Simplifying the complexity: Single enzyme (choline oxidase) inhibition-based biosensor with dual-readout method for organophosphorus pesticide detection

Organophosphorus pesticides (OPs) are widely used in agricultural production, but their residues could cause pollution to the environment and living organisms. In this paper, a simple dual-readout method for OPs detection was proposed based on ChOx single enzyme inhibition. Firstly, ChOx can catalyze the production of H₂O₂ from choline chloride (Ch-Cl). Bifunctional iron-doped carbon dots (Fe-CDs) with good peroxidase-like activity and superior fluorescence properties can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) by H₂O₂ formed, and oxTMB could quench the fluorescence of Fe-CDs. In light of the fact that OPs exhibited activity in inhibiting ChOx, less H₂O₂ and the decreasing oxTMB led to a result that the fluorescence of the system recovered and the solution became lighter in blue color. Moreover, the process of ChOx inhibition by OPs was analyzed by molecular docking technique and it was found that OPs interact with key amino acid residues catalyzed by ChOx (Asn510, His466, Ser101, His351, Phe357, Trp331, Glu312). Finally, a dual-mode (colorimetry and fluorescence) sensor was created for the detection of OPs with the detection limit of 6 ng/L, and was successfully used in the quantitative determination of OPs in actual samples with satisfactory results.