Highly sensitive and selective amperometric microbial biosensor for direct determination of p-nitrophenyl-substituted organophosphate nerve agents.
We report herein a whole cell-based amperometric biosensor for highly selective, highly sensitive, direct, single-step, rapid, and cost-effective determination of organophosphate pesticides with a p-nitrophenyl substituent. The biosensor was comprised of a p-nitrophenol degrader, Pseudomonas putida JS444, genetically engineered to express organophosphorus hydrolase (OPH) on the cell surface immobilized on the carbon paste electrode. Surface-expressed OPH catalyzed hydrolysis of the p-nitrophenyl substituent organophosphorus pesticides such as paraoxon, parathion, and methyl parathion to release p-nitrophenol, which was subsequently degraded by the enzymatic machinery of P. putida JS444. The electrooxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.086 mg dry weight of cells operating at 600 mV applied potential (vs Ag/AgCl reference) in 50 mM citrate--phosphate pH 7.5 buffer with 50 microM CoCl2 at room temperature. Under optimum operating conditions the biosensor measured as low as 0.28 ppb of paraoxon, 0.26 ppb of methyl parathion, and 0.29 ppb parathion. These detection limits are comparable to cholinesterase inhibition-based biosensors. Unlike the inhibition-based format, this biosensor manifests a selective response to organophosphate pesticides with a p-nitrophenyl substituent only, has a simplified single-step protocol with short response time, and can be used for repetitive/multiple and on-line analysis. The service life of the microbial amperometric biosensor was 5 days when stored in the operating buffer at 4 degrees C. The new biosensor offers great promise for rapid environmental monitoring of OP pesticides with nitrophenyl substituent.
Lei
Yu
Y
Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, USA.
Mulchandani
Priti
P
Wang
Joseph
J
Chen
Wilfred
W
Mulchandani
Ashok
A
D043303:Aryldialkylphosphatase / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002244:Carbon; D002619:Chemical Warfare Agents / Q000009:adverse effects / Q000032:analysis*; D004566:Electrodes; D006868:Hydrolysis; D009468:Neuromuscular Diseases / Q000139:chemically induced; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000009:adverse effects / Q000032:analysis*; D010575:Pesticides / Q000009:adverse effects / Q000032:analysis*; D016958:Pseudomonas putida / Q000378:metabolism; D012680:Sensitivity and Specificity
D002619:Chemical Warfare Agents; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010575:Pesticides; D002244:Carbon; D043303:Aryldialkylphosphatase; C024836:4-nitrophenol