Sequential injection/electrochemical immunoassay for quantifying the pesticide metabolite 3,5,6-trichloro-2-pyridinol

An automated and sensitive sequential injection electrochemical immunoassay was developed to monitor a potential insecticide biomarker, 3,5,6-trichloro-2-pyridinol. The current method involved a sequential injection analysis (SIA) system equipped with a thin-layer electrochemical flow cell and permanent magnet, which was used to fix 3,5,6-trichloro-2-pyridinol (TCP) antibody coated magnetic beads (TCP-Ab-MBs) in the reaction zone. After competitive immunoreactions among TCP-Ab-MBs, TCP analyte, and horseradish peroxidase (HRP) labeled TCP, a 3,3′,5,5′-tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB-H₂O₂) substrate solution was injected to produce an electroactive enzymatic product. The activity of HRP tracers was monitored by a square wave voltammetric scanning electroactive enzymatic product in the thin-layer flow cell. The voltammetric characteristics of the substrate and the enzymatic product were investigated under batch conditions, and the parameters of the immunoassay were optimized in the SIA system. Under the optimal conditions, the system was used to measure as low as 6 ng L⁻¹(ppt) TCP, which is around 50-fold lower than the value indicated by the manufacturer of the TCP RaPID Assay^® kit (0.25 μg/L, colorimetric detection). The performance of the developed immunoassay system was successfully evaluated on tap water and river water samples spiked with TCP. This technique could be readily used for detecting other environmental contaminants by developing specific antibodies against contaminants and is expected to open new opportunities for environmental and biological monitoring.