Matrix interference-resistant acetylcholinesterase probing: Toward reliable pesticide detection in complex food matrices.

Matrix interference remains a critical obstacle to reliable pesticide screening in real samples by acetylcholinesterase (AChE) inhibition assays. However, due to the lack of a holistic mechanistic understanding of matrix interferences, it is inherently difficult to identify and mitigate this issue in practice. Drawing inspiration from the electron distribution analysis of conventional AChE probes, the authors argued for the viability of mitigating the matrix-induced probe reduction by computationally-guided probe development. The xanthene-quinolinium hemicyanine acetate probe with a uniformly conjugated fluorophore was developed, which exhibited excellent stability in common fruit- and vegetable-derived reductants. The employment of newly developed probe further led to the successful identification of two major matrix-probe interactions in conventional AChE inhibition assays, including direct probe reduction and endogenous esterase-induced probe hydrolysis. Accordingly, a novel heterogeneous AChE inhibition assay was established for anti-interference screening of pesticides in real samples. This assay integrated a reduction-resistant near-infrared AChE activity probe, the organic solvent-based sample extraction, and a paper-chip platform that facilitates both sampling and the enzyme inhibition procedures involving organic solvents. It effectively eliminated matrix interferences in real sample measurement, showing only -13.71% to +0.18% signal variation (vs. +93.69% to +1121.36% with conventional method) for AChE activity measurement across nine different sample matrices compared to the blank control. Owing to its excellent anti-interference capability, this method enabled the pesticide quantification across different matrices without matrix-matched calibration. The new method proved robust and reliable for pesticide assessment in real samples in both spike-and-recovery and non-targeted screening modes.