Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detectionElectronic supplementary information (ESI) available. See DOI: 10.1039/c6an02575g

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 (PTE 3 ), 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 PTE 3 onto the AuNP surface via metal affinity coordination. This occurs between the PTE 3 -His 6 termini and the AuNP-displayed Ni 2+ -nitrilotriacetic acid end groups and was confirmed with gel electrophoresis. The enzymatic efficiency of the resultant PTE 3 -AuNP bioconjugates was analyzed via enzyme progress curves acquired from two distinct assay formats that compared free unbound PTE 3 with the following PTE 3 -AuNP bioconjugates: (1) fixed concentration of AuNPs while increasing the bioconjugate molar ratio of PTE 3 displayed around the AuNP and (2) fixed concentration of PTE 3 while increasing the bioconjugate molar ratio of PTE 3 -AuNP by decreasing the AuNP concentration. Both assay formats monitored the absorbance of p -nitrophenol that was produced as PTE 3 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 PTE 3 -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. A significant enzymatic enhancement of phosphotriesterase has been demonstrated when immobilized on various sized gold nanoparticles.