Competitive flow immunoassay with fluorescence detection for determination of 4-nitrophenol Original Research Article

A selective competitive flow-immunoassay for monitoring of 4-nitrophenol, a demonstrated carcinogenic and mutagenic compound, is presented. The immunochemical detection principle is based on the competition between a fixed amount of labelled 4-nitrophenol derivative (tracer) and the analyte for a limited amount of antibody binding sites. The separation of free and bound tracer fractions is performed in a restricted-access (RA) column with subsequent fluorescence detection of the labelled immune complex. Optimisation of parameters, e.g. incubation time, flow rate, column size, type of antibody and tracer and their concentrations, was performed. The cross-reactivity for about 30 phenols, nitro-derivatives and other compounds was estimated. No major interferences except for 2,4-dinitrophenol and mercury(II) ions were observed. The optimised assay allowed a detection limit of 0.5 μg l−1 4-nitrophenol (3.5 nM) with a sensitivity of 5.7 mg−1 l, a sample throughput of 30 injections h−1 and a dynamic range between 5 and 1000 μg l−1 4-nitrophenol. The assay was further tested on different sample matrices such as phosphate buffer, tap-, and rainwater without the need for any pre-treatment step.