Biosensors and Bioelectronics 29 (2011) 82–88 Contents lists available at ScienceDirect Biosensors and Bioelectronics jou rn al h om epa ge: www.elsevier.com/locate/bios Immobilization of rat brain acetylcholinesterase on ZnS and poly(indole-5-carboxylic acid) modified Au electrode for detection of organophosphorus insecticides Nidhi Chauhan, Jagriti Narang, C.S. Pundir ∗ Department of Biochemistry, M. D. University, Rohtak 124 001, Haryana, India a r t i c l e i n f o Article history: Received 5 June 2011 Received in revised form 13 July 2011 Accepted 27 July 2011 Available online 3 August 2011 Keywords: Acetylcholinesterase ZnS nanoparticles Poly(indole-5-carboxylic acid) Organophosphorus compounds Amperometric biosensor a b s t r a c t 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). © 2011 Elsevier B.V. All rights reserved. 1. Introduction Acetylcholinesterase (AChE) (acetylcholine acetylhydrolase; EC 3.1.1.7) has attracted the attention of several workers, as it is inhib- ited 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 accu- mulated, 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 pesti- cides 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 ∗ Corresponding author. Tel.: +91 9416492413; fax: +91 126274640. E-mail addresses: pundircs@rediffmail.com, pundircs@gmail.com (C.S. Pundir). thus overcome the shortcomings of the conventional chromato- graphic 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), ZrO 2 /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 tetrafluorob- orate/multiwalled carbon nanotube composite gel (Zamfir et al., 2011), nanostructured films of acetylcholinesterase and CdTe quan- tum 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 stor- age 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 promis- ing 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 con- ductivity and chemical stability (Periasamy et al., 2009). Further, 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.07.070