Biosensors for Field Monitoring of Organophosphate PesticidesEPA Grant Number: R828160
Title: Biosensors for Field Monitoring of Organophosphate Pesticides
Investigators: Mulchandani, Ashok , Chen, Wilfred , Wang, Joseph
Institution: University of California - Riverside , New Mexico State University - Main Campus
EPA Project Officer: Lasat, Mitch
Project Period: June 1, 2000 through May 31, 2002 (Extended to June 30, 2003)
Project Amount: $227,169
RFA: Exploratory Research - Engineering, Chemistry, and Physics) (1999) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Air , Engineering and Environmental Chemistry
The lack of sensors to perform discrete and real-time in-situ measurement/detection of organophosphates (OPs) in the field has limited the ability to routinely monitor these highly neurotoxic but widely used pesticides/insecticides. The overall objective of this research is to develop, optimize, characterize and validate biosensors for rapid, selective, sensitive, precise, accurate, simple and low-cost discrete and real-time in-situ monitoring of OPs in the field. The biosensors will be based on screen-printed electrodes (SPE), constructed using thick-film screen printing technology, modified with Escherichia coli cells displaying organophosphorus hydrolase (OPH) on the cell surface alone and together with pNP-monooxygenase. OPH catalyzes the hydrolysis of paraoxon, parathion, methyl parathion, fenitrothion, EPN, etc., at high rate and selectively to p-nitrophenol (pNP), which will be detected directly at the SPE or converted to hydroquinone by pNP-monooxygenase and detected at SPE. Additionally, the biosensors will be coupled with micromachined electrophoresis chips for selective determination of different OPs in a mixture and real-time in-situ measurement.
Preliminary work in our research laboratory has demonstrated the potential of combining the biosensing capability of OPH-modified SPE for sensitive, selective, rapid, precise, accurate and low-cost field monitoring of OPs. Experiments will be conducted to: (1) Optimize the fabrication conditions -- type of ink, ink additives, curing temperature and time -- optimize method and condition of cell (E. coli displaying OPH on the cell surface) immobilization and operating conditions-- operating potential, weight of cells immobilized, buffer ionic strength and pH, organic solvent type and concentration and temperature--for the determination of OPs. (2) Evaluate the effect of pNP-monooxygenase incorporation with E. coli displaying OPH on cell surface in the SPE on the operating potential and optimize the operating conditions for the new biosensor. (3) Determine the sensitivity, linearity, detection limit, selectivity (for OPs such as paraoxon, parathion, methyl-parathion, EPN, fenitrothion, etc., that produce pNP when hydrolyzed) and life-times (shelf-storage and operational) of the biosensors. (4) Critically evaluate and validate the new biosensors for monitoring OPs by investigating the precision and accuracy (comparing the biosensor measurement to the EPA standard method for pesticide analysis) on simulated and real samples. (5) Optimize operating parameters of the coupled SPE biosensors - micromachined electrophoresis chip for selective determination of individual OP in a mixture, and evaluate and validate the microsystem. (6) Adapt, evaluate and validate the microsystem of SPE biosensors for real-time in-situ OP monitoring.
The combining of the attractive biocatalytic actions of OPH (selective and fast hydrolysis) and pNP monooxygenase (conversion to hydroquinone) with SPEs (sensitive, low-cost, reproducible mass production and portable) and electrophoretic separation on a micromachined electrophoresis chip will result in the development of a simple, sensitive, selective, fast and low-cost analytical tool for OPs suitable for discrete and real-time in-situ field monitoring needs.