A ratiometric fluorescence biosensor based on recombinant carboxylesterase and dual-excitation N,S-CQDs for robust and sensitive detection of organophosphorus pesticides

A ratiometric enzymatic fluorescence biosensor for OP detection was developed. • Recombinant carboxylesterase PvCarE1 enhances stability and reproducibility over conventional AChE. • Dual-excitation N,S-CQDs enable self-calibrated and robust signal output. • Satisfactory recoveries (82.0%–109.9%) in eight food matrices. • Results agreed well with LC-MS/MS, supporting practical food analysis. The widespread use of organophosphorus pesticides (OPs) poses significant threats to food safety and environmental health, necessitating the development of robust and sensitive detection platforms. We present a novel ratiometric biosensor based on a recombinant carboxylesterase (PvCarE1) integrated with dual-excitation nitrogen and sulfur co-doped carbon quantum dots (N,S-CQDs). The sensing mechanism relies on an inner filter effect (IFE), where the enzymatic hydrolysis product ( p -nitrophenol) selectively quenches 370 nm-excited fluorescence, while 255 nm-excited emission remains less affected. OP inhibition reduces p -nitrophenol generation, yielding a dose-dependent F₃₇₀/F₂₅₅ response that provides intrinsic self-calibration. The biosensor exhibited high sensitivity for dichlorvos, trichlorfon, and profenofos with detection limits of 0.07, 0.38, and 0.45 μg/L, respectively. Successful application in eight representative fruit and vegetable samples yielded satisfactory recoveries of 82.0%–109.9% with RSDs of 0.4%–11.9%, confirming its potential for real-world food safety monitoring. Download: Download high-res image (57KB) Download: Download full-size image