Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection

The rapid detection of organophosphates (OPs), a class of strong neurotoxins, is critically important for monitoring acute insecticide exposure and potential chemical warfare agent use. Herein, we improve the enzymatic activity of a phosphotriesterase trimer (PTE₃), an enzyme that selectively recognizes OPs directly, by conjugation with distinctly sized (i.e., 5, 10, and 20 nm diameter) gold nanoparticles (AuNPs). The number of enzymes immobilized on the AuNP was controlled by conjugating increasing molar ratios of PTE₃ onto the AuNP surface via metal affinity coordination. This occurs between the PTE₃-His₆ termini and the AuNP-displayed Ni²⁺-nitrilotriacetic acid end groups and was confirmed with gel electrophoresis. The enzymatic efficiency of the resultant PTE₃-AuNP bioconjugates was analyzed via enzyme progress curves acquired from two distinct assay formats that compared free unbound PTE₃ with the following PTE₃-AuNP bioconjugates: (1) fixed concentration of AuNPs while increasing the bioconjugate molar ratio of PTE₃ displayed around the AuNP and (2) fixed concentration of PTE₃ while increasing the bioconjugate molar ratio of PTE₃-AuNP by decreasing the AuNP concentration. Both assay formats monitored the absorbance of p-nitrophenol that was produced as PTE₃ hydrolyzed the substrate paraoxon, a commercial insecticide and OP nerve agent simulant. Results demonstrate a general equivalent trend between the two formats. For all experiments, a maximum enzymatic velocity (V_max) increased by 17-fold over free enzyme for the lowest PTE₃-AuNP ratio and the largest AuNP (i.e., ratio of 1 : 1, 20 nm dia. AuNP). This work provides a route to improve enzymatic OP detection strategies with enzyme-NP bioconjugates.