Biosensor for Direct Determination of Organophosphate Nerve Agents Using RecombinantEscherichia coliwith Surface-Expressed Organophosphorus Hydrolase. 1. Potentiometric Microbial Electrode

Abstract

A potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed by modifying a pH electrode with an immobilized layer of Escherichia coli cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosporus pesticides to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and weight of cells immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 2.5 mg of cells and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, methyl parathion, and diazinon. The biosensor had very good storage and multiple use stability. The use of cells with the metabolic enzyme expressed on cell surface as a biological transducer provides advantages of no resistances to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively.

Enzyme vs. Bacterial Electrochemical Sensors for Organophosphorus Pesticides Quantification

“…Only few bacterial electrochemical sensors for OP pesticides quantification have been developed until now. They include, as biological recognition element, genetically engineered Moraxella sp., Pseudomonas putida or Escherichia coli with surface-expressed OPH (Mulchandani et al, 1998;Mulchandani et al, 2001c;Mulchandani et al, 2006;Richins et al, 1997). The detection principle is identical to the described above, when employing the isolated and purified enzyme.…”

The Microbial Cell Based Biosensors

“…Another method allowing to reduce the sensor's reaction time suppose the application of genetic engineering technique in order to ensure the exposing of the particular enzymes on the outer surface of cell wall; in this case the diffusion obstacles are eliminated. Thus, in (Rainina et al, 1996;Mulchandani et al, 1998) the recombinant Escherichia coli strain containing the surface-expressed organophosphate hydrolase had been utilized. The cells carried out the substrate degradation with higher rate over the cells with the intracellular expression of the enzyme.…”

“…The attempts to solve the problem by means of incubation of the sensor between measurements in the solution of inductor or inhibitor are known (Kobos et al, 1979;Suzuki et al, 1992) but this can cause a contamination of the culture and altering the analytical characteristics of the sensor. A promising approach is related to genetic manipulations allowing to enhance the expression of genes responsible to an analyte conversion or eliminate or block the genes of interfering compounds metabolism (Korpan et al, 1993;Mulchandani et al, 1998). The modification ensures actually the same result as in the case of metabolic activation or inhibition but eliminates the possibility of reverting the initial activity of enzymes after inductor or inhibitor removal.…”

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

“…With the discovery of organophosphorus hydrolase (OPH), an enzyme that can hydrolyze a wide range of organophosphate pesticides releasing detectable products [16,17], several enzyme and microbial biosensors based on OPH for rapid, simple, selective monitoring of these neurotoxic pesticides with the potential for in the field analysis have been reported [18][19][20][21][22][23][24][25][26][27][28][29].…”