Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets

Abstract

An improved biomonitoring system for the analysis of 2,4,6-trichlorophenol (TCP) in urine samples has been developed. The principle of the biosensor device is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d = 58,4 μm) produced by a piezoelectric generator system with 10-μm-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 × 512 cooled, charge-coupled device camera. Fluorescence is quenched by specific binding of TCP polyclonal antibodies to the fluorescent tracer (hapten A−fluorescein); the quenching effect is diminished by the presence of the analyte. Thus, an increase in the signal is produced in a positive dose-dependent manner when TCP is present in the sample. In 10 mM PBS buffer, the IC50 of the LIF-microdroplet QFIA is 0.45 μg L-1 reaching a LOD of 0.04 μg L-1. The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 μg L-1 in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 μg L-1, with a dynamic range between 4 and 149.5 μg L-1 in urine, was obtained without any sample treatment other than dilution with the assay buffer. The detectability achieved is sufficient for occupational exposure risk assessment.