Biocompatible ZrO2- reduced graphene oxide

Materials and Design 111 (2016) 312–320

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Biocompatible ZrO2- reduced graphene oxide immobilized AChE biosensor for chlorpyrifos detection Navin Kumar Mogha a, Vikrant Sahu a, Meenakshi Sharma b, Raj Kishore Sharma a,⁎, Dhanraj T. Masram a,⁎ a b

Department of Chemistry, University of Delhi, Delhi 110007, India Dr B.R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110007,India

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• RGO supported ZrO2 nanocomposite is investigate as AChE based biosensor. • RGO/ZrO2 is an efficient matrix to detect pesticide in ultralow concentration. • Proposed electrode in biocompatible, highly sensitive and cost effective. • AChE/ZrO2/RGO biosensor is portable and can be used at remote places.

a r t i c l e

i n f o

Article history: Received 4 July 2016 Received in revised form 2 September 2016 Accepted 5 September 2016 Available online 06 September 2016 Keywords: Biosensor Graphene ZrO2 AChE Nanocomposite Chlorpyrifos Pesticide

a b s t r a c t Based on Acetylcholinestrase (AChE) inhibition, a novel biosensing electrode involving Reduced Graphene Oxide (RGO) supported Zirconium Oxide (ZrO2/RGO) nanoparticles is fabricated for Chlorpyrifos (pesticide) detection. AChE/ZrO2/RGO biosensor when used for pesticide detection, exhibited high sensitivity towards Chlorpyrifos. Typically AChE/ZrO2/RGO biosensing electrode is capable of detecting Chlorpyrifos concentration as low as 10−13 M (with 28% enzyme inhibition). Furthermore the enzyme inhibition is remarkable high (N 70%) in case of high Chlorpyrifos concentration (up to 10−4 M). Our results indicate that AChE/ZrO2/RGO is an efficient and biocompatible electrode that can be used for Chlorpyrifos detection in ultra low concentrations. Electrochemical sensing response of AChE/ZrO2/RGO sensor revealed two different linear ranges of chlorpyrifos detection. The first linear response was observed from 10−13 M to 10−9 M whereas the second linear range was observed between 10−9 M to 10−4 M revealing complete saturation of enzyme active sites by Chlorpyrifos. Ameperometric response of AChE/ZrO2/RGO electrode demonstrates constant decrease in the current with respect to increase in the Chlorpyrifos concentration. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction

⁎ Corresponding authors. E-mail addresses: [email protected] (R.K. Sharma), [email protected] (D.T. Masram).

http://dx.doi.org/10.1016/j.matdes.2016.09.019 0264-1275/© 2016 Elsevier Ltd. All rights reserved.

Pesticides are widely used in modern agriculture [1], however their acute toxicity and hazardous effect on animal health and environment [2] are found to be a matter of concern. Among widely used pesticides, organophosphorus compounds (OPs) like Chloropyrifos exhibit

N.K. Mogha et al. / Materials and Design 111 (2016) 312–320

catalytic inhibition of Acetylcholinesterase (AChE), which leads to in vivo accumulation of acetylcholine, resulting in nervous system break down and cell death [3,4]. Highly toxic character of OPs can cause severe threat to public safety and health, as observed in subway sarin incident, Tokyo in 1995, and Ghouta chemical attack, Syria in 2013. In order to monitor the overuse and analysis of the permissible levels in food, detection of pesticides with high precision is needed. Traditional methods for pesticides detection are based on gas chromatography (GC) [5], high performance liquid chromatography (HPLC) [6], enzymelinked immuno absorbant assays [7] etc. Despite the established procedures, major drawbacks of these include the cost and the testing time with operational difficulties. Biosensors can substitute current analytical techniques by eliminating sample preparation and therefore significantly reduces the analysis time and cost [8]. Among them, enzyme-based electrochemical biosensors are particularly attractive because of their high sensitivity, rapid response and portability [9]. AChE biosensors based on inhibition of AChE enzyme have shown interesting result in OPs determination [10]. Here enzyme activity is directly linked with quantitative detection of Pesticide. In the process, an electro active molecule, thiocholine is produced during the interaction of the substrate Acetylthiocholine (ATCl) with AChE immobilized electrode [11]. Thiocholine detection is used to assess the inhibition of AChE activity in presence of Ops [12]. Analysis of ATCl has therefore attracted great attention, especially in the development of biosensors for OPs detection [13]. Owing to efficient catalytic character, high electrolyte accessible area, easy fabrication and many other interesting properties, metal oxide nanoparticles are extensively used in a variety of electro-analytical processes [14]. Metal oxide nanoparticles immobilized over a conductive and large area support like graphene is shown to further enhance the performance [15,16]. Electrochemical sensing using graphene based nanomaterials was first reported by Papakonstantinou [17] and later Dong et.al. [18] and Kim et al. [19]. demonstrated fast electron-transfer in graphene sheet. Consequent to the good electronic conductivity and efficient electrocatalytic property, different type graphene based biosensors are reported [20–26]. On the other hand, due to high chemical inertness and low toxicity, Zirconium oxide (ZrO2) is recognized as an environment friendly material [27]. Properties of ZrO2 like thermal stability, biocompatibility, cost effective production and electrochemical activity pave its way to superior electrode material in electrocatalysis [28–30], photocatalysis [31,32], urea sensing [33], glucose sensing [34], humidity sensing [35] and more importantly OPs detection [28,29]. In the present article, we demonstrate ZrO2 immobilized graphene nanocomposite electrode for chloropyrifos detection. In amperometric detection of chloropyrifos using graphene supported ZrO2 electrode, our result indicate extended linear detection range many orders higher to the reported values in literature. Synthetic methodology of Reduced Graphene Oxide (RGO) supported ZrO2 nanocomposite and its biosensing mechanism for Chlorpyrifos detection is shown in Scheme 1. Pesticide detection methodology is based on the measurement of reduction in AChE activity by means of Faraday current. AChE breaks down the ATCl present in electrolyte and consequently releases two electrons in the process. So generated electronic current, is monitored as the electro-activity of ZrO2/RGO electrode. In the presence of Chlorpyrifos, activity of enzyme slows down because of irreversible binding that consequently results in decreased current.

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was purchased from Sigma Aldrich, USA, The standard technical grade Chlorpyrifos (C9H11Cl3NO3PS) was purchased from Merck, Germany. All other materials used were of high quality. Deionized (DI) water was used throughout this study. 2.2. Characterization X-ray diffraction patterns of RGO, ZrO2/RGO nanocomposite were recorded using X-ray diffractometer (Model No. D8 DISCOVER). Morphological features were recorded using Zeiss Ultra 55 field emission scanning electron microscope (FESEM) and TECNAI 200 kV TEM (Fei, Electron Optics). Electrochemical sensing tests using AChE/ZrO2/RGO electrodes were carried out using CH-604D electrochemical workstation. Raman spectra were recorded by Renishaw Invia Reflex MicroRaman spectrometer in which the sample was excited by 514 nm wavelength Ar+ laser. 2.3. Synthesis of Graphene oxide (GO) and Reduced graphene oxide (RGO) GO was prepared by the improved synthesis method of graphene oxide [36,37]. Obtained GO was hydrothermally reduced by taking 100 mg GO powder in 100 ml NaOH (1 M) in Teflon-lined autoclave maintained at 150 °C for 24 h. After 24 h, mixture was filtered and washed thoroughly with DI water and kept for drying at 100 °C overnight in vacuum. The dried black powder obtained was used as RGO. 2.4. Synthesis of ZrO2/RGO nanocomposites Zr(NO3)4·5H2O (20 mg) and Poly ethylene glycol (0.2 g) were ultrasonically mixed in 20 ml DI water for 1 h, to this mixture 200 mg GO powder was added and ultrasonically mixed for 30 min. So formed suspension was loaded into 50 ml Teflon-lined autoclave maintained at 150 °C for 24 h. After cooling to ambient, the precipitate was filtered, washed using absolute ethanol and DI water several times followed by drying at 80 °C for 1 h in vacuum and then put for annealing at 250 °C for 3 h. Resulted material (ZrO2/RGO nanocomposite) was spray deposited on Indium Tin Oxide (ITO) glass as reported in our earlier article [37]. The preparation of AChE/ZrO2/RGO electrode involved dropping (20 μl, optimization of AChE amount shown in supporting information S1) of AChE solution (2 mg/ml) onto pre-deposited ZrO2/RGO film on ITO plate and incubating it at 4 °C for 24 h. The electrode was rinsed with DI-water, dried and stored at 4 °C prior to use. Stability studies were also performed for four weeks as reported in supporting information (S2). 2.5. Biocompatibility of ZrO2/RGO nanocomposite

2. Materials and methods

SiHa cells were grown as adherent monolayers in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic cocktail. The cultures were maintained in 25 ml flasks in an incubator at 37 °C with a humidified atmosphere composed of 5% CO2 and 95% air. The media was changed twice and were trypsinized two times weekly for maintaining the lineages. To check the bio-compatibility of ZrO2/RGO nanocomposite, a stock concentration of 1 mg/ml in incomplete Dulbecco's modified eagle medium was prepared. This was used to treat the 24 h grown cells with 200 μg/ml as the final concentration. The treated cells and appropriate control flask were kept for another 48 h to observe the cell morphology and growth of culture. The treated and control flasks were photographed at 200× magnification under a Nikon inverted microscope.

2.1. Materials and reagents

2.6. Cytotoxicity assay

Graphite powder (~ 200 mesh, 99.9%) was purchased from Alfa Aesar, India, Zr(NO3)4·5H2O was obtained from Merck Millipore, India, AChE (500 U/mg), and Poly (ethylene glycol), MW ~ 35,000,

The colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to determine the cytotoxicity of ZrO2/RGO nanocomposite. In brief, trypsinized SiHa cells were seeded

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Scheme 1. Synthetic methodology of ZrO2/RGO nanocomposite and AChE adsorption for Chlorpyrifos biosensing electrode.

into a 96-well plate at cell densities of 1000 cells/well, in 200 μl of growth medium and were pre-incubated for 24 h at 37 °C in an atmosphere of 5% CO2 and 100% relative humidity to allow adaptation of cells prior to the addition of the test compounds. The medium was then removed, and 200 μl of new growth medium containing various concentrations of the ZrO2/RGO nanocomposite was added. After 48 h, the medium was removed, 200 μl of a 0.8 mg/ml solution of MTT in DMEM was added, and the plate was incubated for an additional 4 h. The DMEM/MTT mixture was aspirated, and 150 μl DMSO was added

to dissolve the purple formazan crystals. The absorbance of the plates was read at 570 nm. 2.7. Statistical analysis All experiments were performed in at least three replicates per compound and results shown are the average of three independent experiments. Data are represented as mean ± SEM (Standard error in Mean). Significance was tested by the Student's test t.

Fig. 1. (a) X-ray diffraction patterns of ZrO2/RGO, GO, pure ZrO2 and (b) Raman spectra of ZrO2/RGO, GO, pure ZrO2.


315

Fig. 2. FESEM image (a) GO, (b) RGO, (c) ZrO2 and (d) ZrO2/RGO nanocomposite.

3. Result and discussion 3.1. X-ray diffraction and Raman analysis The X-ray diffraction patterns of ZrO2/RGO nanocomposite, graphene oxide and pure ZrO2 are depicted in Fig. 1a. Diffraction peaks of ZrO2/RGO nanocomposite are in good agreement with those of the standard data (JCPDS 37-1484) [38] for ZrO2. Most intense diffraction peak at ~ 28.2° corresponding to ⟨111⟩ plane was observed in both (nanocomposite and bare ZrO2) the samples. The broad and intense peak at ~ 10.2° corresponds to 〈001〉 plane of GO [39]. Disappearance of this peak from diffraction pattern of ZrO2/RGO nanocomposite indicate the reduction of GO to RGO under hydrothermal conditions. The Raman spectrum traces of graphene oxide, pure ZrO2, and ZrO2/ RGO nanocomposite are shown in Fig. 1b. Intense peaks in ZrO2/RGO spectrum located at 178, 189 and 476 cm− 1 correspond to the

characteristic peak of monoclinic phase of ZrO2. This spectrum showed total 14 vibration modes with a small shift (in all the 14 peaks) due to the interactions with ZrO2 and GO. Raman spectrum of GO exhibited characteristic D and G peaks at 1345 cm−1 and 1630 cm−1 respectively. ID/IG ratio (0.86) of the spectrum support the ordered structure of GO, however the same (ID/IG = 0.69) for ZrO2/RGO indicate regaining of conjugation in graphene after reduction. 3.2. Morphological characterization by FESEM and HRTEM FESEM image of GO in Fig. 2a, exhibit few layer stacked large area graphene oxide sheets. After reduction the stacking of RGO (Fig. 2b) is reduced due to exfoliation. Fig. 2c depicts the dense morphological features of bulk ZrO2. Interestingly, when ZrO2 is allowed to grow on graphene sheets, the grain size of ZrO2 is significantly reduced, (Fig. 2d). Cross-sectional SEM image in supporting information S3

Fig. 3. TEM micrographs (a) GO (b-d) ZrO2/RGO nanocomposite at different magnifications, HRTEM image showing lattice plane h111i of ZrO2, Inset in (c) showing SAED pattern for the formation of ZrO2 nanoparticles.

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Fig. 4. (a) pH-enzyme activity relationship of AChE/ZrO2/RGO electrode, (b) comparative voltammograms of different electrodes (i) RGO (ii)ZrO2/RGO (iii) AChE/ZrO2/RGO, (c and d) optimization of ATCl concentration.

Fig. 5. (a & b) Voltammetric response of AChE/ZrO2/RGO electrode as a function of Chlorpyrifos concentration, (c) chronoamperometric analysis of different Chlorpyrifos concentrations (black line in the absence of Chlorpyrifos) (d) percentage enzyme inhibition by Chlorpyrifos.


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Fig. 6. Mechanism of AChE (a) action on ATCL (b) inhibition by Chlorpyrifos, where phosphate group of Chlorpyrifos binds irreversibly to serine hydroxyl group in active catalytic site.

suggest the thickness of biosensing electrode (AChE/ZrO2/RGO) in the range of 5–6 μm. The HRTEM micrograph of GO in Fig. 3a exhibits characteristic wrinkled sheet like morphology, whereas uniform embedment of fine ZrO2 nanoclusters on RGO sheet can be seen, Fig. 3b. High magnification micrograph of the selected area shows clustering of granular ZrO2 nanoparticles (Fig. 3b,c). The lattice fringes of ZrO2 in Fig. 3d suggest 0.32 nm d spacing for h111i plane in agreement with the JCPDS 371484 card of monoclinic ZrO2. Selected Area Electron Diffraction (SAED) pattern as inset in Fig. 3c further demonstrate the formation of ZrO2 nanoparticles indicated by presence of its different crystal planes.

3.3. Electrochemical characterization Electrochemical properties of GO, ZrO2/RGO and AChE/ZrO2/RGO electrodes were evaluated in 3 electrode cell containing, Pt plate as auxiliary and saturated Ag/AgCl as reference electrode while the composite

coated film on ITO as working electrode. In a typical experiment, ATCl (1.5 mM) solution was injected into the cell containing 20 ml phosphate buffer saline as electrolyte (0.1 mol l−1, pH 7). The substrate (ATCl) was enzymatically hydrolyzed to thiocholine, which undergoes electrocatalytic oxidative dimerization at 0.36 V to yield disulfide compound. The activity of AChE generated during this process at working electrode was measured by cyclic voltammetry and chronoamperometry. Each experiment was performed in triplicates and calculated error is shown in figures using error bars. In order to establish the relationship between enzyme activity and electrolyte pH, voltammogram of AChE/ZrO2/RGO electrodes were recorded with substrate (1.5 mM) at different pH (4.0 to 10.0) buffer solutions. The variation in peak current at different pH in Fig. 4a suggests highest activity at pH ~ 7. Fig. 4b depicts the comparative voltammograms of AChE/ZrO2/RGO, ZrO2/RGO, and bare RGO at pH 7. Interestingly the ZrO2/RGO electrode with AChE shows maximum peak current among the three. Further to study the AChE/ZrO2/RGO electrode sensing characteristics, concentration of ATCl was varied (from 1 μM–

Fig. 7. Biocompatibility studies of ZrO2/RGO nanocomposite.

318


1.5 mM) in different steps. Upon adding the ATCl, expectedly the voltammogram of AChE/ZrO2/RGO electrode showed increase in current, peaking at 0.36 V. This peak corresponds to the ATCl oxidation (Fig. 4c) and peak current increases linearly with ATCl (from 1 μM to 100 μM) concentration, following the regression equation y = 1.588 + 12,530.13x, and R2 = 0.935. Upon further increasing the ATCl concentration (100 μM to 1.5 mM), peak current showed a deviation in slope with a different linear range (y = 2.986 + 862.31x, R2 = 0.946), where y is current in μA, x is concentration of ATCl in μM and R is regression coefficient. Obviously, the increase in ATCl concentration causes a saturation of the active sites on enzyme by ATCl, leaving fewer sites available for new molecules to bind. Consequently the rate of increase in peak current showed a decrement and therefore a decrease in slope. Based on the above observations, 1.5 mM ATCl was selected as the optimum concentration in subsequent enzyme inhibition experiments. 3.4. Electrochemical detection of Chlorpyrifos Chlorpyrifos detection was performed by first dipping AChE/ZrO2/ RGO electrodes in 0.1 mol l−1 PBS solutions (pH 7) of different chlorpyrifos concentrations. After soaking for 10 min in the above solution, cyclic voltammograms of AChE/ZrO2/RGO electrode in 20 ml PBS solution (pH 7) with 1.5 mM ATCl electrolyte were recorded. Inhibition of enzyme activity (expressed as a percentage) was calculated as follows:

Inhibition% ¼

Io −It 100 Io

Where, I0 is the oxidation peak current in the absence of inhibitor and It is the peak current consequent to chlorpyrifos inhibition. Chlorpyrifos concentration was determined by the inhibition of immobilized AChE. Firstly, AChE/ZrO2/RGO electrode was dipped into Chlorpyrifos leading to the formation of enzyme – inhibitor complex. Electrode was then dipped into ATCl solution with predetermined concentration to form enzyme–substrate–inhibitor complexes (ESIs). Chlorpyrifos like other organophosphorous compounds has intrinsic property of binding irreversibly to AChE that subsequently results in ESI formation. Fig. 5a with the help of cyclic voltammograms depicts the AChE activity inhibition as a function of Chlorpyrifos concentration. A continuous decrease in peak current with Chlorpyrifos concentration is attributed to the reduction of overall charge on the catalytic site. CV response revealed two different linear detection ranges (Fig. 5b). The first linear response was observed from 10−13 M to 10−9 M, following the regression equation y = −(1.2786 + 0.328x), and R2 = 0.996, where y is current in μA, x is concentration of Chlorpyrifos in M and R is regression coefficient. This response has highest slope and indicate that large number of free active sites are present over the enzyme surface which allow more Chlorpyrifos molecules to inhibit the enzyme, hence, a rapid decrease in the current was noted. In the second linear response (y = −(0.9005 + 0.087x), R2 = 0.968), from 10−9 M to 10−4 M, the decrease in current indicates the saturation of enzyme active sites by Chlorpyrifos, because of irreversible binding. Under the optimized conditions, amperometric response of AChE/ ZrO2/RGO electrode to different Chlorpyrifos concentrations was studied. Amperometric current–time response (I-t) of Chlorpyrifos at 0.36 V in Fig. 5c, similar to CV study revealed that as concentration of Chlorpyrifos increases, a decrease in the current in noticed. Percentage inhibition curve of AChE against Chlorpyrifos in Fig. 5b, suggest that 10−13 M pesticide inhibit more than 28% enzyme. This further can be enhanced to 70% if the Chlorpyrifos concentration is increased (~ 10−4). For the comparison purpose electrode without AChE was also studied, to determine whether nanocomposite has some OPs sensing character. Results indicate that ZrO2/RGO does not show any sensitivity towards Chlorpyrifos. It is understood that ZrO2/RGO

Fig. 8. Cytotoxic effect of ZrO2/RGO nanocomposite on SiHa cell line. Cells were treated with ZrO2/RGO nanocomposite for 48 h and the cell proliferation/cell survival was measured by the MTT assay as described in the experimental section.

nanocomposite provides a conducting and highly accessible matrix for the strong binding of enzyme AChE. 3.5. Mechanism of AChE inhibition AChE action on ATCl and its subsequent inhibition is shown in Fig.6. Active catalytic center of AChE have two sites viz. cationic and anionic [40–42]. Fig. 6a, depicts the mechanism of action of AChE, where carbonyl group of ATCl binds to cationic site having free serine hydroxyl group, whereas positively charged Nitrogen binds anionic site of enzyme through electrostatic interactions. AChE acts on ATCl to break the molecule in acetic acid and electroactive thiocholine. On the other hand trace amount of Chlorpyrifos can phosphorylate AChE by binding to cationic site via phosphate group (Fig. 6b). Therefore rather than getting hydrolysed, Chlorpyrifos undergoes dealkylation making its binding to AChE irreversible. 3.6. Biocompatible studies Bio-compatibility studies (Fig. 7) of ZrO2/RGO nanocomposite were performed on SiHa cell line. Both the treated and control flask when observed under 100 × magnification showed no significant change in growth percentages (75–85% confluent culture). Similar size, morphology, and adherent property, was observed which suggest that ZrO2/RGO nanocomposite is bio-compatible and do not cause any morphological change or cell death. The cytotoxicity of ZrO2/RGO nanocomposite against SiHa cell line was tested using colorimetric MTT assay. The MTT assay is a standard colorimetric assay, in which mitochondrial activity is measured by splitting tetrazolium salts with mitochondrial dehydrogenases from viable cells [43]. The yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form insoluble purple formazan crystals, which are solubilized by the addition of a solvent. The color can then be quantified colorimetrically. The samples are read using an ELISA plate reader at a wavelength of 570 nm. The amount of purple color produced is directly proportional to the number of viable cells. Fig. 8 shows the percentage of cell proliferation in SiHa cells treated with ZrO2/RGO nanocomposite versus control cells. The tested ZrO2/RGO nanocomposite showed similar cell viability and the difference was not significant when compared

Table 1 Real Sample monitoring by AChE/ZrO2/RGO biosensor. Water spiked sample (ppm)

Pesticide added (ppm)

Recovered (ppm)

% % RSD (relative Recovery standard deviation)

0.06 0.08 0.1

0.01 0.02 0.05

0.0683 0.0963 0.0983

97.57 96.33 93.64

3.69 1.58 3.85


319

Table 2 Comparison of the reported biosensors with present work.

Electrode material

Type of technique

Cellophanemembrane/AuE Amperometric PEI-coated GCE Potentiometric flow injection Zinc oxide sol–gel/SPE Amperometric PB modified SPE

Amperometric

CdTe QDs/AuNPs/CHIT/GCE

Amperometric

ZrO2/CHIT composite film/GCE

Amperometric

ZrO2/RGO nanocomposite

Amperometric

Immobilization method

Limit of detection (M)

Linear range (M)

Analyte

Ref.

Crosslinking Covalent

1.45 × 10−6 1 × 10−6

1.45 × 10−6–7.26 × 10−6 Not reported

Paraoxon Dichlorvos

[44] [45]

Electrostatic interactions Not reported

1.27 × 10−7

1.27 × 10−7–5.010 × 10−6

Paraoxon

[46]

Aldicarb, Carbaryl

[47]

Monocrotophos

[48]

Phoxim, and imethoate

[49]

Chlorpyrifos

This work

1.26 × 10

−7

, 1.24 ×

6.3 × 10

−8

–3.15 × 10

−7

, 1.24 ×

10−7 1.34 × 10−6

10−7–4.97 × 10−7 4.4 × 10−9–4.48 × 10−6, 8.96 ×

Absorption

1.3 × 10−6, 1.7 ×

10−6–6.72 × 10−5 6.6 × 10−6–4.4 × 10−4, 8.6 × 10−6–5.2 ×

Adsorption

10−6 10−13

10−4 10−13–10−9, 10−9–10−4

Covalent

to the control cells suggesting that the tested compound is biocompatible and is not causing cytotoxicity in the SiHa cell lines. 3.7. Real sample monitoring Chlorpyrifos spiked water samples were analyzed for application in AChE/ZrO2/RGO biosensing electrode. Standard addition method was applied for analysis, in which a known amount of Chlorpyrifos was added to the test solution. Table 1 suggest an excellent chlorpyrifos recovery percentage from 93.64–97.57% recovery. Each experiment was performed in three replicates and then mean value was calculated to determine the recovered percentage. Results demonstrate that the AChE/ ZrO2/RGO is an efficient and sensitive electrode for Chlorpyrifos detection and quantification. 4. Conclusion Nanostructured Reduced Graphene Oxide (RGO) supported ZrO2 nanocomposite (ZrO2/RGO) was synthesized via hydrothermal route and tested as an efficient electrode matrix for enzyme immobilization. AChE/ZrO2/RGO biosensor based on electrochemical principles exhibited cost efficiency and high sensitivity towards Chlorpyrifos detection. Our results demonstrate the Chlorpyrifos detection as low as 10−13 M concentration with 28.27% enzyme inhibition. The enzyme inhibition was remarkably high (72.94%) for the samples containing 10−4 M concentration of Chlorpyrifos. Biocompatibility tests of ZrO2/RGO matrix further demonstrate the application of biosensor in the conditions where living cells are involved. A quick literature survey as presented in Table 2, suggests that the AChE immobilized ZrO2/RGO matrix constitute a high performance Chlorpyrifos biosensor. Which is many orders of magnitude higher compared to other materials reported for pesticide detection. Literature reports mentioned in Table 2, support that the AChE/ZrO2/RGO biosensor is most sensitive and cost effective with fabrication and operational convenience reported till date. Acknowledgements Authors are grateful to the University of Delhi, Delhi, India for providing financial assistance through R & D fund [RC/2014/6820], SERBDST (SR/FT/CS-123/2010) and Sophisticated Analytical Instrument Facility (SAIF) – AIIMS, New Delhi, under the SAIF Program of DST for providing TEM facility. V. Sahu greatly acknowledges CSIR (Council of Scientific & Industrial Research) for providing SRF. References [1] R. Xue, T.F. Kang, L.P. Lu, S.Y. Cheng, Immobilization of acetylcholinesterase via biocompatible interface of silk fibroin for detection of organophosphate and carbamate pesticides, Appl. Surf. Sci. 258 (2012) 6040–6045, http://dx.doi.org/10.1016/j. apsusc.2012.02.123.

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