A novel nanobiosensor for the detection of paraoxon using chitosan-embedded organophosphorus hydrolase immobilized on Au nanoparticles

Abstract

Organophosphorus (OP) compounds are one of the most hazardous chemicals used as insecticides/pesticide in agricultural practices. A large variety of OP compounds are hydrolyzed by organophosphorus hydrolases (OPH; EC 3.1.8.1). Therefore, OPHs are among the most suitable candidates that could be used in designing enzyme-based sensors for detecting OP compounds. In this work, a novel nanobiosensor for the detection of paraoxon was designed and fabricated. More specifically, OPH was covalently embedded onto chitosan and the enzyme-chitosan bioconjugate was then immobilized on negatively charged gold nanoparticles (AuNPs) electrostatically. The enzyme was immobilized on AuNPs without chitosan as well, to compare the two systems in terms of detection limit and enzyme stability under different pH and temperature conditions. Coumarin 1, a competitive inhibitor of the enzyme, was used as a fluorogenic probe. The emission of coumarin 1 was effectively quenched by the immobilized Au-NPs when bound to the developed nanobioconjugates. However, in the presence of paraoxon, coumarin 1 left the nanobioconjugate, leading to enhanced fluorescence intensity. Moreover, compared to the immobilized enzyme without chitosan, the chitosan-immobilized enzyme was found to possess decreased Km value by more than 50%, and increased Vmax and Kcat values by around 15% and 74%, respectively. Higher stability within a wider range of pH (2-12) and temperature (25-90°C) was also achieved. The method worked in the 0 to 1050 nM concentration ranges, and had a detection limit as low as 5 × 10(-11) M.

Enzymatic Bioremediation of Organophosphate Compounds—Progress and Remaining Challenges

“…To illustrate the potential for increased functionality of PTE when immobilized to a solid surface, Raynes et al cross-linked the enzyme to amyloid fibrils generated from insulin and crystallin and observed a marked improvement in thermal stability (Raynes et al, 2011). Similarly, covalently conjugating PTE to negatively charged gold nanoparticles not only decreased Km value and increased Vmax and Kcat values, but also achieved stability within a wider range of pH (2–12) and temperature (25–90°C) (Karami et al, 2016). This phenomenon of improved enzymatic and biophysical properties is not unique to PTE as highlighted in numerous review articles and recent publications (Chen M. et al, 2017; Kreuzer et al, 2017; Arsalan and Younus, 2018; Wang et al, 2018).…”

Encapsulation of Gold Nanorods with Porphyrins for the Potential Treatment of Cancer and Bacterial Diseases: A Critical Review

“…The first synthesis was performed by Rothermund in 1936, and the method starts with the reaction of an aldehyde and pyrrole with both reactants at high temperature and concentration in a bomb reactor with no added oxidant. The addition of zinc acetate to the reaction only increases the yield of tetraphenylporphyrins two-fold, but this is not observed with any other porphyrins [153–157].…”

Enhancing organophosphate hydrolase efficacy via protein engineering and immobilization strategies

“…Even with such reduced activities, immobilized PTE has been shown to hydrolyze dilute solutions of paraoxon or ethyl parathion and attain partial hydrolysis of DFP. 90 Several other substrates, including cellulose microfibers, 91 chitosan beads, 92 quantum dots, 93 carbon nanotubes, 94 gold NPs, 95 and agarose beds, 90 have been utilized to immobilize OPH. Some of these substrates are best suited for fabricating novel biosensors for OP detection and are discussed elsewhere.…”

“…Several other substrates, including cellulose microfibers, 91 chitosan beads, 92 quantum dots, 93 carbon nanotubes, 94 gold NPs, 95 and agarose beds, 90 have been utilized to immobilize OPH. Some of these substrates are best suited for fabricating novel biosensors for OP detection and are discussed elsewhere 2,96 .…”