Short communicationA novel disposable electrochemical immunosensor for phenyl urea herbicide diuron
Introduction
Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a substituted phenyl urea herbicide is used as a broad spectrum pre-emergence weed control in a wide variety of crops. It is one of the prime ecological analytical targets in terms of perseverance and stability in soil and water. The absorption of diuron from the soil takes place by the root system of plants and gets translocated into the stem and leaves through their vascular system by transpiration. It blocks the light reaction in photosynthetic plants by selective binding at the quinone binding (Qb) site thus disallowing the electron flow from where it is generated, in photosystem II, to plastoquinone (Sorensen et al., 2008). The prolonged use of diuron and other phenyl urea herbicides is a big concern since their undegraded residues remain in surface and ground water in a limit that is not permissible for healthy environment (Eriksson et al., 2007, Lapworth and Gooddy, 2006). Phenyl urea herbicides show ecotoxic effects by acting as endocrine disruptors and thus adversely affect the human health (Tixier et al., 2001). It is moderately continual in the soil with a half-life of 3–6 months that further varies from few weeks to years depending specifically on the environment (Giacomazzi and Cochet, 2004, Louchart and Voltz, 2007). Due to its potential toxic and mutagenic effects to plants and animals, it is important to have diuron detection system to check soil and water contamination with many rigid requirements, such as specificity, sensitivity, speediness, stability, and simplification (Cox, 2001, Marriage et al., 2004). Numerous analytical methods have been developed for its detection that include chromatography and mass spectrometry (Kramer, 1996, Liu et al., 2007, Scheyer et al., 2007). In a different analytical approach, a highly conductive silicon-based nanocomplex, i.e., gold nanoparticles-coated silicon nanowires were used for the improvement of acetylcholinesterase (AChE) based electrochemical sensors for pesticides detection (Shao et al., 2008). Electrochemically deposited zirconia (ZrO2) nanoparticles have been used as selective sorbents for the detection of organophosphate pesticides (Liu and Lin, 2005). Electrochemical sensors using antibody as receptor molecules have gained much importance to become common methods for monitoring environmental pollutants such as pesticides. But the generation of antibodies against small molecules is a very tedious task, as they do not elicit an immune response. Small molecules are usually modified with additional functional groups (–NH2 or –COOH) for carrier linkage to generate highly specific antibodies. The authors have previously reported the development of highly specific antibody against herbicide diuron using a metabolic intermediate of its microbial degradation pathway i.e. DCPU (3,4-dichlorophenylurea) as a novel hapten (Sharma and Suri, 2011).
Despite the need for a portable device that allows the fast determination of pesticide for human health programme or agriculture management, a sensitive immuno-sensor built on a disposable and low cost electrochemical chip is very much in demand. Prussian blue (PB) used as an electron transfer mediator has been used to improve the analytical performance of electrochemical sensors (Guan et al., 2004). The structural aspects of PB suggest the transition between PB and its reduced form, prussian white (PW) is associated with relatively faster electron-transfer kinetics which works as the redox mediator in catalysis and electroanalysis. PB film embedded with nanoparticles has the advantage of large surface to volume ratio, adhesion and increased surface activity in the form of polycrystalline-electrodeposited films (Karyakin et al., 2004, Kumar et al., 2007). The present report describes technical advancement in the construction of a disposable low cost electrochemical immunosensor for rapid screening of phenyl herbicides. The detection scheme is based on the modification of LC-LAGE sensors by electrodeposition of PB-GNP film that enhances electron transfer in the vicinity of the gold electrode increasing the sensitivity of the assay as compared to unmodified gold electrodes. To our knowledge this is the first report wherein highly cost effective, disposable electrode sensors based on sputtering of gold on polyester substrate and PB-GNP film deposition has been used in an electrochemical platform for monitoring diuron.
Section snippets
Materials
Analytical standards diuron, DCPU, alkaline phosphatase labelled anti-rabbit IgG, gold chloride, gold cleaning solution, bovine serum albumin (BSA), ovalbumin (OVA), 1-naphthyl phosphate, casein, gelatin, tween 20 polyvinyl alcohol (PVA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), sulfo N-hydroxysuccinimide (sulfo-NHS), Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) was procured from Sigma Chemical Co. (USA). HRP labeled anti-rabbit IgG and
Anti-diuron antibody generation
Generation of specific antibody against low molecular substances such as pesticides is a critical aspect for the development of sensitive immunosensor system. By selecting an appropriate protein–hapten conjugate with optimum molar ratio (1:20), significantly high titers of anti-diuron antisera was obtained. Generated antibody showed good reactivity against BSA-DCPU conjugate while showing significantly low reactivity against carrier protein (BSA). The specificity of the antibodies was measured
Conclusion
A novel low cost laser ablated gold electrodes functionalised with specific anti-diuron antibodies were fabricated on polystyrene substrate. The electrodes exhibited high sensitivity for diuron because of large surface activity of GNPs embedded in PB film that increases the efficiency of electron delocalization in the vicinity of LC-LAGE. The study presents highly cost effective disposable gold sputtered polyester substrate electrodes embedded with PB-GNP film for monitoring diuron with
Acknowledgements
We greatly acknowledge the financial assistance for this research programme from joint Indo-Russia Integrated Long Term Programme, under their ILTP programme. Authors also acknowledge the technical support of Mr. Gaurav Sharma for TEM imaging at DST funded TEM facility, NIPER, Mohali, Mr. Vijay Mathur for electrode design and UGC India for the Research Fellowship granted to Ms. Priyanka Sharma.
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Both authors contributed equally.