DOI: 10.1002/celc.201402255

Application of Cationic Poly(lactic-co-glycolic acid) IronOxide/Chitosan-Based Nanocomposite for theDetermination of ParaoxonIda Tiwari,* Mandakini Gupta, and Chandra Mouli Pandey[a]

1. Introduction

Pesticides are widely used in agriculture to increase yield, con-trol micro-organisms that might produce toxic or carcinogenicmetabolites, and reduce the cost of food production. Accord-ing to statistical data published by the Food and AgricultureOrganization of the United Nations, more than 1.2 millionmetric tons of pesticides were sold to the agricultural sectorworldwide in 1995. These pesticides could be divided into26 % insecticides, 31 % fungicides, and 43 % herbicides. Themajority of the insecticides used were acetylcholinesterase(AChE) inhibitors, 55 % of which belonged to the group of or-ganophosphates and 11 % to carbamates, whereas the otherswere pyrethroids, chlorinated hydrocarbons, or other insecti-cides.[1] Organophosphate (OP) pesticides (e.g. paraoxon, para-thion, and chlorophos) that are widely used in agricultureshow high effectiveness for insect eradication. Their residues inagricultural products and water pose a potential hazard tohuman health, such as eye pain, abdominal pain, convulsions,respiratory failure, paralysis, and even death.[2–6] So, they arenow rarely used as insecticides in developed countries, butthey still remain the major insecticides for agricultural pestcontrol in developing countries. Nowadays, OPs have also at-tracted attention for their illegal use as pesticides, especially inprivate homes. Therefore, it is necessary to develop a rapidand effective method for the determination of OPs. Generally,most widely used techniques for OP measurements are chro-matography, including gas chromatography, liquid chromatog-raphy, and thin-layer chromatography.[5] Biological techniques

such as immunoassays and biosensors are also used for the de-termination of OPs.[7–9] Among these techniques, chromatogra-phy is time consuming, expensive, and cannot easily be ap-plied to the continuous monitoring of OPs. Electrochemicalmethods based on chemically modified electrodes have showngreat potential over other techniques for the detection of OPs,because of the simplicity, fast response, good sensitivity, highselectivity, and excellent long-term calibration stability. Subse-quently, there has been growing interest in the developmentof electrochemical sensors for the determination of OPs.[10] Inthese sensors, the most popular enzyme inhibition mechanismuses pesticides as inhibitors, which can be determined bymeans of measuring the kinetic performance of the initial ve-locity of the reaction catalyzed by the enzyme, before andafter an incubation step with the pesticide.

There are numerous studies reported for pesticide detectioninternationally, using different enzymes such as tyrosinase,[11–14]

alkaline phosphates,[15] ascorbate oxidase,[16] and luciferase[17]

as inhibitors against pesticides and bi-enzyme systems thatuse butylcholinesterase and choline oxidase.[18, 19] But, the ma-jority of biosensors for OPs, to date, are based on the direct in-activation of the enzyme AChE for the detection of pesticides,and the substrate used for this purpose is acetylthiocholine(ATCl), which is a redox-active compound.[20–27] Generally, theproblem encountered for the adsorption of enzymes directlyonto naked surfaces of bulk materials is frequently attributedto their denaturation, and loss of bioactivity occurs. Moreover,reports have shown that the addition of electronic mediatorsto electrode composite mixtures lowers the potential for elec-trocatalytic oxidation of thiocholine,[28–31] but only to a certainextent.

Taking all of this into account, it is essential to design a mate-rial that is highly stable, can retain enzymatic activity forlonger periods of time, and can be used to detect OP with

The preparation of stable oleic-acid-capped iron-oxide nano-particles is reported, which were further coated with cationicpoly (d,l-lactic-co-glycolic acid) and chitosan in aqueous mediaby using a nano-emulsion technique. The prepared compositewas used to modify a glassy carbon electrode and the electro-chemical behavior and stability of modified electrode were in-vestigated by using cyclic voltammetry. This platform was thenutilized to prepare an electrochemical pesticide biosensor

based on the inhibition study of the enzyme acetylcholinester-ase. Furthermore, the response characteristics show that thisfabricated electrode has a shelf life of about 3–4 months andhas good adhesion properties along with homogeneous dis-persion at the electrode surface. The linear range and detec-tion limit for paraoxon pesticide detection was found to be9.55–37.1 nm and 8.91 nm, respectively.

[a] Dr. I. Tiwari, M. Gupta, C. M. PandeyCentre of Advanced Study in Chemistry, Faculty of ScienceBanaras Hindu University, Varanasi 221005 (India)Fax. : + 91-5422368174E-mail : sensorsbhu@yahoo.co.in

Supporting Information for this article is available on the WWW underhttp://dx.doi.org/10.1002/celc.201402255.

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high sensitivity and selectivity. However, various biosensorshave already been reported in the literature, using differentmaterials such as Prussian blue,[32] mesoporous materials,[33]

nanoparticles,[22, 34] hybrid nanoparticles decorated graphene,[35]

and polyaniline–carbon nanotubes[36] for various pesticides in-cluding monocrotophos, parathion, carbamate, endosulfan,malathion, and chlorpyrifos. Among these reports, only a fewcan be used for paraoxon insecticide detection. Yin et al. re-ported an amperometric biosensor for paraoxon based on im-mobilized AChE on gold nanoparticles and silk fibroin.[37] Chol-ine-oxidase-based paraoxon determination has been reportedby Sajjadi et al.[38] and Chauhan et al. have reported a goldnanoparticle–CaCO3 hybrid material for this purpose.[39] Self-as-sembly of AChE on a gold nanoparticle–graphene nanosheethybrid, using a polyelectrolyte as the linker, has been reportedby Wang et al.[40] Du et al. used Fe3O4/AuNPs magnetic nano-composites for the highly sensitive determination of AChE ac-tivity in the determination of paraoxon in human serum.[41]

Iron-oxide nanoparticles (IONPs) possess unique size-depen-dent magnetic properties, thermal stability, and chemical inert-ness with a large specific surface area and high surface energy,representing a suitable material for strongly absorbing biomol-ecules, including enzymes, DNA, RNA, antibodies, and so forth.Furthermore, the immobilization of enzymes onto the magnet-ic nanoparticles enhances enzyme activity, facilitates rapid con-tact between the enzyme and its substrate, and reduces mass-transfer limitations.[42, 43] They can also be retained and re-moved with a magnet without affecting the transducer surface,thus creating possibilities for regeneration and reuse.[44] Re-cently, IONPs have been used for the immobilization of en-zymes with retained bioactivity for construction of enzyme-based biosensors.[43, 45, 46] Although IONPs represent a suitablematerial for the preparation of biosensors, owing to the highchemical reactivity and large surface-to-volume ratio of uncoat-ed IONPs, they are easily agglomerated. To overcome theseissues, the IONPs can be modified by using inorganic layers(e.g. carbon, silicon), conducting polymers, biopolymers (poly-saccharides), surfactants, long-chain carboxylic acids, and phos-phates, as they give thermodynamically stable dispersions ofIONPs.[47] For this purpose, we utilized poly(d,l-lactic-co-glycol-ic acid) (PLGA) as a stabilizing agent for IONPs througha strong coordination interaction of the carboxylate groupwith that of Fe(III).[48] As reported earlier,[49] PLGA-coated IONPmicrospheres (PlgNPs) contain negative charges, which can beshifted to neutral or positive charges through surface modifica-tion. To further stabilize these PlgNP microspheres, chitosan(CS), an abundant biopolymer,[46] was used. The polysaccharideof CS possesses free amino groups that are protonated andpositively charged in slightly acidic media. These amino groupsof CS are effective in stabilizing PlgNP microspheres. There arevery few reports available for the use of Fe2O3 nanoparticlesfor pesticide detection, using AchE as the enzyme inhibitor. Inthose available reports,[50, 51] the attachment of the enzyme hasbeen achieved by covalent binding, resulting in a decrease inenzyme activity for the substrate.

Hence, in this paper, cationic PLGA iron oxide with a CS-modified glassy carbon electrode (CS/PlgNPs/GCE) was used to

prepare a sensitive, stable, and highly reproducible sensor forparaoxon determination through AChE enzyme inhibition, inwhich the enzyme was attached through electrostaticinteractions.

Experimental Section

Materials

Enzyme AChE (857 units mg�1, from electric eel E.C. 3.1.1.7), ATCl,iron(III) acetylacetonate, 1,2-hexadecanediol, oleic acid, oleylamine,glutaraldehyde, PLGA (Mw = 40 000–75 000), 1-ethyl-(dimethylami-nopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), poly-vinyl alcohol (PVA), and CS (Mw = 15 000–20 000) were purchasedfrom Sigma–Aldrich (USA). Acetylthiocholine iodide was purchasedfrom Himedia (Mumbai, India). All aqueous solutions were pre-pared by using triple-distilled water, and the chemicals employedwere of analytical grade.

Enzyme Assay

The enzyme assay test was performed by using a photometricmethod for determining AChE activity, according to Ellaman’smethod.[52] The enzyme activity was measured by following the in-crease in yellow color produced from thiocholine when it reactswith the dithiobisnitrobenzoate ion (cf. Figure S1 in the SupportingInformation). A typical run used pH 8.0 buffer (3.0 mL), substrate(20.0 mL), dithiobisnitrobenzoic acid reagent (DTNB; 100.0 mL), andenzyme (50.0 mL). The blank for such a run consisted only of buffer,substrate, and DTNB solutions. The absorbances were recorded,the best line was drawn through the points, and the slope wasmeasured. In a run such as that described above, the linear portionof the curve, describing the hydrolysis, was observed during thefirst 15–20 min of the reaction; the slope was then considered asthe rate in absorbance units per minute, and the rate was calculat-ed by using Equation (1):

rate ðmole l�1 min�1Þ ¼ Dabsorbance min�1=1:36� 104 ð1Þ

Instrumentation

Electrochemical characterization was performed by using an elec-trochemical analyzer (CHI 630 C) with a three-electrode system,using a GCE as the working electrode, Ag/AgCl as the referenceelectrode, and a platinum wire as the counter electrode in phos-phate buffer solution (PBS). All electrochemical measurementswere carried out in 0.1 m PBS (4 mL) at pH 8.0, which was deaerat-ed by bubbling nitrogen for 15 min prior to the experiments. Scan-ning electron microscopy (SEM) of the nanocomposite films wasperformed with a JEOL840 A microscope (Japan).

Synthesis of PlgNPs and CS/PlgNPs

Preparation of IONPs and PLGA-capped IONPs (PlgNPs) was carriedout as reported earlier with slight modification.[49, 53] In short,2 mmol iron(III) acetylacetonate, 5 mmol 1,2-hexadecanediol,6 mmol oleic acid, 3 mmol oleylamine, and 10 mL phenyl etherwere mixed with constant mechanical stirring at 200 8C for 2 hunder a nitrogen atmosphere. The obtained black-colored mixturewas cooled to room temperature (25 8C) and purified by repeatedlywashing with pure ethanol followed by separation through centri-fugation. The formed product was dispersed in hexane; the partial-

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ly dispersed mixture was centrifuged at 10 000 rpm for about10 min to remove any undispersed residue. PlgNPs were preparedusing a nano-emulsion method. For this, a mixture of PLGA(50 mg) and IONPs (7.5 mg) was dissolved in dichloromethane(2.5 mL) under continuous stirring. This organic solution was thenadded to an aqueous solution (5 mL) containing PVA (1.0 %) andCS (0.3 %) as the stabilizer mixture, followed by addition of EDC(0.4 m) and NHS (0.05 m) to obtain the CS/PlgNPs composite solu-tion. Thereafter, the organic–aqueous solution mixture was emulsi-fied for 15 min by using an ultrasonicator (Vibronics Pvt. Ltd,Mumbai, India) operated at 24 kHz. Furthermore, the organic sol-vent was evaporated by using a water bath at 40 8C, and the solu-tion was subjected to centrifugation (20 000 rpm) for three repeti-tive cycles. Thus, the prepared CS/PlgNPs were re-suspended in5 mL of water and were stored at 4 8C.

Modification of GCE with AChE/CS/PlgNPs Composite

The GCE was polished with alumina slurry (0.5 m) and then washedwith acetone, sonicated in distilled water, and allowed to dry atroom temperature. For electrode modification, the prepared com-posite was sonicated prior to casting the composite on the GCE.Then, 5 mL (optimized concentration) of the prepared PlgNPs/CScomposite solution was dropped onto the pretreated GCE and al-lowed to dry at 50 8C in a vacuum oven for 2 h. To this modifiedelectrode, 5 mL (optimized concentration) of enzyme AChE (5 units)was dropped. The negatively charged AChE enzyme interacts elec-trostatically with the PlgNPs/CS composite film (AChE/CS/PlgNPs/GCE). The prepared electrode was washed with triple-distilledwater twice to remove non-adsorbed nanocomposite material.

Electrochemical Detection of Paraxon Pesticide

The AChE/CS/PlgNPs/GCE biosensor was employed for the determi-nation of pesticides by using cyclic voltammetry (CV). The per-formance of the biosensor was studied by its CV response in 0.1 m

PBS (pH 8.0) containing 0.15 mm ATCl. Then, the electrode wasrinsed with water and incubated in an aqueous solution containingthe desired concentration of pesticide (paraoxon) for 10 min. Final-ly, it was transferred into 0.15 mm ATCl solution for CV measure-ments under the same conditions. The inhibition rate of pesticideswas calculated as follows:

Inhibition ð%Þ ¼ ðIp0�IpÞ=Ip

0 � 100 ð2Þ

where Ip0 and Ip are the anodic peak current of 0.15 mm ATCl

before and after treatment with paraoxon pesticide on AChE/CS/PlgNPs/GCE, respectively. Inhibition (%) was plotted against thepesticide concentration as a calibration plot.

Preparation of a Real Sample, using Cabbage for theAnalysis of Paraoxon

For studying the paraoxon detection in the vegetable cabbage,a bundle of leaves (1.213 g) was cut and mixed in a blender with0.1 m PBS (pH 7.0), resulting in a final volume of 1.0 L. The analysisof paraoxon was carried out directly in the extracts of the cabbagesample by using the standard addition method, as reportedearlier.[36]

2. Results and Discussion

2.1. Morphological and Structural Characterization

2.1.1. Transmission Electron Microscopy (TEM) of PlgNPs

Figure 1 A shows the TEM image of PLGA-IONPs, which indi-cates that PLGA-IONPs are monodispersed and nearly sphericalin shape, having particle sizes of 8–12 nm. Figure 1 B shows

the TEM image of the CS/PlgNPs composite, clearly indicatingthe CS coating over the PLGA-IONPs, which further results inthe formation of stable PlgNPs. The detailed morphologicaland structural characterizations are similar to the work previ-ously reported.[49]

2.1.2. Scanning Electron Microscopy (SEM)

SEM images of CS/PlgNPs (Figure 2 A) indicate uniform distribu-tion of PLGA-capped IONPs in the CS matrix. These nanoparti-cles do not aggregate and maintain their spherical structure

after formation of PlgNPs on the electrode surface. Figure 2 Bshows the SEM images of the AChE/CS/PlgNPs-modified GCE,at which the attachment of enzyme AChE can be seen clearly.The negatively charged AChE enzyme is attached to highlystable, positively charged PlgNPs through strong electrostaticinteractions, as discussed above.

Figure 1. TEM image of A) PLGA-IONPs and B) CS/PlgNP composite; scalebar = 20 nm.

Figure 2. SEM images of a) CS/PlgNPs/GCE and b) AChE/CS/PlgNPs/GCE(inset: enlarged image of enzyme AChE attached to CS/PlgNPs/GCE surface.

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2.2. Electrochemical Characterization

Electrochemical impedance spectroscopy (EIS) was employedto investigate the charge-transfer process occurring at an elec-trode/solution interface. EIS measures real Zre(w) versus imagi-nary Zim(w) impedance that can be plotted in a Nyquist dia-gram, which typically exhibits two important regions for a re-versible reaction at a solid interface.

As indicated in Figure 3, the semicircle obtained shows thechange in electronic transfer resistance (RCT) at high modula-

tion frequency, describing the faradic electron-transfer processat the electrode. Whereas, the straight region obtained forlower modulation frequency contains information about thediffusion-limited transport of the redox species in the electro-lyte to the electrode interface. It was observed that, after thedeposition of PlgNPs onto the GCE surface, the RCT value forthe PlgNPs/GCE decreased almost twofold (0.42 kW; Figure 3,curve i) in comparison to that of CS/GCE (0.99 kW; Figure 3,curve ii). This shows that electron transfer in the PlgNPs nano-composites film is easier between the medium and the elec-trode. Furthermore, the Fe3O4 nanoparticles increase the elec-troactive surface area, resulting in more diffusion of the redoxprobe [FeIII/FeIV] onto the cationic PlgNPs nanocomposite film,which enhances the electron-transfer kinetics from themedium to the electrode. When the AChE is immobilized onthe electrode surface, the electron transfer through the nega-tively charged redox marker, [Fe(CN)6]3�/4�, is hindered, result-ing in an increased RCT (1.9 kW; Figure 3, curve iii) compared tothat of the PlgNPs/GCE.

The electrochemical studies of AChE/CS/PlgNPs/GCE wereperformed by using CV. The cyclic voltammograms of AChE/CS/PlgNPs (cf. Figure 4) and CS/PlgNPs (cf. Figure S2) modifiedGCE were obtained in 10 mm ferricyanide buffer solution asa function of scan rate (n= 10–400 mV s�1). The positive andnegative peaks correspond to the oxidation and reduction re-

actions of the [Fe(CN)6]3�/4� redox pair, respectively. As dis-cussed above, CS is a natural polymer and has a positivecharge under acidic conditions, which is attributed to the pro-tonation of the amino groups, resulting in the formation ofa positively charged film of CS. The enzyme AChE is negativelycharged in nature, owing to the presence of carboxylic groupsof the amino acids. Hence, the positively charged CS interactselectrostatically with negatively charged enzyme AChE withoutaffecting its activity. At CS/PlgNPs/GCE, anodic (Epa) and catho-dic peak potentials (Epc) were observed to be 275 and 134 mV,respectively, with a peak separation of 141 mV (cf. Figure S2),whereas the AChE/CS/PlgNPs-modified GCE shows Epa and Epc

at 316 and 141 mV, respectively, with a peak separation of175 mV. The increase in peak separation, DEp, for AChE/CS/PlgNPs- compared to that of CS/PlgNPs-modified GCE is indica-tive of AChE loading over the CS/PlgNPs-modified electrodesurface, which may be attributed to the fact that binding ofa biomolecule to the surface of a working electrode leads tothe progressive increase in electron-transfer resistance, as dis-cussed earlier.[54] The inset to Figure 4 shows the variation ofpeak current Ip with scan rate n for both oxidation and reduc-tion, following a linear relation with respect to the square rootof the scan rate, indicating the existence of diffusion-controlled

Figure 3. Nyquist diagram (Zim vs. Zre) for the faradaic impedance measuredin PBS solution (pH 7.4) containing 5 mm [Fe(CN)6]3�/4� in the frequencyrange 105–1 Hz for i) CS/PlgNPs/GCE, ii) CS/GCE, and iii) AChE/CS/PlgNPs/GCE.

Figure 4. A) Cyclic voltammograms of AChE/CS/PlgNPs-modified GCE stud-ied in 10 mm ferricyanide solution as a function of scan rate (n = 10–400 mV s�1). B) Randles–Sevcik plot (Ip vs. square root of n).

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electrochemical reaction rates for the redox pair at the elec-trode. The plot of the log scan rates versus the peak potential(Epa and Epc) showed linear relationships (cf. Figure 5), whichalso agrees well with Laviron’s theory and may be given byEquations (3)–(5):

Epa ¼ Eo þ X ln½ð1�aÞFn=RTks� ð3Þ

Epc ¼ Eo þ Y ln½ðaÞFn=RTks� ð4Þ

ln K s ¼ a lnð1�aÞþð1�aÞ ln a�lnðRT=nFnÞ�að1�aÞnF DEp=2:3 RT

ð5Þ

where F is the Faraday constant, R is the ideal gas constant, Tis the absolute temperature and a and ks are the electron-transfer coefficient and charge-transfer rate constant, respec-tively. On the basis of Laviron’s equations [Eqs. (3)–(5)] , thevalues of a and ks were determined to be 0.48 and 1.38 s�1, re-spectively. The values of the slopes of the plot of ln n versusEpa and Epc were found to be as 0.114 and 0.121 [where X = RT/(1�a)nF and Y = RT/anF] , respectively. In this work, surfacecoverage area of the surface-active species was calculatedfrom Equation (6):[49]

Ip ¼ n2F2nAG=4 RT ð6Þ

where n is the number of electrons transferred for the redoxreaction; here, n is 10 mV s�1. Taking the average of both thecathodic and anodic results, G of electroactive species at theelectrode surface was found to be 1.98 � 10�7 mol cm�2. On thebasis of the linear slope of the anodic peak currents, we obtainthe Randles–Sevcik equation [Eq. (7)]:

Ip ¼ ð2:99� 105Þa1=2n3=2ACD1=2n1=2 ð7Þ

where the diffusion coefficient (D) is calculated as 1.15 �10�10 cm2 s�1 and C is the molar concentration of [Fe(CN)6]3�/4� ;here, n is 50 mV s�1.

2.3. CV Response to ATCl

Figure 6 A shows the CV response investigated in the presenceand absence of 0.15 mm ATCl. To discern the effect of eachcomponent, a control experiment was also performed at CS/GCE, CS/PlgNPs/GCE, and AChE/CS/PlgNPs/GCE. In Figure 6 A,curve a shows the cyclic voltammogram for CS/GCE in the ab-sence of ATCl. In the presence of ATCl (cf. Figure 6 A, curve b),

Figure 5. Plot of log n versus Epa and Epc.

Figure 6. A) Cyclic voltammograms of CS/GCE, CS/PlgNPs/GCE and AChE/CS/PlgNPs/GCE in the absence (a, c, e) and presence (b, d, f) of 0.15 mm ATCl, re-spectively, under optimal experimental conditions. B) DPV response of ATCloxidation on AChE/CS/PlgNPs/GCE at concentrations of 125, 150, 175, 240,277.5, 314.5, 351.5, 388.5, 425.5, 481.5, 518.5, 555, 629, 703, 777, and830 mm. C) Calibration plot of Ipa vs. concentration of ATCl.

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no appreciable change was observed. For CS/PlgNPs/GCE inthe absence of ATCl (cf. Figure 6 A, curve c), no current is ob-served, but in presence of ATCl (cf. Figure 6 A, curve d), en-hancement of anodic current was found to be more than CS/GCE, which clearly indicates that the PlgNPs enhances the oxi-dation of ATCl. But, on AChE/CS/PlgNPs/GCE (Figure 6 A,curve e, in absence of ATCl), there is an almost threefold in-crease in oxidation peak current upon addition of ATCl (cf. Fig-ure 6 A, curve f) compared to CS/PlgNPs/GCE, which indicatesthat there is catalytic oxidation process occurring because ofthe immobilized PlgNPs and AChE. Differential-pulse voltam-metry (DPV) was used to study the detection of ATCl at differ-ent concentrations (cf. Figure 6 B). The oxidation current ofATCl was proportional to the concentration and was found tobe linear (cf. inset of Figure 6 B).

2.4. Pesticide Determination on PlgNP/CS/AChE Composite-Modified Electrode

The CV (Figure S3, curves b–f) and DPV (Figure 7) responseswere examined before and after exposure to different concen-trations of pesticide paraoxon, which is associated with inhibi-

tion of enzymatic activity of AChE, with increased concentra-tion of paraoxon. The decrease in anodic current of ATCl(0.830 nm) on exposure to increased pesticide concentration isattributed to the blocking of the serine residue of AChE.Figure 8 shows the percentage inhibition of the AChE/PlgNPs/

CS-modified GCE at different concentrations of paraoxon. Thelinear range was found to be 9.55 to 37.1 nm, with a correlationcoefficient of 0.995 and detection limit of 8.91 nm, witha signal-to-noise ratio of 3. The detection limits and other char-acteristics of the biosensor were better/on par with works(Table 1).

2.5. Kinetic Study of Enzyme-Modified Electrode

The apparent Michaelis–Menten constant (Kappm) is an impor-

tant quantity, describing enzyme affinity towards a given sub-strate. Kapp

m was calculated from the electrochemical version ofthe Lineweaver–Burk equation [Eq. (8)]:

1=Iss ¼ 1=Imax þ K appm=ImaxC ð8Þ

Figure 7. DPV responses of ATCl oxidation (0.83 mm) in the presence of dif-ferent concentrations of paraoxon (9.55, 13.8, 17.7, 22.0, 26.21, 29.18, 34.03,and 36.81 nm) associated with the inhibition of AChE activity.

Figure 8. Plot of percentage inhibition of AChE activity versus the concentra-tion of paraoxon.

Table 1. Comparison of linear range and detection limit of some modified electrodes and th PlgNPs/CS/AChEs composite-modified GCE for the determina-tion of pesticides.

Mode ofdetection

Transducer Enzyme immobili-zation method

Linearrange [mM]

Detectionlimit [mM]

Storagestability [days]

Correlationcoefficient

Ref.

CV PDMS-PDDA/AuNPs/ChO/AchEs/GCE hydrophobic 0.0005[a] [22]amperometry MC/CB/AchEs/GCE entrapment 0.120 30 [33]chronoamperometry ChOx/PB/SPE cross linking 0.1–1 0.10 30 0.999 [38]amperometry poly(acrylonitrile–methylmethacrylatesodium vinyl

sulfonate)/AchE/GCEcovalent andcross linking

10�7–10�10 0.0739[a] 50 0.996 [55]

amperometry PVA/SbQ/Pt electrode entrapment 0.0055 30 [56]amperometry cellophane/Au crosslinking 1.45–7.26 1.45 [57]optical sol-gel/glass entrapment 0.098–0.55 0.0980 0.980 [58]DPV AChE/CS/PlgNPs/GCE electrostatic

interaction0.00955–0.0370

0.00891 90 0.995 presentwork

[a] Units = [ng mL�1] .

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where Iss is the steady-state current after the addition of a sub-strate, C is the bulk concentration of the substrate, and Imax isthe maximum current measured under saturated substrateconditions (Figure S4). According to this equation, once theoptimal conditions were obtained, the inhibitory effect of para-oxon on the response of the AChE biosensors was investigatedfollowing the method of Lineweaver–Burk. The Kapp

m value(5.8�0.3) � 10�3

m found in presence of paraoxon is higherthan that obtained without paraoxon (4.7�0.3) � 10�3

m. There-fore, the presence of paraoxon diminishes the enzyme sub-strate affinity, which is in agreement with other reportedwork.[59, 60] The Kapp

m value obtained by using the immobilizedPlgNPs/CS/AChE composite biosensor was lower than that re-ported by Doong and Tsai[58] and, on the other hand, it washigher than some earlier reported work.[33, 38, 55] Several reasonscan account for the low activity of the enzyme over the PlgNP/CS nanocomposite-modified GCE, but most probably, it is dueto the denaturation of a small number of active sites in AChEduring the preparation procedure, and the enzyme activitymay be significantly hindered through the polymeric media;consequently, a higher concentration of substrate is neededcompared to the solution.

3. Parameter Optimization for BiosensorPerformance

3.1. Effect of pH Value

The pH value of the buffer has an important role in the activa-tion of enzyme AChE, and the optimum working range of theenzyme is reported to be between pH 7.0 and 8.0.[22] We stud-ied the effect of pH on the AChE/CS/PlgNPs/GC biosensor inthe presence of 0.1 m PBS containing 0.15 mm ATCl, and themaximum value of response current was found at pH 8.0 (cf.Figure S5 A). Hence, pH 8.0 was chosen as the optimum pHvalue.

3.2. Effect of Enzyme Concentration

The effect of enzyme concentration was also studied. As theconcentration of the enzyme increased, the current responsefor ATCl increased gradually until the concentration of theenzyme reached a value of 5 U and then the response de-creased gradually (cf. Figure S5 B). Therefore, an enzyme con-centration of 5 U has been selected as the optimum enzymeconcentration for AChE/CS/PlgNPs/GC biosensor fabrication.

4. Performance of Sensor

4.1. Accuracy, Precision, and Recovery

The intra-assay accuracy and precision was estimated by deter-mining the response of 0.83 mm ATCl at one enzyme electrodefor three replicate determinations, using DPV. Inter-assay accu-racy and precision of the proposed procedure were estimatedby analyzing 0.83 mm ATCl solutions at three different electro-des, which were immersed in 9.55 nm paraoxon solution, and

the relative standard deviations (RSDs) were calculated to be1.59–3.10 %, indicating high accuracy and precision of the pro-posed sensor (Table 2).

Recovery studies were performed in the concentration rangeof 10–26 nm by adding different amounts of pesticides intothe vegetable sample (cabbage). The results show that the re-coveries vary in the range of 97.3–103.8 %.

4.2. Stability, Reproducibility; and Interferences of theBiosensor

The modified electrode stability was stability was measured byrecording cyclic voltammograms. The stability of the AChE/CS/PlgNPs-modified GCE could be maintained by storing the elec-trode at 4 8C when not in use. The response of pesticides wastested intermittently. No obvious decrease in the current re-sponse was observed during the first four consecutive dayswhen performing four measurements on each day for thesame concentration of ATCl after immersion in 9.55 nm of pes-ticide solution; the RSD value was found to be 1.25 %, whichdemonstrates good reproducibility of the biosensor. After a 60-day storage period, the sensor retained 70 % of its initial cur-rent response. The responses of the sensors decreased withthe number of measurements. Interferences from the otherelectroactive nitrophenyl derivatives, such as nitrobenzene, ni-trophenol, and other oxygen-containing inorganic ions (PO4

3�,

SO42�, and NO3

�), were investigated (Figure S6). No obvious in-hibition behavior could be observed after addition of a 100-fold increase in the concentration of interferences, but thepeak currents of ATCl varied slightly.

5. Conclusions

The present work indicates that the use of IONPs capped withcationic PLGA, iron oxide, and CS results in the preparation ofa material that facilitates the loading of enzyme AChE and de-termination of the pesticide paraoxon at low potentials withincreased sensitivity and selectivity.

Table 2. Accuracy, precision, and recovery for the determination ofpesticide (n = 3).[a]

Paraoxon concentrationadded [nM]

Paraoxon concentrationfound [nM]

RSD[%]

Recovery[%]

Intra-assay10 9.41 2.20 98.114 14.20 1.59 101.418 17.82 1.94 99.022 21.73 3.20 98.726 26.91 2.25 103.5Inter-assay10 9.73 1.82 97.314 13.94 3.10 99.518 18.7 2.80 103.822 22.6 1.30 102.726 26.1 2.13 100.3

[a] Average of three replicates.

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Acknowledgement

This work was performed under the Board of Research in NuclearSciences (BRNS), Mumbai, India. funded project no. 2009/37/43/BRNS. The author is grateful for this financial assistance.

Keywords: biosensors · iron-oxide nanoparticles ·nanocomposites · paraoxon · voltammetry

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Received: July 26, 2014Published online on && &&, 2014

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ARTICLES

I. Tiwari,* M. Gupta, C. M. Pandey

&& –&&

Application of Cationic Poly(lactic-co-glycolic acid) Iron Oxide/Chitosan-Based Nanocomposite for theDetermination of Paraoxon

A paraoxon universe: Iron-oxide nano-particles are prepared by using a nano-emulsion technique with poly(lactic-co-glycolic acid) as the stabilizing agent,which are very stable and found to begood for enzyme immobilization.Hence, a pesticide biosensor is preparedwith this material, based on an inhibi-tion study of the enzyme acetylcholines-terase by using differential-pulsevoltammetry.

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