Fabrication of Organophosphorus Biosensor Using ZnO Nanoparticle

Jun 14, 2013 - Rajesh Madhu , Bose Dinesh , Shen-Ming Chen , Ramiah Saraswathi , Veerappan Mani. RSC Advances 2015 5 (67), 54379-54386 ...Missing:...
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Fabrication of Organophosphorus Biosensor Using ZnO Nanoparticle-Decorated Carbon Nanotube−Graphene Hybrid Composite Prepared by a Novel Green Technique Pranati Nayak, B. Anbarasan, and S. Ramaprabhu* Alternative Energy and Nanotechnology Laboratory (AENL), Nano Functional Materials Technology Centre (NFMTC), Department of Physics, Indian Institute of Technology Madras, Chennai 600036, India ABSTRACT: In this work, we report a green synthesis procedure for preparing zinc oxide nanoparticle (NP)decorated multiwalled carbon nanotube−graphene hybrid composite (ZnO−MWCNTs-sG) by using solar energy and its application as a transducer candidate for organophosphorus biosensor. This hybrid composite shows a good electrochemical activity due to large electrochemical active surface area in comparison to ZnONP-decorated graphene (ZnO-sG) prepared by the same root. This can be due to the presence of MWCNTs between the graphene layers, which effectively prevents its restacking, thereby increasing the accessible surface area. The fabricated biosensor using this composite as a transducer candidate shows a high affinity to acetyl cholinesterase (AChE) enzyme with a Michaelis−Menten constant (Km) value of 0.8 mM. Further, it exhibits a linear response for Paraoxon detection from 1 to 26 nM with a detection limit of 1 pM (S/ N = 3). On the basis of the above synergetic electrochemical sensing and easy scale up green synthesis procedure, the hybrid material can be recommended as a robust material for sensor related applications.

1. INTRODUCTION For the last two decades graphene, the two-dimensional architecture of sp2 hybridized carbon atoms, has attracted enormous scientific attention affording remarkable physicochemical properties and promising applications.1 It holds outstanding properties including high surface area, excellent thermal and electrical conductivity, high mechanical strength, and enhanced electrochemical behavior, which makes it a forefront potential material for actuators, solar cells, super capacitors, fuel cells, batteries, electron field emitters, and electrochemical biosensors.2−7 Incorporation of different metal, metal oxide, and polymer nanostructures with graphene also significantly enhances its properties due to its unique surface chemistry.8 Owing to its excellent electronic properties, catalytic activity, and huge accessible surface area, people have reported graphene as well as its surface modified derivatives as an advanced transducer candidate for electrochemical biosensing applications.9 In particular, graphene-based nanostructures have displayed outstanding sensing platform for glucose, cholesterol, dopamine, cytochrome, and organophosphorus compounds.10−13 The theoretical surface area of a single-layer graphene sheet is 2630 m2/g, which is multifold higher than that for activated carbon.14 For application purposes, large scale synthesis of single-layer graphene is very difficult, and also, it is challenging to physically separate the graphene sheets from restacking due to the van der Walls force of attraction between the layers. This decreases significantly the accessible surface area of graphene layers. For practical © XXXX American Chemical Society

fabrication of biosensors, high accessible surface area of the transducer element is desirable for effective enzyme immobilization and to enhance electrochemical response. In this regard, the integration of one-dimensional multiwalled carbon nanotubes (MWCNTs) in between graphene sheets is highly enviable and can be applicable in order to preserve its huge accessible surface area.15 ZnO nanostructures are one of the promising metal oxides for biosensors due to its electrochemical catalytic activity, superior surface properties, biocompatibility, and nontoxicity. Decorating ZnO nanostructures over huge surface area of graphene as well as carbon nanotubes helps in preserving its surface area. This enhances the catalytic activity of the composite material, which makes it promising for biosensing applications.16 Also from an electrochemical point of view, ZnO possesses a high isoelectric point (IEP ≈ 9.5). This makes it robust for immobilization of low IEP enzymes such as acetyl cholinesterase (AChE, IEP∼5.5) by strong electrostatic binding.17 In pH 7 buffer medium, the positively charged ZnO creates a favorable platform for adsorption of negatively charged enzyme and promotesthe electron transfer mechanism between enzyme and electrode. In recent literature, there are many techniques for decorating metal oxide nanostructures over huge surface area of graphene. Received: December 29, 2012 Revised: April 28, 2013

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using 5,5′-dithiobis(2-nitro benzoic acid) (DTNB, 98% from SRL India Pvt ltd.) as Ellamans reagent, which was found to be 3 mM.26 2.2. Synthesis of GO and MWCNT. Graphite oxide was synthesized by Hummers method using flake graphite as the precursor.27 Flake graphite powder was refluxed in concentrated H2SO4 and continuously stirred in an ice bath. Then, KMnO4 and NaNO3 were added to the suspension and allowed to cool down to room temperature by removing from ice bath followed by adding water to it. The above suspension was again diluted by adding warm water. Then, H2O2 (3%) was added to it until it turned into a bright yellow color. It was filtered and washed thoroughly by copious amount of DI water. Finally, the residue was dried in vacuum at 60 °C temperature. MWCNTs were prepared by catalytic chemical vapor deposition (CVD) method using acetylene as a carbon source using rare earth based MmNi3 (Mm = Misch metal) type alloy catalyst.28 The synthesized MWCNTs were air oxidized at 400 °C followed by acid treatment to remove amorphous carbon impurities and remaining catalyst particles. 2.3. Synthesis of ZnO-MWCNT-sG Hybrid Nanomaterial. The ZnO-MWCNT-sG was prepared by using focused sunlight as the energy source. About 100 mg of GO, 5 mg of purified MWCNTs, and 100 mg of zinc acetate was ground well using a mortar and sprinkled in a glass petridish. Focus sunlight was allowed to fall directly on the mixture for 1−2 min by using a convex lens of 90 mm diameter. The high intensity of focused sunlight creates a rapid heating rate (about 100 °C/sec), which decomposes Zn(CH3COO)2 and simultaneously reduces GO to solar graphene resulting in a visible color change from brown to dark black. During synthesis, an apparent volume expansion was also observed with a release of gaseous byproducts in the form of fumes. This confirms conversion of GO to solar graphene.29 The temperature generated ranges from 250 to 300 °C. As zinc is highly reactive to form oxide easily, it is expected that the formation of ZnO can be due to the decomposition of Zn(CH3COO)2 and its reaction with oxygen present in acetate salt, oxygen containing functional groups, or open air atmosphere. For a comparative study, ZnO-sG was also prepared taking a calculated amount of Zn(CH3COO)2 and GO mixture without MWCNTs following the same root. The synthesis procedure is described in the schematic shown in Figure 1.

Liu et al. synthesized functionalized graphene sheet (FGS)/ ZnO composites by thermally heating graphite oxide (GO), Zn(NH3)4CO3 with poly(vinyl pyrolidone) at a high reaction temperature of 300 °C, and showed enhanced photocatalytic activity for rhodamine 6G decomposition.18 Sun et al. prepared ZnO−graphene composite by microwave-assisted reaction of ZnSO4 and GO suspension at 150 °C in 10 min reaction time.19 They have explained that the heating effect by microwave radiation decorates ZnO over graphene and that it exhibited enhanced photocatalytic performance for Cr (IV) reduction. Recently, another report from the same group came with UV-assisted synthesis of ZnO-reduced graphene oxide (RGO) composite by reduction of GO in the presence of presynthesized ZnONPs in ethanol medium.20 Other than these techniques, many other techniques such as solvothermal, hydrothermal, chemical reduction, and different solution based syntheses have been reported for different applications.21−25 Despite of enhanced performance for various applications, the above synthesis techniques are multistep, both energy and timeconsuming, and less environmentally friendly due to use of harsh chemicals. Also, it requires specific equipment like microwave and UV lamps, but for application purposes, a lowcost, less time-consuming, large-scale, and environmentally friendly technique is highly desirable. In this article, a single-step green technique is employed to decorate ZnO nanoparticles over graphene and carbon nanotubes using solar energy. Incorporation of MWCNTs in the synthesis is done with an idea to physically separate graphene layers from restacking when applied for biosensor fabrication. The synthesis technique is facile, inexpensive, environmentally friendly, easy to scale up, and requires only a few minutes without using any harsh chemical reducing agents. The detailed characteristics of the hybrid nanomaterial are studied and compared with ZnO-decorated graphene prepared by the same root. On the basis of the enhanced electrochemical performance studies, the hybrid nanomaterial was employed as a potential transducer candidate for the fabrication of an organophosphorus biosensor. The overall performance of the hybrid nonmaterial in biosensing could combine the individual performance of each component. First, the incorporation of MWCNTs can physically separate the graphene layers and prevent from restacking thereby increasing exposed surface area, and second, uniformly dispersed ZnONPs over graphene and MWCNTs can create a favorable electrostatic interaction for acetyl cholinesterase (AChE) enzyme immobilization.

2. EXPERIMENTAL METHODS 2.1. Materials. Flake graphite powder (99.99% SP-1, Bay carbon, average particle size 45 μm), zinc acetate (C4H10O6Zn·2H2O, 99.5%), concentrated sulphuric acid (H2SO4, 99%), concentrated nitric acid (HNO3, 98%), N,Ndimethyl formamide (DMF, 99%) (Rankem Chemicals, India) were used as received. Potassium phosphate buffer solution (PBS, pH 7.4) was prepared using potassium dihydrogen phosphate (KH2PO4, 99.5%) and di-potassium hydrogen phosphate (K2HPO4, 98%) using deionized water (DI water). Acetyl cholinesterase (AChE, 500 UN) and acetylcholine iodide (AChI, 98%) were purchased from Sigma Aldrich. The substrate solution of AChI (0.1 M) was prepared using PBS and stored at 4 °C for further use. The thiocholine solution was prepared each time before study by enzymatic reaction of AChI stock solution with the enzyme AChE. The exact concentration of thiocholine was calculated spectroscopically by Ellamans test

Figure 1. Schematic of synthesis procedure for the synthesized ZnOMWCNTs-sG hybrid nanomaterial.

2.4. Material Characterization Techniques. The abovesynthesized material was characterized by field emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM, FEI Technie), and Xray diffractometery (PANalytica Cu−Kα as X-ray source). Raman spectra analysis was done by confocal Raman B

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to 11° corresponding to C(002) plane as well as a peak broadening starting from 15° to 30° is observed, which confirms the complete conversion of GO to sG. Also, the peaks corresponding to zinc salts completely disappear; instead, nine highly crystalline ZnO peaks in addition to a combined broaden and sharp peak corresponding to C(002) plane of sG and MWCNTs appears. The XRD pattern was analyzed and fits well to the hexagonal lattice pattern of Wurtzite ZnO (JCPDS 36-1451), which reveals the complete conversion of zinc salt to ZnO nanoparticles over two-dimensional graphene and onedimensional MWCNT nanostructures. The surface morphology of the hybrid nanomaterial was analyzed by electron microscopy technique. Figure 3 shows the

spectrometer (WiTech) with Nd:YAG laser (532 nm) as excitation source. The thermogravimetric analysis (TGA) was recorded by Q500 Hi-Res TGA instruments with a heating rate of 20 °C/min in air atmosphere. All the electrochemical studies were carried out by CH Instrument, electrochemical workstation with a three electrode electrochemical cell comprising a glassy carbon electrode GCE(3 mm dia.) as working electrode, Ag/AgCl (3 mM KCl solution) as reference electrode, and Pt wire counter electrode. Potassium phosphate buffer solution (PBS, PH 7.4) was used as supporting electrolyte for all electrochemical studies at ambient temperature. 2.5. Working Electrode Fabrication. Before fabrication, the GCE was polished mirror-like by 0.05 μm alumina slurry followed by rinsing thoroughly by DI water. For preparing the modified electrode, 20 μL of solution containing ZnOMWCNTs-sG (1 mg/mL in DMF) was drop-casted on the signing surface of GCE and allowed to dry at room temperature. The modified electrode can be named as ZnOMWCNTs-sG@GCE for further studies. For comparison purposes, ZnO-sG modified electrode (ZnO-sG@GCE) was also made taking the same weight percentage of material as taken in ZnO-MWCNTs-sG@GCE. To prepare enzymemodified electrode,10 μL of AChE enzyme solution was overcoated on above prepared electrodes by drop casting and was allowed to dry and kept at 4 °C in order to preserve the enzyme activity before use.

3. RESULTS AND DISCUSSION 3.1. Materials Characterization. The crystallinity of the synthesized hybrid nanomaterial was studied by powder X-ray diffraction technique in the range from 5° to 90° with a step size of 0.016°. Figure 2 shows the X-ray diffraction pattern of

Figure 3. Field emission scanning electron micrograph (FESEM) of ZnO-MWCNTs-sG (a,b) and ZnO-sG (c,d).

FESEM image of ZnO-MWCNTs-sG hybrid (a,b) and ZnOsG(c,d). As shown, here a uniform decoration of ZnO nanostructures over one-dimensional MWCNTs and twodimensional sG was clearly seen. The side view and top view (c,d) FESEM images of layer structure of exfoliated solar graphene with ZnO nanoparticles scattered over the surface are observed. The HRTEM images of ZnO-MWCNTs-sG (a,d) and ZnO-sG (e,f) are shown in Figure 4 at different

Figure 2. XRD pattern of (a) pure MWCNT, (b) GO, (c) mechanically mixed GO and zinc acetate, (d) ZnO-sG, (e) mechanically mixed MWCNTs, GO, and zinc acetate, and (f) ZnOMWCNTs-sG. Figure 4. High-resolution transmission electron microscope (HRTEM) image of ZnO-MWCNTs-sG (a,b) and ZnO-sG (c,d) at different resolution.

(a) pure MWCNTs, (b) GO, (c) mechanically mixed GO and zinc acetate, (d) ZnO-sG, (e) mechanically mixed GO, MWCNTs, and zinc acetate, and (f) ZnO-MWCNTs-sG hybrid nanomaterial. The characteristic peak for pure MWCNTs is observed at 26° corresponding to C(002) plane of hexagonal lattice. For GO, the characteristic peak appears at 11°. In the presence of metal salt in the mechanical mixture of zinc acetate, MWCNTs and GO (c,e), many additional peaks with the peaks corresponding to MWCNTs and GO appear. After treating under focused sunlight, a shift in peak from 26°

magnification confirming uniformity of ZnO nanostructure of average particle size 10−60 nm over MWCNTs and graphene. The particle appears to be spherical in shape over MWCNTs and sG. Figure 5a,b shows the magnified HRTEM image of a single ZnO nanoparticle at 5 nm resolution and the energy dispersive X-ray spectrometry (EDX) results. A clear lattice spacing of 0.24 nm depicts the crystallinity of ZnO NPs, which C

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Figure 5. High magnification HRTEM image of (a) a single ZnO nanoparticle in the hybrid material and (b) EDX of ZnO-MWCNT-sG hybrid material.

peaks corresponding to E2 high frequency phonon mode (∼437 cm−1) and first order longitudinal-optical (E1 LO) phonon mode (∼582 cm−1) of ZnO nanocrystals appear along with D band and G band of MWCNTs and graphene. Again, the peak assigned to A1 symmetry in the transmission optical (TO) mode (∼330 cm−1) and second order longitudinal-optical (E2 LO) phonon mode (∼1124 cm−1) appears in plots c and d, which are basically sensitive to the multiple phonon scattering process in ZnO.32,33 This confirms the decoration over sG and MWCNTs.34 The D/G band intensity ratio for ZnOMWCNTs-sG and ZnO-sG was calculated to be ID/IG = 0.72 and 0.64, respectively. Another important aspect of the synthesis method is the utilization of the whole precursor salt used for synthesis without giving any ionic residue or chemical byproducts. In most chemical synthesis procedures, many side products form, which need postsynthesis treatments in order to be removed. To confirm this, the TGA of the hybrid material has been done. Figure 7 shows the TGA spectra of ZnO-MWCNTs-sG hybrid nanomaterial and the mechanical mixture of zinc acetate, MWCNTs, and GO. The weight loss below 100 °C in mechanical mixture can be due to the decomposition of water present in hydrated zinc salt. The major weight loss occurs at about 220 °C, which can be due to the decomposition of zinc acetate and oxygen containing functional groups of GO.35 However, in TGA spectra of ZnO-MWCNTs-sG, there are no signs of major weight loss below 450 °C, which confirms the complete reduction of zinc salt in the synthesized hybrid nanomaterial. Excluding these features, a two-step weight loses starts from 480 °C in both plots, which can be due to decomposition of graphene followed by MWCNTs. The loading of ZnO in the hybrid nanomaterial is about 50% from TGA spectra. 3.2. Electrochemical Activity Study. To study the electro catalytic activity of the hybrid nanomaterial, cyclic voltammetry was employed as a good appliance. Figure 8A shows the voltammogram of (a) bare, (b) ZnO-sG, and (c) ZnOMWCNTs-sG modified GCE for 5 mM K4[Fe(CN)6] at 10 mV/s scan rate using 1 mM KCl as supporting electrolyte. As shown in the figure, the redox peak due to Fe3+/Fe2+ redox couple for ZnO-MWCNTs-sG@GCE is the most prominent peak in comparison to bare and ZnO-sG@GCE exhibiting more redox current. This can be due to high electron transfer kinetics and high electrocatalytic active surface area of the hybrid nanomaterial. The active surface area of the material was calculated using Randles−Sevsic equation36

were previously discussed in XRD analysis. The presence of MWCNTs in the hybrid material can act as a spacer between graphene layers while doing fabrication of biosensor and preventing agglomeration. This may enhance the overall exposed surface area and hence the electrocatalytic activity of the hybrid nanomaterial, which would be favorable for biosensing and also different energy, related applications. Figure 6 shows Raman spectra of (d) ZnO-MWCNTs-sG compared with (a) pure MWCNTs, (b) GO, and (c) ZnO-sG,

Figure 6. Raman spectra of (a) pure MWCNT, (b) GO, (c) ZnO-sG, and (d) ZnO-MWCNTs-sG.

respectively. For pure MWCNTs, the peak at 1582 cm−1 corresponds to the E2g vibration mode corresponding to graphitic carbon.30 The defect induced D band appears at ∼1345 cm−1, which are particularly sensitive to the presence of disorder carbon atoms and surface modification by doping, etc. The peak intensity of D band is a measure to scale the defects on the modified structures. The D band peak for GO exhibits dominance over G band with (ID/IG = 1.25) as expected, which can be due to the presence of oxygen containing functional groups in the edges as well as in-plane sp2 domains. The peak around 2700 cm−1 in plots a, c, and d corresponds to second harmonics of D band called 2D band, which is sensitive mainly to the number of layers and structural changes along the c axis. The peak intensity of 2D band decreases with an increase in the number of layers. As in GO, the graphitic planes are highly disordered and the number of graphene layers is more; the peak intensity becomes very less or sometimes disappears in the Raman spectra.31 In the hybrid nanomaterials (plots c and d), D

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This equation relates the redox peak current (Ip) with the concentration (C) of the probe molecule (K4[Fe(CN)6]), scan rate γ (mV/s), diffusion coefficient D(= (6.70 ± 0.02) × 10−6 cm2/sec), number of electrons participating in reaction n, and electrochemical active surface area of electrode A. The A value for ZnO-MWCNTs-sG@GCE is calculated to be (30.88 ± 1) mm2, and that for ZnO-sG@GCE is (11.60 ± 1) mm2, which is more than that for bare GCE (A = 7.067 mm2). Figure 8B shows the redox peak current versus square root of scan rate at bare, ZnO-sG@GCE, and ZnO-MWCNT-sG@GCE, which increases linearly revealing the charge transport to be diffusion controlled.37 Again, the slope of the line for hybrid nanomaterial is more in comparison to ZnO-sG@GCE as well as bare GCE. This explores the high electrocatalytic activity of the hybrid material, which can be used as a transducer for fabrication of an organophosphorus biosensor. 3.3. Catalytic Activity toward Thiocholine Oxidation. Thiocholine is an enzymatic hydrolysis product of the neurotransmitter acetylcholine by acetyl cholinesterase (AChE), an important enzyme found in neuromuscular junction and cholinergic brain synapses of mammals. The OP compounds (Paraoxon) are highly toxic because it inhibits the activity of AChE and thereby limits the production of thiocholine. This leads to severe health threats to humanbeings as well as other mamals.38 Because of the acute toxicity and hence health concerns, a low cost, sensitive, and easy monitoring Paraoxon biosensor fabrication is of great importance. Since enzyme inhibition in the presence of Paraoxon leads to a decrease in thiocholine production, the inhibited oxidation current of thiocholine can act as a probe to monitor the Paraoxon concentration. Therefore, the electrochemical activity of thiocholine for fabricated biosensorshas been studied. Figure 9A shows the cyclic voltammogram at ZnO-sG and ZnO-MWCNTs-sG@ GCE in for 3 mM thiocholine solution with pH 7.4 PBS as supporting electrolyte. A sharp oxidation peak appears at 0.67 V for ZnO-MWCNTssG in comparison to ZnO-sG@GCE, which reveals its catalytic activity toward thiocholine oxidation. Further the oxidation current is about 4-fold more than that of ZnO-sG modified electrode. Also a pair of redox peaks appears at lower potential, which can be due to the presence of an iodide redox couple in accordance to the following equations:39

Figure 7. TGA spectra of (a) mechanical mixture of zinc acetate, MWCNTs, and GO, and (b) ZnO-MWCNTs-sG.

AChI → TCh + acetic acid + I TCh → TCh(ox) + 2H+ + 2e−

3.4. Calibration of Biosensor at Different Substrate Concentration. The response of the enzyme-modified electrode toward different concentrations of the AChI solution was studied, which gives linearity from 0.2 to 6.4 mM as shown in Figure 9B. Using the following Michelson−Menton equation, the Michelson−Menton constant is calculated to be 0.8 mM:40 I = Imax − K m(I /[S])

where Imax is the maximum current obtained, I is the steadystate current, [S] is the substrate concentration, and Km is the Michelson−Menton constant. The lower value of Km is a measure of how efficiently an enzyme converts a substrate to product, which could be due to better enzyme adhesion on the hybrid nanomaterial retaining its bioactivity. Also, as ZnO has a high isoelectic point (IEP = 9.5), it could efficiently bind the AChE enzyme (IEP = 4.5) electrostatically in a pH 7.4

Figure 8. (A) Cyclic voltammetry obtained for 5 mM K4[Fe(CN)6] in 0.1 M KCl solution at 10 mV/s scan rate. (B) Plot of peak current vs square root of scan rate at (a) bare, (b) ZnO-sG, and (c) ZnOMWCNTs-sG@GCE.

Ip = 2.69 × 105AD1/2n3/2γ 1/2C E

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Figure 10. Plot of inhibition current at different concentrations of Paraoxon. Inset: plot of % inhibition against Paraoxon concentration.

advantage of this method lies in a uniform distribution of ZnO NPs over graphene and MWCNT hybrid composite surface without using any chemical oxidizer in a few minutes synthesis time. The hybrid nanomaterial has an increased electrochemical active surface area due to the presence of MWCNTs, which act like a spacer between the graphene layers preventing from restacking. Besides, this composite as a transducer candidate shows a high catalytic activity to thiocholine oxidation and a good response toward the AChI over a wide linear range from 0.2 to 6.4 mM, which can be attributed to high electron transfer kinetics and better immobilization of AChE enzyme over the large surface area of the hybrid composite. In addition, the biosensor for Paraoxon detection shows linearity over the 1 to 26 nM range with a low detection limit of 1 pM (S/N = 3). On the basis of the overall significantly enhanced activity of ZnO NP-decorated MWCNTs-sG hybrid composite and its largescale green synthesis technique, it can be recommended as a promising candidate for sensors and energy related applications.

Figure 9. (A) Cyclic voltammogram at 3 mM thiocholine for (a) ZnOsG and (b) ZnO-MWCNTs-sG@GCE. Inset: Response for only phosphate buffer solution at (c) ZnO-sG and (d) ZnO-MWCNTssG@GCE. (B) Response toward different concentrations of AChI substrate solution for AChE-ZnO-MWCNTs-sG@GCE.

phosphate buffer environment, which withholds its bioactivity.41 3.5. Paraoxon Detection. On the basis of the good response to different substrate concentration, the biosensor was employed for the detection of different molar concentration of Paraoxon. The mechanism of detection comprises the loss of activity of AChE enzyme in the presence of Paraoxon, thereby decreasing the current due to thiocholine oxidation, which is the enzymatic hydrolysis product of AChI. The inhibited current for AChE-ZnO-MWCNT-sG@GCE was recorded after successive addition of different molar concentrations of Paraoxon for an incubation time of 5 minutes. Figure 10 shows the plot of inhibition current with different concentrations of Paraoxon, which shows a sharp decrease with successive addition of Paraoxon. This reveals the decrease in catalytic activity of the enzyme AChE after exposure to Paraoxon. The inset shows the percentage of inhibition calculated by [(Imax − Iin)/Imax] × 100, where Imax is the maximum current and Iin is the current obtained after incubation with Paraoxon. A linear response from 1 to 26 nM was obtained with a detection limit of 1 pM (S/N = 3). The lower detection limit and good response can be attributed to the functionality of the hybrid matrix to hold the enzyme.

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AUTHOR INFORMATION

Corresponding Author

*(S.R.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Indian Institute of Technology Madras, Chennai, for financial support and IITM, Chennai, and Intellectual Ventures, Bangalore, for their support in filing this work as a patent.

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REFERENCES

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dx.doi.org/10.1021/jp312824b | J. Phys. Chem. C XXXX, XXX, XXX−XXX