dsCT-DNA-PPy-PVS/ITO bioelectrodes with each ofhe pesticides [10].

. Results and discussion

.1. Cyclic voltammetric studies

Fig. 1 shows the cyclic voltammograms (CVs) ofPy-PVS/ITO and disposable dsCT-DNA-PPy-PVS/ITO bio-lectrodes in phosphate buffer (0.05 M) with pH 7.0 at 25 ◦C.he oxidation peaks can be clearly seen at about 0.6 V and 1.1 V

or disposable PPy-PVS/ITO and dsCT-DNA-PPy-PVS/ITO

Fig. 1) bioelectrodes, respectively. The observed oxidationeak of disposable dsCT-DNA-PPy-PVS/ITO bioelectrode cane attributed to the irreversible guanine oxidation of both theandomly distributed dsCT-DNA molecules stationed at the PPy-

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ig. 1. Cyclic voltammogram of dsCT-DNA-PPy-PVS/ITO bioelectrode andf PPy-PVS/ITO electrode obtained at 20 mV s−1 (−600 mV to 1400 mV) inhosphate buffer 0.05 M, pH 7.0.

VS surface and those entrapped in the PPy-PVS matrix. Theuanine oxidation peak (Fig. 1) may arise from the lone pair oflectrons of NH2 group present at the C-2 carbon of the guanineolecules in the dsCT-DNA on application of potential as these

lectrons are the most loosely bound due to sp3 hybridization ofhe concerned nitrogen atoms resulting in the minimum stericindrance to these electrons. Both the PPy-PVS and dsCT-DNA-Py-PVS films are irreversible electron transfer systems; hence,

he surface concentration of the ionic species can be estimatedsing following equation [21,22].

p = 0.227nFAC∗0k0 exp

[−αnaF

RT(Ep − E0′

)

](1)

here n is the number of electrons transferred (2), F isaraday constant (96584 C mol−1), R is the gas constant8.314 J mol−1 K−1), C∗

0 is the surface concentration of thesCT-DNA-PPy-PVS films (mol cm−2), k0 is the rate constant0.5), −αnaF/RT is the slope of the ln(ip) versus (Ep − Eo′) atifferent scan rates. Ep is the cathodic peak potential and Eo′ ishe formal potential and has been found to be as 5.52 × 10−9

nd 63.53 × 10−9 mol, respectively.

.2. FTIR studies

Fig. 2 shows the FTIR spectra of PPy-PVS/ITO andsCT-DNA-PPy-PVS/ITO bioelectrodes, respectively. Theharacteristic peaks seen in the range of 500–1300 cm−1 (590,77, 778, 810, 856, 961, 1019, 1082, 1130, 1278 cm−1) haveeen ascribed to polypyrrole [23]. The 3200 cm−1 peak in theTIR spectra of PPy-PVS/ITO electrodes is due to NH stretchingibration while the enhancement of peak intensity at 3200 cm−1

n the spectra of dsCT-DNA-PPy-PVS/ITO electrode can per-aps be attributed to the association of nucleic acid bases [24].

he peaks seen at 1716, 1492, 1609, 1663 cm−1 correspond

o the guanine, cytosine, adenine and thymine, respectively25,26]. The peaks observed at 1717, 1507, 1609, 1683 cm−1

an be ascribed to the presence of guanine, cytosine, adenine

pri

ig. 2. FTIR spectra: % transmittance vs. wave number (1 cm−1) of PPy-VS/ITO (· · ·) and dsCT-DNA-PPy-PVS/ITO (—) bioelectrode.

nd thymine, respectively and the slight shift in these peaks maye attributed to the interaction of the DNA molecule with theolypyrrole backbone. The small peak obtained at 2750 cm−1

s attributed to the NH stretching vibration in NH N hydrogenonds. Peaks observed at 1690 cm−1 (C N), 1360 cm−1 (C N)nd the 1034 cm−1 peak are due to the O N vibrations of theitrogenous bases revealing the incorporation of the DNA. Thencreased peak intensity of the 3200 cm−1 peak (Fig. 2) furtherestifies the attachment of DNA to the polypyrrole-PVS back-one. The 1540 cm−1 vibration band is due to the C C bondssociated with C N stretching and bending vibration.

.3. AFM studies

Fig. 3a and b shows the AFM pictures of PPy-PVS and dsCT-NA-PPy-PVS/ITO bioelectrodes, respectively. The roughnessarameters of PPy-PVS/ITO and dsCT-DNA-PPy-PVS/ITOave been found to be about 40 nm and 23 nm, respectively.he decreased value of the roughness parameter (Fig. 3b) indi-ates entrapment of DNA onto smoother dsCT-DNA-PPy-PVSurface [27,28].

.4. Response studies

The cyclic voltametric response of disposable dsCT-DNA-Py-PVS/ITO bioelectrodes have been studied after interactionith different concentrations of chlorpyrifos (0.0016–0.02 ppm)

Fig. 4) and malathion (0.17–6.0 ppm) (Fig. 5). It has beenbserved that the peak height of the guanine oxidation ofisposable dsCT-DNA-PPy-PVS/ITO bioelectrodes in cyclicoltammograms decreases with the increase in the concentrationf respective pesticide. The percentage reduction in the variousuanine oxidation peak areas has been calculated and has beenlotted as a function of respective concentration of chlorpyrifosnd malathion (Figs. 4 and 5).

Fig. 4 shows some typical cyclic voltammograms of dis-osable dsCT-DNA-PPy-PVS/ITO bioelectrodes revealing theeduction in the oxidation peak of the DNA (guanine) with thencrease in chlorpyrifos concentration (0.0, 0.0016, 0.005, 0.01,

(b) d

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Fig. 3. AFM pictures of (a) PPy-PVS and

.025 ppm). Reduction in the guanine oxidation peak area haseen found to be 100% at the 0.02 ppm concentration of chlor-yrifos and the detection limit of the bioelectrode has been founds 0.0016 ppm. It has been found that there is linear increase inercentage reduction in the guanine oxidation peak area of dis-osable dsCT-DNA-PPy-PVS/ITO bioelectrode follows the Eq.2) for the chlorpyrifos within the concentration range (0.0016-.025 ppm).

eduction in peak area (%)

= 18.42 × concentration of chlorpyrifos (ppm) − 0.216

(2)

Similarly, there is linear increase in percentage reduction inhe guanine oxidation peak area of disposable dsCT-DNA-PPy-VS/ITO bioelectrode follows Eq. (3) for malathion within the

ig. 4. Typical cyclic voltammograms of dsCT-DNA-PPy-PVS films showingecrease in the peak height of guanine oxidation for increasing concentrationf chlorpyrifos (0.0016–0.025 ppm) at scan rate 20 mV s−1 in phosphate buffer.05 M, pH 7.0.

avo

Fdm2

sCT-DNA-PPy-PVS/ITO bioelectrodes.

oncentration range (0.17–5.0 ppm).

eduction in peak area (%)

= 18.42 × concentration of malathion (ppm) + 5.9 (3)

The observed linear decrease in the guanine oxidation cur-ent (Figs. 4 and 5) of disposable dsCT-DNA-PPy-PVS/ITOioelectrode arises due to the respective interaction of chlor-yrifos and malathion with DNA. It has been reported thatrganophosphates being phosphorylating agents may damagehe entrapped DNA through nucleophilic attack [29]. Both

alathion and chlorpyrifos bearing phosphorothioate group arexpected to phosphorylate the amino group at C-2 position of

ttack (Scheme 1). These bulkier groups of the pesticides pro-ide resistance to the electron flow resulting in the reduced valuef the guanine oxidation current since the lone pair of amino

ig. 5. Typical cyclic voltammograms of dsCT-DNA-PPy-PVS films showingecrease in the peak height of guanine oxidation for increasing concentration ofalathion (0.0, 0.17, 0.52, 1.0, 2.0, 3.0, 4.0, 5.0 ppm, respectively) at scan rate

0 mV s−1 in phosphate buffer 0.05 M, pH 7.0.

Scheme 1. The mechanism of the interaction of chlorpyrifos and malathion with the DNA entrapped into the polypyrrole-polyvinyl sulphonate films.

Fig. 6. FTIR spectra of dsCT-DNA (a) 400–4000 cm−1; (b) 1600–1800 cm−1; (c) dsCT-DNA + chlorpyrifos; (d) dsCT-DNA + malathion in Tris (10 mM)–EDTA(1 mM), pH 8.0.

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Table 1Response characteristics of dsCT-DNA entrapped polypyrrole-polyvinyl sulphonate (dsCT-DNA-PPy-PVS/ITO) bioelectrodes for detection of organophosphatesMatrix Sensing element Method of

immobilizationMethod of sensing Analyte Detection range

(ppm)Detection limit Response time Correlation

coeffiecient (r)Shelf life (drycondition)

Effect of interferrents(inorganic salts) Na+

(50 ppm) + Ca2+ (80 ppm) + Cl−(520 ppm) + Mg2+ (60 ppm)

Reference

PVA-SbQ polymer Acetylcholineesterase andbutylcholineesterase

Photo-co-polymerization

Amperometric(flow cell)

Chlorpyrifos,methylchlorppyrifos

0.0240–0.24 ppmand 0.25–0.7 ppm,respectively

24 ppb and 25 ppm,respectively

16 min. and 8 min,respectively

Not reported Not reported Not reported [6]

Polyaniline ontoglassy carbonelectrode

Butyl cholineesterase

Covalentimmobilizationusingglutaraldehydecross linking

Potentiometric Caumaphos,Trichlorfon,Mthiocarb,Aldicarb

7 × 10−8 to1 × 10−6 mol L−1;2 × 10−6 to1 × 10−5 mol L−1;1 × 10−7 to8 × 10−5 mol L−1;and 8 × 10−7 to5 × 10−3 mol L−1

4 × 10−8 mol L−1;8 × 10−7 mol L−1;8 × 10−7 mol L−1;and6 × 10−7 mol L−1

1.5–2.0 min. Not reported <20 days Not reported [7]

Polyaniline ontoglassy carbonelectrode

Acetyl cholineesterase

Covalentimmobilizationusingglutaraldehydecross linking

Potentiometric Trichlorfon,Caumaphos,Mthiocarb,Aldicarb

2.5 × 10−7 to5 × 10−6 mol L−1;2 × 10−6 to2 × 10−5 mol L−1;6 × 10−7 to2 × 10−5 mol L−1;and 4 × 10−7 to4 × 10−5 mol L−1

9 × 10−8 mol L−1;1 × 10−6 mol L−1;4 × 10−7 mol L−1;and2 × 10−7 mol L−1

1.0–1.8 min. Not reported <15 days Not reported [7]

Carbon nanotube Organophosphoroushydrolase

Physical adsorption Cyclic voltamery Paraoxon, Methylparathion

2–4 �M 2–10 �M 0.15 �M 0.8 �M Not reported Not reported Not reported Not reported [8]

Carbon pasteelectrode

Geneticallyengineered cells

Carbon pastecontaining cellshavingorganophosphoroushyrolase

Amperometric Paraoxon, Methylparathion

0.2 �M 1 �M 0.2–40 �M1–175 �M

Not reported Not reported 45 days Not reported [9]

PPy-PVS/ITO dsCT-DNA Eletrochemicalentrapment

Cyclic voltametry Chloropyrifos 0.0016–0.02 0.0016 30 s 0.91 5 months No effect Present work

PPy-PVS/ITO dsCT-DNA Eletrochemicalentrapment

Cyclic voltametry Malathion 0.17–5.0 0.17 30 s 1.0 5 months No effect Present work

Fig. 7. Cyclic voltammograms of dsCT-DNA-PPy-PVS/ITO films as a function of Chlorpyrifos (0.005 ppm) + Malathione (2.0 ppm), (b) chlorpyrifos( ospha

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0.005 ppm) + 0.25 ppm, 0.5 ppm, 1.0 ppm, 2.0 ppm, 3.0 ppm of malathion in ph

roup at C-2 of guanine is not easily available for oxidation.he observed linear decrease in the guanine oxidation current

Figs. 4 and 5) can be attributed to the formation of 1:1 product atow concentration and 1:2 at high concentration of chlorpyrifosnd malathion (Scheme 1).

Attempts have been made to delineate the mechanism ofsCT-DNA with pesticides (chlorpyrifos and malathion). FTIReasurements of dsCT-DNA in Tris (10 mM)–EDTA (1 mM)

uffer pH 8.0 have been undertaken (Fig. 6a and b). Thenteraction of chlorpyrifos and malathion with the guanineesidue is likely to lead to the formation of product contain-ng the N P S group (Scheme 1). The 748 and 800 cm−1

eaks (DNA–chlorpyrifos derivative) in Fig. 6c and 745 and10 cm−1 peaks (DNA–malathion derivative) in Fig. 6d repre-enting P N bond stretching indicate the formation of productsf chlorpyrifos and malathion with guanine moiety of DNA,espectively. Besides this, efforts have been made to investi-ate the effect of a mixture of pesticides on the amperometricesponse of dsCT-DNA-PPy-PVS/ITO bioelectrode. It is foundhat enhanced reduction in the guanine oxidation peak of dispos-ble dsCT-DNA-PPy-PVS/ITO bioelectrodes is observed in theresence of a mixture of chlorpyrifos (0.005 ppm) + malathion2 ppm) (Fig. 7a). This result is further affirmed by studying theffect of increasing concentration of malathion (0.25–3 ppm)ith 0.005 ppm of chlorpyrifos (Fig. 7b). It has been found

hat there is no effect of interferents (water-soluble salts likea2+, Mg2+, Cl− and Na+ etc.) on the amperometric response ofsCT-DNA-PPy-PVS/ITO bioelectrode when added alongwithesired pesticide solution. Therefore, disposable dsCT-DNA-Py-PVS/ITO bioelectrodes can be used for screening of theeal time water samples suspected of containing genotoxic pes-icides.

The stability of the disposable dsCT-DNA-PPy-PVS/ITOioelectrode has been investigated using CV measurements. We

repared several dsCT-DNA-PPy-PVS/ITO bioelectrodes andhese were stored under desiccated conditions at 25 ◦C. It maye remarked that CVs of these bioelectrodes recorded at an inter-al of 3 days exhibited no loss in the guanine oxidation current

DACd

te buffer 0.05 M, pH 7.0, scan rate 20 mV s−1.

p to a maximum of 150 days after which about 30% reductionn the guanine oxidation current was observed (data not shown).

Table 1 shows the comparison of the results obtained forsCT-DNA-PPy-PVS/ITO bioelectrodes and those reported forhe enzyme based biosensors and the whole-cell based biosen-ors [6–8]. The dsCT-DNA-PPy-PVS/ITO electrode showsesponse time of about 30 s.

. Conclusions

It has been demonstrated that disposable dsCT-DNA-PPy-VS/ITO bioelectrodes have response time of about 30 s and

hese electrodes can be used to detect chlorpyrifos and malathionp to 0.0016 ppm, 0.17 ppm, respectively. It has been revealedhat there is considerable reduction in the response time asell as detection limit as compared to enzyme-based biosensros

Table 1). The presence of inorganic salts as Ca2+, Mg2+, Cl−,nd Na+ does not affect the observed amperometric responsef the dsCT-DNA-PPy-PVS/ITO bioelectrodes for both pes-icides, though these electrodes are disposable in nature andence can be utilized for rapid and initial screening of geno-oxicants in industrial toxic waste, various food and beverages.fforts are being made to improve the stability of the dispos-ble dsCT-DNA-PPy-PVS/ITO biosensing electrodes beyond 5onths.

cknowledgements

We are grateful to Dr. Vikram Kumar, Director, NPL, Newelhi, India for his interest in this work. NP is grateful to

he University Grant Commission (UGC) and Council of Sci-ntific Industrial Research (CSIR), India for the award ofenior Research Fellowship (SRF). Thanks are due to Dr..P Singh for interesting discussions. Prof. M. Iwamoto and

r. Pratima R. Solanki are gratefully acknowledged for theFM studies. Thanks are due to Mr Sunil K. Arya, Ms.hetna Dhand and other members of the group for helpfuliscussions. Financial support received under the DST spon-

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