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

Abstract

The rapid detection of organophosphates (OPs), a class of strong neurotoxins, is critically important for monitoring acute insecticide exposure and chemical warfare agent use. Herein, we improve the enzymatic activity of phosphotriesterase trimer (PTE3), an enzyme that selectively recognizes OPs without inhibition, by conjugation with distinctly sized (i.e., 5, 10, and 20 nm) gold nanoparticles (AuNPs). The number of enzyme immobilized on the AuNPs was precisely controlled by conjugating increasing molar ratios of PTE3 onto the AuNP surface ratios via metal affinity coordination with PTE3 His6 termini and Ni2+ end groups and verified through agarose gel electrophoresis. The enzymatic efficiency of the resultant PTE3-AuNP bioconjugates was analyzed via full 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 by increasing enzyme concentration 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 (405 nm max.) that was produced as PTE3 hydrolyzed substrate paraoxon—an organophosphate nerve agent simulant. Results demonstrate a general equivalent trend between the two formats. For all case experiments a maximum enzyme velocity (Vmax) and the turn over constant (Kcat) increased by 17 fold over free enzyme for lowest PTE3-AuNP ratio and the largest AuNP (i.e., ratio of 1:1, 20 nm dia. AuNP). Thus this work provides a route to improve enzymatic OP detection strategies with enzyme-nanoparticle bioconjugates.

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Publication details

The article was received on 02 Dec 2016, accepted on 23 Jun 2017 and first published on 26 Jun 2017


Article type: Paper
DOI: 10.1039/C6AN02575G
Citation: Analyst, 2017, Accepted Manuscript
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    Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection

    J. A. Hondred, J. Breger, N. Garland, E. Oh, K. Susumu, S. Walper, I. Medintz and J. C. Claussen, Analyst, 2017, Accepted Manuscript , DOI: 10.1039/C6AN02575G

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