Amperometric biosensing of organophosphate and organocarbamate pesticides utilizing polypyrrole entrapped acetylcholinesterase electrode Rekha Rani Dutta, Panchanan Puzari n Department of Chemical Sciences, Tezpur University, Tezpur, Assam 784028, India article info Article history: Received 21 June 2013 Received in revised form 22 August 2013 Accepted 23 August 2013 Available online 31 August 2013 Keywords: Acetylcholinesterase biosensor Conducting polymer Electroimmobilization Ethylparaoxon Organophosphates Organocarbamates abstract The work presented here describes a novel, easy and low-cost method of fabrication of a highly sensitive acetylcholinesterase biosensor and its application to detect organophosphate and organocarbamate pesticides. Acetylcholinesterase was electro-immobilized into a thick conducting layer of polypyrrole. Porcine skin gelatin and gluteraldehyde mixture was used for stabilizing the system. Acetylthiocholine chloride was used as the substrate. Polypyrrole catalyzed the electrochemical oxidation of thiocholine and promoted the electron transfer, thus lowering the oxidation potential and increasing the detection sensitivity. Electro oxidation of thiocholine in polypyrrole matrix occurred at 0.1 V under low potential scan rate. The thiocholine sensitivity of the electrode was found to be 143 mA/M. The sensor was applied to detect the sample organophosphate pesticide ethylparaoxon and organocarbamate pesticide carbo- furan. The detection limit for paraoxon was found to be 1.1 ppb and that for carbofuran is 0.12ppb. The sensor showed good intra and inter state precision with relative standard deviation (RSD) 0.742% and 6.56% respectively. Both dry and wet storage stability were studied. The sensor stored at 0 1C in dry condition had a good storage stability retaining 70% of its original activity for 4 months. During wet storage, the activity decrease followed the same trend, however, the operational stability at the end of the storage period was found to be less compared to the dry storage case. The developed biosensor is as a promising new tool for analysis of cholinesterase inhibitors. & 2013 Elsevier B.V. All rights reserved. 1. Introduction Organophosphates (OPs) and organocarbamates (OCs) have been widely used as pesticides in modern agriculture due to their low persistence and high insecticidal activity (Amine et al., 2006; Carlsson et al., 2000; Rekha et al., 2000). Although they degrade faster than the organochloride, they have greater acute toxicity, posing risk to people who may be exposed to large amounts (Jeyaratnam, 1990). That is why rapid determination and reliable quantification of OPs and OCs have become of great importance. Numerous analysis methods including gas chromatography (Asensio-Ramos et al., 2010), liquid chromatography (Cappiello et al., 2002), ultraviolet spectroscopy (Ganzera et al., 2006), gas- mass spectroscopy (Wong et al., 2007), fluorimetry (Dams et al., 2002) and surface plasmon resonance (SPR) (Chand and Gupta, 2007) have been developed which can estimate the OPs and OCs in contaminated samples with high sensitivity, reliability and precision. Despite their advantage, these methods require expen- sive instrumentation, highly trained personnel, and long work up time, making them unsuitable for rapid analysis of field samples. Hence, simple, sensitive, selective, and field deployable tools are still highly desired for monitoring of OP and OC contamination. Enzyme based electrochemical biosensors have emerged in the past few years as the most promising alternative to detect pesticides (de Albuquerque, Ferreira, 2007; Marinov et al., 2010; Pohanka et al., 2011; Pohanka et al., 2012; Roepcke, et al., 2010; Skladal, 1996; Xavier et al., 2000). Among them, amperometric acetylcholinesterase (AChE) biosensors based on the inhibition of AChE have shown satisfactory results for pesticides analysis (Crew et al., 2011; Du et al., 2010a,2010b; Raghu et al., 2012; Sun and Wang, 2010; Tuoro et al., 2011) in which the enzyme activity is employed as an indicator of quantitative measurement of insecticides. Effective immobilization of enzyme to solid electrode surface still remains as a great challenge for the fabrication of biosensor. A key consideration for immobilizing enzyme is how to retain its bioactivity. The usual immobilization methods include direct phy- sical adsorption on a solid support (Sotiropoulou et al., (2005)), cross linking (Kandimalla and Ju, 2006), encapsulation into a hydro gel (Anitha et al., 2004; Yadavalli et al., 2004), covalent binding Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.08.050 Abbreviations: Geltn, gelatin; Glut, gluteraldehyde n Corresponding author. Tel.: þ91 3712 275061, þ91 3712 273640, mobile: þ91 9854293344; fax: þ91 3712 267005, þ91 3712 267006. E-mail address: pancha@tezu.ernet.in (P. Puzari). Biosensors and Bioelectronics 52 (2014) 166–172