A novel nanobiosensor for the detection of paraoxon using chitosan-embedded organophosphorus hydrolase immobilized on Au nanoparticles.
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.
a Department of Toxicology, Faculty of Medical Sciences , Tarbiat Modares University , Tehran , Iran.
Rezvan
R
Karami
b Nanosystems Research Team (NRTeam) , Karaj , Iran.|c Research and Development Department , Nanozino , Tehran , Iran.
Afshin
A
Mohsenifar
a Department of Toxicology, Faculty of Medical Sciences , Tarbiat Modares University , Tehran , Iran.
Seyede Maryam
SM
Mesbah Namini
a Department of Toxicology, Faculty of Medical Sciences , Tarbiat Modares University , Tehran , Iran.
Nahid
N
Kamelipour
c Research and Development Department , Nanozino , Tehran , Iran.
Tavoos
T
Rahmani-Cherati
b Nanosystems Research Team (NRTeam) , Karaj , Iran.|d Institute of Advanced Technology (ITMA), Universiti Putra Malaysia , Serdang , Malaysia.
Taha
T
Roodbar Shojaei
b Nanosystems Research Team (NRTeam) , Karaj , Iran.|e Microbial Biotechnology Department , Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education, and Extension Organization (AREEO) , Karaj , Iran.
Meisam
M
Tabatabaei
D015374:Biosensing Techniques; D048271:Chitosan / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D053768:Metal Nanoparticles / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D036103:Nanotechnology; D010261:Paraoxon / Q000032:analysis*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D004800:Enzymes, Immobilized; D006046:Gold; D048271:Chitosan; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases; D010261:Paraoxon
Chitosan; enzyme embedding/immobilization; gold nanoparticles; nanobiosensor; organophosphorous compound