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.

Organophosphorus (OP) compounds are extensively used in agricultural practice for pest management. However, their residues have a long half-life in the ecosystem as well as in the agro-products, posing a serious threat to human and animal health. Aryldialkylphosphatase (EC 3.1.8.1) is widely used in detoxification procedures. In the present study, aryldialkylphosphatase was immobilised on synthesised cross-linked nano-sized gel particles, also known as nanogels, in order to enhance the enzyme's physicochemical properties. Accordingly, a new nanogel consisting of chitosan and myristic acid (CMA nanogel) was synthesised and characterised by way of Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The aryldialkylphosphatase-CMA nanogel conjugate was then assayed by FTIR, and its physicochemical characteristics were also investigated. The data obtained from SEM and TEM showed the nanogels to be homogenous spherical particles less than 50 nm in diameter. The proper formation of the nanogel and nanobioconjugate was also confirmed by FTIR spectra. In comparison with the free enzyme, the pH and thermal stability of the aryldialkylphosphatase were enhanced by the covalent immobilisation. Moreover, the immobilised enzyme could maintain approximately half of its activity over more than one month. The kinetic parameters of the aryldialkylphosphatase-CMA nanogel conjugate were also shown to undergo remarkable improvements, hence the synthesised CMA-nanogel could act as a promising support for aryldialkylphosphatase immobilisation. It is suggested that the aryldialkylphosphatase-CMA nanogel could be used for detoxifying paraoxon; a nerve agent. Further clinical experiments are underway.