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Biochemical Engineering Journal 46 (2009) 132–140

Contents lists available at ScienceDirect

Biochemical Engineering Journal

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ron oxide-chitosan hybrid nanobiocomposite based nucleic acid sensor foryrethroid detection

jeet Kaushik a,b, Pratima R. Solanki b, Anees A. Ansari b, Bansi D. Malhotra b,∗∗, Sharif Ahmad a,∗

Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, IndiaDepartment of Science and Technology Centre on Biomolecular Electronics, K. S. Krishnan Marg, National Physical Laboratory, New Delhi 110012, India

r t i c l e i n f o

rticle history:eceived 28 January 2009ccepted 26 April 2009

a b s t r a c t

Nucleic acid sensor has been fabricated via immobilization of single standard calf thymus deoxyribosenucleic acid (ssCT-DNA) onto chitosan (CH)-iron oxide (Fe3O4) nanoparticles based hybrid nanobiocom-posite film deposited onto indium-tin-oxide (ITO) coated glass for pyrethroids [cypermehtirn (CM) andpermethrin (PM)] detection. The ssCT-DNA immobilized onto CH-Fe3O4 nanocomposite/ITO electrode

eywords:iosensoriocatalysisiopolymerse3O4 nanoparticles

has been characterized using scanning electron microscopy (SEM), UV–visible, Fourier transform infrared(FTIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry(DPV) techniques. This disposable ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode is stable forabout two months under refrigerated conditions and can detect CM (0.0025–2 ppm) and PM (1–300 ppm)using DPV technique within 25 s and 40 s, respectively.

anobiocompositelectrochemical detection

. Introduction

Pyrethroids are man-made pesticides that are being used asousehold insecticides and insect repellents [1]. Among the variousyrethroids, cypermethrin (CM, type I) and permethrin (PM, type II)re being increasingly utilized due to increasing food demand. CMs one of the most widely used pyrethroid since it is a biodegrad-ble and fast-acting neurotoxin [2–5]. It causes neurotoxic effecthrough voltage-dependent sodium channel and integral proteinTPase in the neuronal membrane. In vitro and in vivo studiesave shown that it results in free radical-mediated (reactive oxy-en species) tissue damage in brain, liver, erythrocytes and causesancer [2–4]. PM is also used to control a wide range of insectsn agriculture, forestry and public health. A topical exposure of PM

ay cause reduction of 14 macrophage functions; antibody produc-ion in the spleen that may result in decreased thymus weight andellularity, indicating that exposure may produce systemic immuneffects [5].

It has been reported that minute concentration of pyrethroids0.25–1.5 mg/kg bw/day for CM and 5-2000 mg/kg bw/day forM (acute toxic effect)] may affect immune system and centralerve system resulting in cancer and other associated disorders.

∗ Corresponding author. Tel.: +91 11 26981717x3268; fax: +91 11 2698 0229.∗∗ Corresponding author. Tel.: +91 11 4560 9152; fax: +91 11 4560 9312.

E-mail addresses: bansi.malhotra@gmail.com (B.D. Malhotra),harifahmad jmi@yahoo.co.in (S. Ahmad).

369-703X/$ – see front matter © 2009 Published by Elsevier B.V.oi:10.1016/j.bej.2009.04.021

© 2009 Published by Elsevier B.V.

These insecticides can be detected using UV–visible spectroscopy[6], enzyme linked immunosorbent assay [7,8], Fourier transforminfrared spectroscopy [9], gas chromatography–mass spectroscopy[10–12], coupled column choromatography [13], gas chromatogra-phy with electron capture detection [14], electronic nose modularsensor [15], flow injection analysis [16], high performance liquidchromatography [17] and surface plasma resonance, etc. [18]. Thesetechniques are, however, expensive, complicated and require exper-tise. There is thus an urgent need for availability of a simple, rapidand cost-effective technique for detection of desired pesticides. Inthis context, electrochemical DNA biosensors have been consideredas interesting tools for detection of desired genotoxicants in envi-ronmental and biological samples [4]. The immobilization of DNAon a given matrix is a crucial step for fabricating an electrochemicalDNA biosensor. Various matrices such as graphite, carbon elec-trodes, self-assembled monolayers, nanomaterials, biopolymers,conducting polymers and organic–inorganic hybrid nanocompos-ites have been used for fabrication of DNA biosensors usingphysical adsorption, covalent-binding, self-assembly, entrapmentin a polymer matrix and biotin–avidin interaction for detectionof genotoxicants [19,20]. The effect of thiacarbocynine dye withanionic polypeptides (polyglutamic acid, PLGA and co-polymersof polyglutamic acid and tyrosine) and DNA at air/water inter-

face has been studied [21]. The disposable graphite electrodewith electrochemically adsorbed dsCT-DNA has been employed fordetection of toxic aromatic amines such as 2-naphthylamine, 2-anthramine, 1, 2-diamino anthraquinone and acridine orange [22].Electrochemical DNA biosensor based on screen-printed carbon

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lectrode and polypyrrole-polyvinyl sulfonate (PPy-PVS) films haveeen fabricated for monitoring toxicants/pollutants like bisphe-ol, o-chlorophenol and 2-aminoanthracene [23,24]. Glassy carbonlectrodes have been used to immobilize DNA to investigatenteraction with benznidazole [25]. Prabhakar et al. have electro-hemically entrapped dsCT-DNA onto PPy-PVS film for detectionf chlorpyrifos, malathion, 2-aminoanthracene and 3-chlorophenol26,27]. Solanki et al. have covalently immobilized dsCT-DNA usingDC/NHS chemistry onto perchlorate doped polyaniline film foretection of pesticides [4]. It may be noted that DNA electrodesased on glassy carbon and other materials have poor shelf-lives,

ong response times, limited sensitivity and detection limit. Besideshis, these DNA electrodes cannot be used under harsh experimen-al conditions [4,18].

Iron oxide (Fe3O4) nanoparticles due to good biocompatibility,

uperparamagnetism, low toxicity, high electron transfer capabilitynd high adsorption ability have been used as immobilizing matrixor application to biosensors. Further, Fe3O4 nanoparticles exhibitnteresting properties like high surface area, lower mass transferesistance and selective separation for immobilized biomolecules

Scheme 1. Proposed mechanism of interaction between ssCT-DNA/CH-Fe

ring Journal 46 (2009) 132–140 133

from a reaction mixture on application of magnetic field [28–32].However, aggregation of Fe3O4 nanoparticles due to high surfacearea and magnetic dipole interaction between nanoparticles haslimited their biomedical applications. This problem can perhaps beovercome by dispersing Fe3O4 nanoparticles in a biopolymer matrixof chitosan (CH) to prepare CH-Fe3O4 hybrid nanobiocomposite[32].

CH-Fe3O4 nanobiocomposites can be functionalized in a cova-lent attachment and self-assembly making them suitable forbiosensor applications. Kaushik et al. have fabricated CH-Fe3O4nanocomposite film for immobilization of glucose oxidase to detectglucose with improved sensitivity and stability [32]. Zhang et al.have studied direct electrochemistry of hemoglobin immobilizedon carbon coated Fe3O4 nanoparticles for amperometric estima-tion of H2O2 [33]. Zhao et al. have reported that multilayer thin

film of CH-Fe3O4 promotes direct electron transfer of hemoglobin[34]. Liang et al. have synthesized polysaccharide modified ironoxide nanoparticles as an effective magnetic affinity adsorbent forbovin albumin serum [35]. Wang et al. have reported amperometricimmunosensor based on CH-Fe3O4 composite film for estimation

3O4 nanobiocomposite/ITO bioelectrode and pyrethroids (CM/PM).

1 gineering Journal 46 (2009) 132–140

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50 mM [Fe(CN)6]3−/4−as the electrolyte. UV–vis spectrophotome-ter (Phoenix 2200 DPCV) has been used to estimate loading ofssCT-DNA onto CH-Fe3O4 nanobiocomposite/ITO electrode andinteraction of CM and PM with ssCT-DNA. The surface morpho-

34 A. Kaushik et al. / Biochemical En

f ferritin [36]. Lin et al. have fabricated CH-Fe3O4 nanocompositeased chemical sensor for cathodic determination of hydrogen per-xide (H2O2) [37]. Harbac et al. have fabricated carbon electrodeodified by nanoscopic Fe3O4 particles based chemical sensor for

stimation of H2O2 using amperometric technique [38].CH (�-1,4-linked glucosamine oligomer) is a natural cationic

olymer that shows excellent film forming ability, non-toxicity,iocompatibility and can form stable complex with polyanionichosphodiester backbones of DNA [39–42]. The excellent film form-

ng ability of CH and the affinity of Fe3O4 nanoparticles towardsxygen of DNA can be used to fabricate an electrochemical nucleiccid sensor.

We report results of studies relating to the immobilizationnd characterization of single standard calf thymus DNA (ssCT-NA) immobilized onto CH-Fe3O4 hybrid nanobiocomposte filmeposited onto indium-tin-oxide (ITO) glass plate for detection ofM and PM, respectively.

. Experimental

.1. Reagents

Ferrous chloride, ferric chloride and triethyl amine have beenurchased from Sigma–Aldrich and used for preparation of Fe3O4anoparticles. Chitosan, cypermethrin and permethrin have beenrocured from Sigma, USA. The single stranded calf thymus DNAssCT-DNA) has been procured from Genei, Bangalore Pvt. Ltd.ndium-tin-oxide (ITO) coated glass plates have been obtained fromalzers, UK. All chemicals used are of molecular biology (MB) grade.he deionized water (Milli Q 10 TS) has been used for preparationf reagents. All the solutions and glasswares are autoclaved prior toeing used.

.2. Fabrication CH-Fe3O4 hybrid nanobiocomposite film

Fe3O4 nanoparticles (∼22 nm), prepared using co-precipitationethod [32] are dispersed into 10 mL of CH (0.50%) solution [pre-

ared by dissolving 50 mg of CH in 100 mL of acetate buffer (0.05 M,H 4.2) solution] via magnetic stirring at room temperature afterhich it is sonicated for 4 h. Finally, viscous solution of CH with

niformly dispersed Fe3O4 nanoparticles is obtained. The CH-Fe3O4ybrid nanobiocomposite film is prepared by dispersing 10 �L solu-ion of CH-Fe3O4 composite onto an ITO surface (surface area is.25 cm2) and allowing it to dry at room temperature for 12 h. ThisH-Fe3O4 hybrid nanobiocomposite film is washed with deionizedater to remove any unbound particles.

.3. Immobilization of single standard calf thymus DNAssCT-DNA)

We have optimized concentration of the single standard calf thy-us DNA (ssCT-DNA) covalently immobilized onto both CH/ITO and

H-Fe3O4 nanobiocomposite/ITO electrode and have found that�L ssCT-DNA (1 mg/mL) is sufficient to cover the electrode sur-

ace (data not shown). 7 �L solution of ssCT-DNA is spread on theurface of both the electrodes and is kept for about 2 h in a humidhamber after which it is stored in a refrigerator for about 8 h.hese ssCT-DNA/CH/ITO and ssCT-DNA/CH-Fe3O4 nanobiocompos-

te/ITO bioelectrodes are rinsed with autoclaved deionized water toemove any unbound ssCT-DNA from the bioelectrode surface andre stored at 4 ◦C when not in use.

CH is known to be a cationic amine-rich polysaccharide (pH 4.2).t is anticipated that surface charged Fe3O4 nanoparticles inter-ct with –NH2/OH groups of CH via electrostatic interactions andydrogen bonding. However, the negative charge bearing phos-hate backbone of ssCT-DNA interacts electrostatically with the

Fig. 1. UV–visible absorption spectra of CH (a), CH-Fe3O4 nanocomposite (b),ssCT-DNA/CH-Fe3O4 nanobiocomposite (c) and ssCT-DNA (d), Inset: UV–visibleabsorption spectra of CH (a), ssCT-DNA (b) and ssCT-DNA (c).

positively charged CH matrix and CH-Fe3O4 nanobiocompositematrix (Scheme 1).

2.4. Characterization

The structure and particle size of Fe3O4 nanoparticles havebeen determined using X-ray diffraction (XRD) pattern. FTIR PerkinElmer (Spectrum BX II) spectrophotometer has been used tocharacterize CH-Fe3O4 hybrid nanobiocomposite and its interac-tion with ssCT-DNA. The cyclic voltammetry (CV), electrochemicalimpedance spectroscopy (EIS) and differential pulse voltammetry(DPV) studies have been carried out on an Autolab Potentio-stat/Galvanostat (Eco Chemie, Netherlands). The electrochemicalmeasurements have been conducted using a three-electrode sys-tem in phosphate buffer (50 mM, pH 7.0, 0.9% NaCl) containing

Fig. 2. FTIR spectra of CH (a), CH-Fe3O4 nanocomposite (b), ssCT-DNA and ssCT-DNAimmobilized CH (c) and CH-Fe3O4 nanobiocomposite (d).

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ogical studies have been investigated using scanning electronicroscope (LEO-440).

The response times of the ssCT-DNA/CH/ITO and ssCT-DNA/CH-e3O4 nanobiocomposite/ITO bioelectrodes have been optimizedith different durations of time (20 s to 2 min). It has been found

hat 30 s and 40 s are sufficient for detection of CM and PM, respec-ively for obtaining maximum reduction in magnitude of currentor both pyrethroids. A control experiment has been performed tonvestigate the effect of these toxicants (2 ppm) onto CH/ITO andH-Fe3O4 nanobiocomposite/ITO electrodes. It has been found thatoth these electrodes do not reveal any change in oxidation current.he effect of pyrethroids onto ssCT-DNA/CH/ITO and ssCT-DNA/CH-e3O4 nanobiocomposite/ITO bioelectrodes has been studied byercentage reduction in the guanine oxidation current on interac-ion with the respective toxicant (CM and PM).

Experiments have been carried out using several CH/ITO andH-Fe3O4 nanobiocomposite/ITO electrodes (20) using DPV tech-iques. Both these bioelectrodes have been used several times10) under identical experimental conditions. It is observed thatioelectrodes yield reproducible results within 0.1%. The electro-hemical response of both ssCT-CH/ITO and ssCT-DNA/CH-Fe3O4anobiocomposite/ITO bioelectrodes as a function of pyrethroidoncentration has been repeated in triplet set and a steady stateurrent response is obtained. It has been found that that the elec-rochemical response is reproducible within 0.2%.

. Results and discussion

.1. UV–visible spectroscopic studies

The UV–visible absorption spectroscopy has been employed toscertain interaction of ssCT-DNA with CH and CH-Fe3O4 hybridanobiocomposite (Fig. 1). The absence of UV–visible band at70 nm (Curve a) associated with –C O group is attributed toligomer originating from degradation of the CH indicating that

t is in its natural state [32,43]. A broad and featureless adsorptionump seen at 250–350 nm in the spectra of CH-Fe3O4 (Curve a)riginates primarily from absorption and scattering of light due toe3O4 nanoparticles and is characteristic of the indirect band gapf semiconductors [32,34]. This absorption band is much broadernd shows cut-off at 250 nm. This may be attributed to the load-ng of Fe3O4 nanoparticles in CH resulting in enhanced bindingf Fe atoms with CH molecules via complexation (degradation of

H by Fe3O4 oligosaccharide containing –C O groups) indicatinghe formation of electro-active CH-Fe3O4 hybrid nanobiocomposite32].

Th absorption band seen at 260 nm (Curve d) assigned tosCT-DNA is found to be shifted to higher wavelength region in

Fig. 3. Scanning electron micrographs of ssCT-DNA/CH/ITO bioelectrode (

ring Journal 46 (2009) 132–140 135

the absorption spectra of ssCT-DNA/CH and ssCT-DNA/CH-Fe3O4nanobiocomposite (Curve c) [3]. This may be attributed to theformation of complex between ssCT-DNA and CH network andCH-Fe3O4 nanobiocomposite revealing that natural structure ofssCT-DNA is preserved.

3.2. FTIR spectroscopic studies

The FTIR spectra of CH (Fig. 2, Curve a) displays a broad band at3200–3400 cm−1 due to the stretching vibration mode of OH andNH2 group. The bands in the region from 2850 to 3000 cm−1 areattributed to the CH3 and CH2 groups due to CH antisymmetricand symmetric stretching modes. The band at 1610 cm−1 is due toamide I group (C–O stretching along with N–H deformation mode),1542 cm−1 peak is attributed to the NH2 group, 1428 cm−1 peak isdue to C–N axial deformation (amine group band), 1340 cm−1 peakarises due to COO− group in the carboxylic acid salt, 1152 cm−1 isassigned to broad peak of � (1- 4) glucosidic band in the polysaccha-ride unit, 1086 cm−1 is attributed to the stretching vibration modeof the hydroxyl group, 1030 cm−1 stretching vibration of C–O–C inglucose circle and 1060–1006 cm−1 bands correspond to CH-OH inthe cyclic compounds [32,44].

However, the IR bands corresponding to –NH/OH stretchingmodes in CH are found to be shifted in the spectra of CH-Fe3O4hybrid nanobiocomposite film (Curve b). It appears that aminegroup of CH binds with Fe3O4 nanoparticles via electrostaticinteractions including weak vander Waal forces and hydrogen bond-ing. The IR band corresponding to Fe3O4 nanoparticles occurs at570 cm−1 (Curve b) due to the stretching vibration mode and tor-sional vibration mode of Fe–O bond in the tetrahedral sites and inthe octahedral sites [32,45]. However, the peak position of this bandis shifted to higher wave number in the CH-Fe3O4 nanocompositedue to the formation of a complex between surface charged Fe3O4nanoparticles and cationic CH matrix, indicating the formation ofCH-Fe3O4 hybrid nanocomposite.

On immobilization of ss-DNA onto CH and CH-Fe3O4 nanobio-composite, the absorption bands corresponding to base pairs andphosphate backbone of ssDNA are observed. The FTIR spectra ofthe ssCT-DNA/CH/ITO bioelectrode (Curve c) and ssCT-DNA/CH-Fe3O4 nanobiocomposite film (Curve d) exhibit characteristic bandsat 1656, 1600 and 1414 cm−1 assigned to the base pairs of ssCT-DNA [46]. Infrared spectral features of ssCT-DNA relating to doublehelical structure are seen in the frequency range from 1225 to

950 cm−1 (sugar-phosphate backbone vibration range). The absorp-tion band at 1225 cm−1 corresponds to antisymmetric stretchingvibration mode due to phosphate group of ssCT-DNA. The bandseen at 1150 cm−1 is attributed to the bending vibration of C–Ogroup linked to dioxyribose unit. The IR band seen at 1088 cm−1

a) and ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode (b).

1 gineering Journal 46 (2009) 132–140

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s assigned to symmetric vibration mode of the dioxyribose phos-hate backbone of ssCT-DNA. The presence of bands correspondingo base pairs and phosphate groups of DNA in ssCT-DNA/ITO andsCT-DNA/CH-Fe3O4 nanobiocomposite/ITO indicates immobiliza-ion of ssCT-DNA.

.3. Scanning electron microscopy (SEM) studies

SEM (Fig. 3) studies reveal that porous morphology of CHhanges into globular porous microstructure after the incorpo-ation of Fe3O4 nanoparticles into CH, indicating the formationf CH-Fe3O4 hybrid nanobiocomposite. This may be attributed tolectrostatic interactions between cationic CH and surface chargede3O4 nanoparticles [32]. However, after the immobilization ofsCT-DNA onto CH/ITO (image a) and CH-Fe3O4 nanobiocompos-te/ITO (image b) electrode, changes in surface morphology oflectrodes are observed. Interestingly, the morphology of ssCT-NA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode is denser inomparison to that of ssCT-DNA/CH/ITO bioelectrode revealingigher loading of ssCT-DNA moieties.

.4. Electrochemical impedance spectroscopy (EIS) studies

Electrochemical impedance spectroscopy (EIS) studies ofH/ITO electrode, CH-Fe3O4 nanobiocomposite/ITO electrode,sCT-DNA/CH/ITO bioelectrode and ssCT-DNA/CH-Fe3O4 nanobio-omposite/ITO bioelectrodes have been investigated in phos-hate buffer (50 mM, pH 7.0 and 0.9% NaCl containing 5 mMFe(CN)6]3−/4−) in the frequency range, 0.01–105 Hz. In the EIS, theemicircle part corresponds to electron-transfer limited processnd its diameter is equal to the electron transfer resistance, Rct thatontrols electron transfer kinetics of the redox probe at the elec-rode interface. It can be seen from the Nyquist plots (Fig. 4) thatemicircle of ITO electrode (Rct = 3.82, Curve a), characteristic of aiffusion limiting step of the electrochemical process, decreases

or CH/ITO electrode (Rct = 2.7, Curve b) and it further decreasesor the CH-Fe3O4 nanobiocomposite/ITO electrode (Rct = 2.54, Curve). These results suggest that electron transfer in the CH-Fe O

3 4anobiocomposite film is easier between solution and the elec-rode, i.e., Fe3O4 nanoparticles not only provide the hydrophilicurface, but also promote electron transfer due to permeable struc-ure of CH/ITO. However, on the immobilization of ssCT-DNA onto

ig. 4. Electrochemical impedance spectra of bare ITO electrode (a), CH/ITOlectrode (b), CH-Fe3O4 nanobiocomposite/ITO electrode (c), ssCT-DNA/CH/ITO bio-lectrode (d) and ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode (e) inhosphate buffer (50 mM, pH 7.0) containing 5 mM [Fe(CN)6]3−/4− .

Fig. 5. Differential pulse voltammograms (DPV) of bare ITO electrode (a), CH/ITOelectrode (b), CH-Fe3O4 nanobiocomposite/ITO electrode (c), ssCT-DNA/CH/ITO bio-electrode (d) and ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode (e) inphosphate buffer (50 mM, pH 7.0) containing 5 mM [Fe(CN)6]3−/4− .

CH/ITO and CH-Fe3O4 nanobiocomposite/ITO, the semicircle partof the bioelectrode further decreases (Rct = 2.1 and 2.49, respec-tively). This may be attributed to improved electrical characteristicsof ssCT-DNA resulting in increased electroactive surface area ofboth bioelectrodes and enhanced electron transfer between theelectrode and medium.

3.5. Differential pulse voltammetry (DPV) studies

Fig. 5 shows DPV of the bare ITO electrode (Curve a), CH/ITO elec-trode (Curve b) CH-Fe3O4 nanobiocomposite/ITO electrode (Curvec), ssCT-DNA/CH/ITO bioelectrode (Curve d) and ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode (Curve e), respectivelyin PBS solution {50 mM PBS (pH 7, 0.9% NaCl) containing 5 mM[Fe(CN)6]3−/4−}. The magnitude of current response increases forthe CH/ITO electrode with respect to that of the bare ITO electrode.This suggests that CH/ITO electrode promotes electron transfer dueto cationic characteristics that accept electrons from the mediumand transfer these to the electrode. Moreover, magnitude of thecurrent peak increases for the CH-Fe3O4 nanobiocomposite/ITOelectrode suggesting that Fe3O4 nanoparticles promote electrontransfer due to uniform dispersion throughout CH network on theelectrode. The magnitude of response current is found to increaseboth for ssCT-DNA/CH/ITO and ssCT-DNA/CH-Fe3O4 nanobiocom-posite/ITO bioelectrode due to electrical characteristics of ssCT-DNAresulting in increased number of charge carriers on the bioelectrodeand enhanced electron transfer towards the electrode. Interest-ingly, observed higher magnitude of the current response forssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode suggeststhat Fe3O4 nanoparticles provide favourable environment for theimmobilization of ssCT-DNA onto electrode resulting in enhancedelectroactive surface area and electron transfer.

3.6. Electrochemical response studies of ssCT-DNA/ITO andssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrodes

Electrochemical response (Fig. 6A and B) of ssCT-DNA/CH-Fe3O4nanobiocomposite/ITO bioelectrodes has been measured using dif-

ferential pulse voltammetry (DPV) in phosphate buffer (50 mM,pH 7.0) containing 5 mM [Fe(CN)6]3−/4− both as a function of CMand PM concentration. It can be seen that magnitudes of currentresponse of ssCT-DNA/CH/ITO and ssCT-DNA/CH-Fe3O4 nanobio-composite/ITO bioelectrode decrease on addition of CM. However,

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agnitude of the response current due to ssCT-DNA/CH/ITO bio-lectrode becomes saturated at 0.5 ppm concentration of CM (dataot shown). And in the case of ssCT-DNA/CH-Fe3O4 nanobiocom-osite/ITO bioelectrode, the current decreases with introduction ofM concentration (0.0025–2.0 ppm). The inset in Fig. 6A shows vari-tion in the percentage reduction current response with increasingM concentration. About 70% reduction in current has beenbtained for ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelec-rode at 2 ppm concentration of CM. Inset b in Fig. 6A shows that

agnitude of the current response decreases linearly with increas-ng CM concentration (Eq. (1)).

CM [A] = 1.108 × 10−4A+20 �A/ppm∗ ln[CM concentration (ppm)]

with linear regression as 0.99 (1)

ig. 6. (A) Electrochemical response of ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioeleetween % reduction in current vs CM concentration, inset b) linear curve between currenanobiocomposite/ITO bioelectrode using DPV PM concentration from 1 to 300 ppm, inseetween current response and ln [PM (ppm)].

ring Journal 46 (2009) 132–140 137

The ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrodeshows low detection limit 0.0025 ppm, linear range 0.0025–2 ppmof CM concentration, high sensitivity of 20 �A/�M cm−2 andresponse time of 25 s.

Fig. 6B shows response studies of ssCT-DNA/CH-Fe3O4 nanobio-composite/ITO bioelectrode for detection of PM. The magnitudeof response current is found to decrease on addition of PM con-centration (1.0–300 ppm). The inset in Fig. 6B shows variationin percentage reduction of current response with increasing PM

concentration. We have observed 45% reduction in current for ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode at 300 ppmconcentration of PM. These results suggest that PM interacts withssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode. Inset b inFig. 6B shows that magnitude of current response decreases linearly

ctrode using DPV as function of CM concentration from 0.0025 to 2 ppm, inset a plott response and ln [CM (ppm)]. (B) Electrochemical response of ssCT-DNA/CH-Fe3O4

t a) plot between % reduction in current vs PM concentration, inset b) linear curve

138 A. Kaushik et al. / Biochemical Engineering Journal 46 (2009) 132–140

Table 1Characteristics of ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode along with those reported in literature.

Analytical technique Bioelectrode Genotoxicants Detection limit Response time Ref.

UV-spectroscopy Liquid phase Cypermethrin 0.12 ppm Online 6Enzyme linked immunosorbent assay Liquid phase Cypermethrin 1.3 ± 0.5 �g/L – 7,

Permethrin 2.50 �g/L 8

Fourier transform infrared spectroscopy andhigh pressure liquid chromatography

– Cypermethrin 0.7% and 0.4% (w/w) – 9

Gas chromatography–mass spectroscopy – Cypermethrin 18 �g/kg – 10Permethrin 0.048 �g/L and 0.6 ng/Ml 11,12

Coupled column liquidchromatography–photochemically inducedflourimetry–fluorescence

– Cypermethrin 0.01 ng/kg – 13

Gas chromatography with electron capturedetection

– Cypermethrin 2 �g/kg – 14

Electronic nose MOdular SEnsor System II – Cypermethrin, 0.7% w/w – 15Mixture ofcypermethrin andpermethrin

4% w/w

Sequential injection chromatography – Permethrin l.0 �g mL−1 – 16HPLC – Permethrin – 17Surface plasmon resonance dsCT-DNA-MCE/Au Cypermethrin 0.0005 ppm Online 18EE rmethEv

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lectrochemical (cyclic voltammetric)lectrochemical (square wave voltammetry) dsCT-DNA-PANI Cypelectrochemical (differential pulseoltammetry)

ssCT-DNA/CH-Fe3O4/ITO CypePerm

ith increasing CM concentration according to Eq. (2).

PM [A] = 2.51 × 10−4A + 14 �A/ppm∗ ln[PM concentration (ppm)]

with linear regression as 0.993 (2)

he ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode forM reveals low detection limit (1 ppm), linearity from 1 to 300 ppmf PM concentration, high sensitivity as 14 �A/�M cm−2 and fastesponse time of 40 s.

The decrease in the magnitude of current may be attributed totrong interaction of pyrethroid with ssCT-DNA/CH-Fe3O4 nanobio-omposite/ITO bioelectrode. It has been found that pyrethroidsnhibit oxidation of nitrogeneous bases of purine and result inecreased current response. It may be remarked that structures ofyrethroid molecules exhibit two types of configurations: firstly theexible part that has two negatively charged chloride ions, one esterroup and active dimethyl group. Among these, carbonium ion pre-ominantly interacts with the –NH2 terminal group of purine basesadenine and guanine) due to polarization. Secondly, rigid part ofhe molecule provides support for this interaction. It appears thatM interacts with ssCT-DNA moiety more strongly than PM due tovailability of more electronegative groups on CM molecule thatay bind more strongly with ssCT-DNA. Table 1 shows character-

stics of ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrodelong with those reported in literature.

Efforts have been made to test the ssCT-DNA/CH-Fe3O4 nanobio-omposite/ITO bioelectrode in presence of inorganic salts such asa2+, Mg2+, Cl−, and Na+ and pyrethroids. It has been found thatesponse of this electrode is not affected by presence of inor-anic salt. However, current response decreases in the presence ofyrethroids.

.7. Mechanism of interaction between pyrethroids andanobiocomposite based nucleic acid sensor

Attempts have been made to delineate the mechanism of

nteraction of CM and PM with ssCT-DNA using UV–visible andTIR spectroscopy. Fig. 7A shows UV–visible absorption spectraf ssCT-DNA with and without CM and PM. UV–visible spectraf ssCT-DNA shows absorption band at 260 nm whereas that ofM shows absorption band at 225 nm (Fig. 7A). On addition of

0.0005 ppm 30 srin 0.005 ppm 30 s 4rin 0.0025 ppm 25 s Present work

0.0025 ppm 40 s

CM (0.005–2 ppm), the absorption band of ssCT-DNA at 260 nmshifts to lower wavelength and a new absorption band appearsat about 220 nm. This bathochromic shift and the new absorp-tion band at 220 nm in the spectra of ssCT-DNA caused by CMindicate binding of CM with ssCT-DNA. It is seen that magni-tudes of absorbance of both 225 and 260 nm bands increaseslinearly (inset 7A) with increasing CM concentration followsEq. (3):

Abs(260 nm) = 0.15 Abs + 0.21 Abs/ppm∗CM concentration (ppm)

with linear regression as 0.995 (3)

The addition of PM (1–300 ppm) causes the bathochromic shift inthe spectra of ssCT-DNA indicating binding of PM with ssCT-DNA(Fig. 7B). The absorbance of 260 nm band increases linearly (inset4B) with increasing PM concentration follows Eq. (4):

Abs(260 nm) = 0.227 Abs + 0.014 Abs/ppm∗log

[PM concentration (ppm)] with linear regression as 0.988 (4)

The FTIR studies have been conducted to support the interaction ofpyrethroids (CM and PM) with the ssCT-DNA/CH-Fe3O4 nanobio-composite/ITO bioelectcrode. On addition of CM and PM ontossCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectcrode (Fig. 7C)IR bands corresponding to the base pairs of ssCT-DNA and phos-phodiester linkage between phosphate group of ssCT-DNA anddeoxyribose sugar are found to be shifted, indicating the interactionof pyrethroid with bioelectrode. However, it may be remarked thatboth pyrethroid/biolectrodes show new bands at 1100 cm−1 due toC-N-C antisymmetric stretching vibration mode corresponding tobonding between the amine group of ssCT-DNA and carbonium ionof CM and PM (Scheme 1).

The binding of these pyrethroids to ssCT-DNA can possibly occuras a result of strong polarization associated with the chlorine atomsin pyrethroids. In principle, this polarization may arise due to thechange in electrical polarizability during vibration of interactinggroups of CM affecting IR and UV–vis bands of ssCT-DNA. These

interactions may perhaps be caused by the generation of free radi-cals (carbonium) and damage ssCT-DNA. The formation of a reactivecarbonium ion through hydroxylation and acetylation binding withssCT-DNA has been proposed as a possible explanation for muta-genic and carcinogenic activity. The binding of pyrethroid with

A. Kaushik et al. / Biochemical Enginee

Fig. 7. (A) UV–visible absorption spectra of ssCT-DNA on successive addition of CMconcentration (0.005–2 ppm), inset a) UV–visible absorption spectra of ssCT-DNAand CM, inset b, linear curve of absorbance (260 nm) vs concentration of CM. (B)UV–visible absorption spectra of ssCT-DNA on successive addition of PM concentra-tion (1–300 ppm), inset a) UV–visible absorption spectra of ssCT-DNA and PM, insetb, linear curve of absorbance (260 nm) vs concentration of PM. (C) FTIR spectra ofssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode (a) after addition of PM (b)and CM (c).

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ring Journal 46 (2009) 132–140 139

ssCT-DNA via polarization may cause destabilization of ssCT-DNAstructure and unwinding of the DNA helix, inducing chromosomaldamage.

4. Conclusions

ssCT-DNA has been immobilized onto the CH-Fe3O4 nanobio-composite/ITO electrode film without any mediator. The ssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrode has been usedfor detection of CM and PM. These disposable ssCT-DNA/CH/ITO andssCT-DNA/CH-Fe3O4 nanobiocomposite/ITO bioelectrodes havebeen used for detection of CM (0.0025–2.0 ppm) and PM(1–300 ppm) within 25 s and 40 s, respectively. It has been shownthat incorporation of Fe3O4 nanoparticles reveals improved elec-troactive surface area of CH for immobilization of ssCT-DNAand accelerated electron transport. CH-Fe3O4 nanobiocompositebased ssCT-DNA biosensor shows improved sensing characteristics.Efforts should be made to utilize that bioelectrode for detection ofthese genotoxicants in wastewater. Besides, it should be test inter-actions of this generic bioelectrode for detection of genotoxicantslike malthion, attrazine, 2-aminoanthracene and 3-chlorophenoletc.

Acknowledgements

We thank Dr. Vikram Kumar, Director, National Physical Lab-oratory, New Delhi, India for facilities. A. Kaushik is thankfulto the Council of Scientific Industrial Research (CSIR), India forthe award of Senior Research Fellowship (SRF). Financial supportreceived under the Department of Science and Technology (DST)projects [DST/TSG/ME/2008/18 and GAP- 070932], in-house project(OLP-070632D) and the DBT project (GAP-070832), is gratefullyacknowledged.

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