This work describes the development of an electrochemical immunosensor for the analysis of atrazine using recombinant single-chain antibody (scAb) fragments. The sensors are based on carbon paste screen-printed electrodes incorporating the conducting polymer polyaniline (PANI)/poly(vinylsulphonic acid) (PVSA), which enables direct mediatorless coupling to take place between the redox centres of antigen-labelled horseradish peroxidase (HRP) and the electrode surface. Competitive immunoassays can be performed in real-time using this separation-free system. Analytical measurements based on the pseudo-linear relationship between the slope of a real-time amperometric signal and the concentration of analyte, yield a novel immunosensor setup capable of regenerationless amperometric analysis. Multiple, sequential measurements of standards and samples can be performed on a single scAb-modified surface in a matter of minutes. No separation of bound and unbound species was necessary prior to detection. The system is capable of measuring atrazine to a detection limit of 0.1 ppb (0.1 g l −1). This system offers the potential for rapid, cost-effective immunosensing for the analysis of samples of environmental, medical and pharmaceutical significance.

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The use of antibodies, or more specifically recombinant antibody fragments (scAbs), for the immunodetection of pesticides, could provide a rapid, reliable, and robust method of environmental analysis. The inherent instability of some antibody structures in nonphysiological conditions, however, limits the utility of this approach. We have developed a generic strategy for the stabilization of scAbs in polar and nonpolar solvents, and in a range of other denaturing environments, by the introduction of an interdomain disulfide bond, generating stabilized antibody fragments or stAbs. StAbs specific for the phenoxyacid herbicide mecoprop and the phenylurea herbicide diuron were fully functional when compared to their equivalent unmodified scAbs but significantly more stable in nonphysiological conditions associated with the extraction of organics from environmental samples. Antiatrazine, mecoprop, diuron, and paraquat stAbs were successfully used to accurately identify and quantify pesticides present in solvent and aqueous extracted soil samples. Immunoassay data were validated by HPLC. Recombinant stAbs can be produced readily, cost-effectively, and in quantity from Escherichia coli expression systems and provide robust reagents for immunoassay detection of pollutants present in complex environmental matrices.

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