Immobilization of rat brain acetylcholinesterase on ZnS and poly(indole-5-carboxylic acid) modified Au electrode for detection of organophosphorus insecticides

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Abstract

A novel, highly sensitive amperometric biosensor for detection of organophosphorus (OP) compounds has been constructed, based on rat brain acetylcholinesterase (AChE) immobilized onto nanocomposite of ZnS-nanoparticles (ZnSNPs) and poly(indole-5-carboxylic acid) electrodeposited on Au electrode. In the presence of acetylthiocholine chloride (ATCl) as a substrate, ZnSNPs promoted electron transfer reactions at a lower potential and catalyzed electrochemical oxidation of enzymatically formed thiocholine, thus increasing detection sensitivity. Under optimum conditions (phosphate buffer, pH 7.5 and 30 °C), the inhibition of AChE by malathion and chlorpyrifos was proportional to their concentrations in the range, 0.1–50 nM and 1.5–40 nM, respectively. The biosensor determined malathion and chlorpyrifos in spiked tap water samples with a acceptable accuracy (95–100%). The enzyme electrode had long-storage stability (50% retention of initial activity within 2 months, when stored at 4 °C).

Introduction

Acetylcholinesterase (AChE) (acetylcholine acetylhydrolase; EC 3.1.1.7) has attracted the attention of several workers, as it is inhibited significantly by organophosphorus (OP) compounds, used in medicine, agriculture, industry and as chemical warfare agents (Shi et al., 2006). The enzyme inhibition mechanism proceeds through the formation of a stable complex through reversible/irreversible reaction of OP pesticides with the active site of AChE (Neufeld et al., 2000). As a result, acetylcholine, a neurotransmitter is accumulated, due to reduced activity of AChE and causes the nerve order to be reiterated, leading to exhaustion and paralysis. This ultimately corroborates the need for the detection of OP pesticides in the environment. During the past, OP pesticides have been effectively monitored using various chromatographic techniques (Corcia and Marchetti, 1991) such as gas chromatography (GC), high performance liquid chromatography (HPLC) (Sherma, 1993) and thin layer chromatography (TLC) (Linford, 1990). Although these chromatographic techniques provide fruitful results, these are rather time consuming and need very expensive equipment, highly trained personnel beside complicated sample preparation.

As highly sensitive and highly selective tools, biosensors can be used as disposable sensors in environmental monitoring and thus overcome the shortcomings of the conventional chromatographic methods for OP determination (Rogers and Gerlach, 1996). In recent years, the use of nanomaterials in AChE biosensors has resulted into their improved analytic performance, such as CdS nanoparticles (Pardo-Yissar et al., 2003), ZrO2/chitosan composite film (Yang et al., 2005), multiwalled carbon nanotubes (MWCNTs) (Liu and Lin, 2006), Prussian blue (PB) (Arduini et al., 2006, Sun and Wang, 2010), gold–platinum bimetallic nanoparticles (Upadhyay et al., 2009), one-dimensional gold nanoparticles (YanRong et al., 2010), ionic liquid–multiwalled carbon nanotube (IL–MWCNT) (Rotariu et al., 2010), 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotube composite gel (Zamfir et al., 2011), nanostructured films of acetylcholinesterase and CdTe quantum dots (Zheng et al., 2011) and MWCNTs, gold nanoparticles (GNPs) and PB (Sun et al., 2011). However, all these biosensor had some common drawbacks such as low sensitivity, low storage stability and reusability. ZnS nano materials are chemically more stable and technologically better than other chalcogenides (such as ZnSe). These nanomaterials are considered to be a promising host material due to their excellent prospective in catalysis owing to their quantum size and magnetic functionality (Peng et al., 2006).

Among various conducting polymers used for immobilization of enzymes, such as polyacetylene, polythiophene, polypyrrole and polyaniline, polyindole (Pin) is better polymer, as it can be easily polymerized, with ease of membrane formation and has high conductivity and chemical stability (Periasamy et al., 2009). Further, the polyindole films have fairly good thermal stability, high redox activity and slow degradation rate in comparison with polypyrrole and polyaniline (Diehl-Faxon et al., 1996, Billaud et al., 1995). The present report describes the use of nanocomposite of Pin5COOH and ZnSNPs to promote the electron transfer and thus increase the detection sensitivity of an AChE biosensor.

Section snippets

Chemical and reagents

Acetylthiocholine chloride, indole-5-carboxylic acid, acetonitrile and 2-pyridine aldoxime methiodide [2-PAM] from Sigma–Aldrich Chemical Co. USA were used. α-Methyl-d-mannoside, Triton X-100, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), zinc acetate and sodium sulphide, K3Fe(CN)6, K4Fe(CN)6, FeCl3, NaCl, KCl, sodium phosphate, N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were from Sisco Research Laboratory, Mumbai. The pesticides (malathion and

Results and discussions

The yield of the purified rat brain AChE (specific activity: 2507 U/mg protein) was 51%.

Conclusion

The use of Pin5COOH/ZnS nanocomposites modified Au electrode has resulted into an improved analytical performance of acetylcholinesterase (AChE) biosensor for detection of OP compounds in terms of low working potential (+0.2 V vs. Ag/AgCl), high sensitivity, wide linear range (0.1–50 nM and 1.5–40 nM for malathion and chlorpyrifos respectively) and low detection limit (0.1 and 1.5 nM). The biosensor could be used for the direct determination of pesticides in real samples e.g. soils extract,

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