Highly Sensitive and Selective Amperometric Microbial Biosensor for Direct Determination of p-Nitrophenyl-Substituted Organophosphate Nerve Agents

Abstract

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.

Detection of Organophosphorus Pesticides Using Potentiometric Enzymatic Membrane Biosensor Based on Methylcellulose Immobilization

“…Compared with other biosensor methods, the linear ranges obtained by this study are wider than those obtained by OPH-modified amperometric biosensor method (0 -2 mmol/L for parathion) 17 and the microbial biosensor method (0.2 -50 mmol/L for parathion). 18 The detection limits obtained by this study are lower than that obtained by the microbial biosensor method (58 ppb for parathion), 18 and higher than that obtained by OPH-modified amperometric biosensor method (0.29 ppb for parathion) 17 and AChE-modified amperometric biosensor method (10 -8 -10 -9 mol/L for chlorpyrifos). 9 However, other enzyme modified biosensors need special auxiliary reagents, and the preparation processes are also complicated.…”

“…6 So far, some OPs biosensors have been reported, including those based on acetylcholinesterase (AChE) inhibition tests using AChE-modified amperometric and potentiometric transducers, [7][8][9][10] those based on hydrolytic tests with organophosphorus hydrolase (OPH) using OPH-modified amperometric and potentiometric transducers, [11][12][13][14][15] those based on hydrolytic tests with alkaline phosphatase (ALP) using ALPmodified fluorescent transducer, 16 those based on oxidation current measurements using recombinant microorganisms with surface expressed OPH, 17,18 and those based on the changes in resonant frequency of a quartz crystal microbalance (QCM) owing to the mass loading using QCM-enzyme sensor. 19 However, these biosensors have some disadvantages, including complicated preparations, unsteady response, poor reproducibility, and low sensitivity.…”

Cellular bioelectric profiling: a novel method to differentially assess the in vitro toxicity of pesticides

“…For example, P. putida has been used for the detection of aromatic hydrocarbons and organophosphate nerve agents [24], while S. cerevisiae Y190 has been used for determining endocrine disruptor compounds [25]. In the same context, amperometric measurements have been used with E.coli, Salmonella typhimurium and Ralstonia eutropha in genotoxicity assays [26][27][28].…”

Biosensor for direct determination of fenitrothion and EPN using recombinant Pseudomonas putida JS444 with surface-expressed organophosphorous hydrolase. 2. Modified carbon paste electrode

“…All chemical reagents and the details of the recombinant PNP-degrader Pseudomonas putida JS444 anchoring and displaying OPH on the cell surface and its growth conditions used in this study have been described elsewhere [30].…”

“…Recently, OPH was functionally expressed onto the surface of a natural p-nitrophenol (PNP) degrader, Pseudomonas putida JS444, using an ice nucleation protein (INP) anchor, creating a single microorganism with the capability to rapidly degrade organophosphate pesticides and PNP simultaneously while consuming oxygen [30]. Using this genetically engineered Pseudomonas putida we modified the Clark dissolved oxygen electrode, to construct a simple microbial biosensor for rapid, cost-effective, selective, precise and accurate determination of organophosphates with PNP substituent and demonstrated the application to paraoxon, parathion and methyl parathion monitoring [31].…”