Development of an immunochromatographic assay for the rapid detection of chlorpyrifos-methyl in water samples

Abstract

A rapid (less than 10 min), qualitative and semi-quantitative immunochromatography using colloidal gold-antibody probe was successfully developed and applied in determination of chlorpyrifos-methyl (a wide-spectrum organophosphorus pesticide) in water samples. The qualitative detection limit of chlorpyrifos-methyl was determined as 0.6 μg ml⁻¹ by using immunochromatography. In the semi-quantitative experiment, the detection results of chlorpyrifos-methyl were scanned by a membrane strip reader, and a detection curve representing the scanned data average was obtained. After conversion, it was observed that in the range of 50 – 12,150 ng ml⁻¹, the graph between logit(B/B₀) and logarithm of concentration of chlorpyrifos-methyl was linear, from which, the regression equation (y = −2.5229x + 7.5951, R² = 0.9889) and IC₅₀ value (1024.39 ng ml⁻¹) was obtained, respectively. Meanwhile, the detection limit was calculated as 132.91 ng ml⁻¹ by the extrapolation of B₀ − 2SD. In addition, the cross-reactivities were less than 1% with tested analog compounds and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to water samples were 102.5–107.6%. Overall, to our knowledge, this is the first report of qualitative and semi-quantitative detection of chlorpyrifos-methyl by immunochromatography.

Introduction

In recent years, there is a growing concern about harmful effects of pesticide on human health and environment due to its widespread use (Kim et al., 2003). Among them, organophosphorus pesticides (OPs) are described as a group of highly toxic compounds (Costa, 1988) and widely used in agriculture for protecting plants. It is reported that OP toxicants could elicit their effects by inhibition of acetylcholinesterase, which leads to the accumulation of the neurotransmitter acetylcholine (Ach) in synapses, over stimulates the post-synaptic cholinergic receptors with consequent signs of neurotoxicity (Gallo and Lawryk, 1991, Ecobichon, 1996). At present, they are still widely used, resulting in more and more non-target organisms, including human, fish and birds are threatened by OPs (Sankararamakrishnan et al., 2005, Pazou et al., 2006, Reinecke and Reinecke, 2007, Revankar and Shyama, 2009). Therefore, it is urgent and necessary to detect pesticide (e.g. OP) residues in real samples by using rapid and sensitive methods, which could prevent non-target organisms and environment from damage.

Chlorpyrifos-methyl (O,O-dimethyl O-(3,5,6-trichloro-2-pyridyl)phosphorthioate) is a broad-spectrum OP used to control pests in grain storage, water and a variety of leafy crop (Daglish et al., 1995, Daglish et al., 1996, Daglish, 2008). Because of its toxicity and extensive application to crops and cattle, chlorpyrifos-methyl was classified as a restricted pesticide with a strict maximum residue limited standard (MRLs) for use in farming in many countries, including China.

Currently, pesticide residue analysis mainly depends on instrument-based detection methods, such as high performance liquid chromatography (HPLC) and gas chromatography (GC) (Farran et al., 1996, Yao et al., 2001, Padrón-Sanz et al., 2005, Tahboub et al., 2005). These analysis methods can achieve a higher detection sensitivity, however, they generally require complex and expensive equipments and professional trained operators, and are time-consuming and expensive, which is difficult to meet the high-throughput, rapid, on-site testing requirements. Therefore, there is an increasing demand for more rapid and economical methods for determining pesticide residues. Immunoassays have recently emerged as an alternative to the traditional methods that can meet such demands (Morozova et al., 2005). Among them, enzyme-linked immunosorbent assay (ELISA) was widely used to detect trace pesticides, heavy metals and other harmful chemicals (Hammock et al., 1982, Wylie et al., 1992, Zhu et al., 2007). To date, it is reported that a number of monoclonal and polyclonal antibody-based ELISAs were developed and successfully applied in detecting pesticides (Kolosova et al., 2004, Kim et al., 2007). However, generally speaking, ELISA detection method depends on laboratory platform and takes a relatively long assay time, which, at least partially, limits the rapid detection of pesticide residue in non-laboratory sites, such as supermarkets, farmer's market and field test.

Recently, colloidal gold has been introduced into immunochemistry and their particles could replace the enzyme to label antibody, and gold-labeled antibody responds to the corresponding antigen and leads to a visible color reaction (Hayat, 1989, Nagatani et al., 2006). Therefore, colloidal gold immunochromatography has been developed as a rapid (8–10 min) and simple test tool to detect chemical in non-laboratory sites and applied increasingly to various research fields (Zhang et al., 2006, Gui et al., 2008, Gandhi et al., 2009, Li et al., 2009, Zhou et al., 2009, Gas et al., 2010, Xu et al., 2010). However, available reports mainly described the qualitative detection of analyte by using the colloidal gold immunochromatographic assay; little was known about quantitative or semi-quantitative detection of analyte by using such a method.

In a previous work, a sensitive monoclonal antibody (3G7/F5)-based ELISA for determination chlorpyrifos-methyl in real samples has been successfully developed (Qian et al., 2009). In this study, we developed a rapid (8–10 min) and sensitive colloidal gold immunochromatographic assay, resulting in realizing qualitative and semi-quantitative detection for chlorpyrifos-methyl in water samples. To our knowledge, this is the first report of successful qualitative and semi-quantitative detection of chlorpyrifos-methyl by using a colloidal gold immunochromatography.