Priti Mulchandani

Biosensors for direct determination of organophosphate pesticides.

Direct, selective, rapid and simple determination of organophosphate pesticides has been achieved by integrating organophosphorus hydrolase with electrochemical and opitical transducers. Organophosphorus hydrolase catalyzes the hydrolysis of a wide range of organophosphate compounds, releasing an acid and an alcohol that can be detected directly. This article reviews development, characterization and applications of organophosphorus hydrolase-based potentiometric, amperometric and optical biosensors.

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH 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, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.

Amperometric Thick-Film Strip Electrodes for Monitoring Organophosphate Nerve Agents Based on Immobilized Organophosphorus Hydrolase

An amperometric biosensor based on the immobilization of organophosphorus hydrolase (OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and rapid anodic detection of the enzymatically generated p-nitrophenol product at the OPH/Nafion layer immobilized onto the thick-film electrode in the presence of the OP substrate. The amperometric signals are linearly proportional to the concentration of the hydrolyzed paraoxon and methyl parathion substrates up to 40 and 5 μM, showing detection limits of 9 × 10(-)(8) and 7 × 10(-)(8) M, respectively. Such detection limits are substantially lower compared to the (2-5) × 10(-)(6) M values reported for OPH-based potentiometric and fiber-optic devices. The high sensitivity is coupled to a faster and simplified operation, and the sensor manifests a selective response compared to analogous enzyme inhibition biosensors. The applicability to river water sampling is illustrated. The attractive performance and greatly simplified operation holds great promise for on-site monitoring of OP pesticides.

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.

Remote Biosensor for In-Situ MOnitoring of Organophosphate Nerve Agents

A remote electrochemical biosensor for field monitoring of organophosphate nerve agents is described. The new sensor relies on the coupling of the effective biocatalytic action of organophosphorus hydrolase (OPH) with a submersible amperometric probe design. This combination results in a fast, sensitive, selective, and stable response at large sample-instrument distances. Such attractive performance is illustrated for direct measurements of micromolar levels of paraoxon and methyl parathion in untreated river water samples. Unlike multi-step inhibition biosensors, the remote OPH probe offers single-step direct measurements, and is thus highly suitable for the continuous monitoring task. Variables relevant to field operations are discussed, along with prospects for remote monitoring and early detection of nerve agents.

Organophosphorus Hydrolase‐Based Assay for Organophosphate Pesticides

We report a rapid and versatile organophosphorus hydrolase (OPH) ‐based method for measurement of organophosphates. This assay is based on a substrate‐dependent change in pH at the local vicinity of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC), which is covalently immobilized to the enzyme. This method employs the use of poly(methyl methacrylate) beads to which the FITC‐labeled enzyme is adsorbed. Analytes were then measured using a microbead fluorescence analyzer. The dynamic concentration range for the assay extends from 25 to 400 μM for paraoxon with a detection limit of 8 μM. Organophosphorus insecticides measured using this technique included ethylparathion, methylparathion, dursban, fensulfothion, crotoxyphos, diazinon, mevinphos, dichlorvos, and coumaphos. This technique was used to measure coumaphos in biodegradation samples of cattle dip wastes and showed a high correlation (r2 = 0.998) to an HPLC method.

Dual amperometric-potentiometric biosensor detection system for monitoring organophosphorus neurotoxins

A dual-transducer flow-injection biosensor detection system for monitoring organophosphorus (OP) neurotoxins is described. Such simultaneous use of different physical transducers in connection to the same (organophosphorous hydrolase (OPH)) enzyme enhances the information content and provides discrimination between various subclasses of OP compounds. While the potentiometric biosensor responds favorably to all OP compounds, reflecting the pH changes associated with the OPH activity, the amperometric device displays well-defined signals only towards OP substrates (pesticides) liberating the oxidizable p-nitrophenol product. The potentiometric detection has been accomplished with a silicon-based pH-sensitive electrolyte-insulator-semiconductor (EIS) transducer, operated in the constant-capacitance (ConCap) mode. Both transducers are prepared by a thin-film fabrication technology, and respond rapidly and independently to sudden changes in the level of the corresponding OP compound, with no apparent cross reactivity. Relevant experimental variables were evaluated and optimized. Such development holds great promise for field screening of OP neurotoxins in connection to various defense and environmental scenarios. The multiple-transduction concept could be extended for increasing the information content of other ‘class-enzyme’ biosensor systems.

A Potentiometric Microbial Biosensor for Direct Determination of Organophosphate Nerve Agents

An easy to construct and inexpensive potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed. The biological sensing element of this biosensor was recombinant Escherichia coli cells containing the plasmid pJK33 that expressed organophosphorus hydrolase (OPH) intracellularly. The cells were immobilized by entrapment behind a microporus polycarbonate membrane on the top of the hydrogen ion sensing glass membrane pH electrode. OPH catalyzes the hydrolysis of organophosphorus 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 and temperature, using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor operating in pH 8.5, 1 mM HEPES buffer and 378C. The biosensor was applied for measurement of paraoxon, ethyl parathion, methyl parathion and diazinon.

Ag(I)-binding to phytochelatins☆

Phytochelatins (PCs) are glutathione-derived peptides with the general structure (gamma-Glu-Cys)nGly, where n varies from 2 to 11. A variety of metal ions such as Cu(II), Cd(II), Pb(II), Zn(II), and Ag(I) induce PC synthesis in plants and some yeasts. It has generally been assumed that the inducer metals also bind PCs. However, very little information is available on the binding of metals other than Cu(I) and Cd(II) to PCs. In this paper, we describe the Ag(I)-binding characteristics of PCs with the structure (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly. The Ag(I)-binding stoichiometries of these three peptides were determined by (i) UV/VIS spectrophotometry, (ii) luminescence spectroscopy at 77 K, and (iii) reverse-phase HPLC. The three techniques yielded similar results. ApoPCs exhibit featureless absorption in the 220-340 nm range. The binding of Ag(I) to PCs induced the appearance of specific absorption shoulders. The titration end point was indicated by the flattening of the characteristic absorption shoulders. Similarly, luminescence at 77 K due to Ag(I)-thiolate clusters increased with the addition of graded Ag(I) equivalents. The luminescence declined when Ag(I) equivalents in excess of the saturating amounts were added to the peptides. At neutral pH, (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly bind 1.0, 1.5, and 4.0 equivalents of Ag(I), respectively. The Ag(I)-binding capacity of (gamma-Glu-Cys)2Gly and (gamma-Glu-Cys)3Gly was increased at pH 5.0 and below so that Ag(I)/-SH ratio approached 1.0. A similar pH-dependent binding of Ag(I) to glutathione was also observed. The increased Ag(I)-binding to PCs at lower pH is of physiological significance as these peptides accumulate in acidic vacuoles. We also report lifetime data on Ag(I)-PCs. The relatively long decay-times (approximately 0.1-0.3 msec) accompanied with a large Stokes shift in the emission band are indicative of spin-forbidden phosphorescence.

A capacitive field-effect sensor for the direct determination of organophosphorus pesticides

Abstract A novel capacitive field-effect sensor for the direct determination of organophosphorus pesticides by the enzyme organophosphorus hydrolase (OPH) is presented. The enzyme OPH hydrolyses organophosphorus compounds catalytically, thus releasing H+ ions. By means of a silicon-based field-effect sensor, which consists of a layer sequence of Al/p-Si/SiO2 with alternative pH-sensitive materials (Ta2O5, Al2O3 and Si3N4), this pH shift can be recorded directly in a potentiometric measuring arrangement. To obtain the enzyme biosensor, thus the OPH has been immobilised on top of the pH-sensitive layer structure. Typical sensor characteristics, like pH sensitivity, sensitivity towards paraoxon, reversibility of the sensor signal, response time, detection limit, long-term stability and selectivity to various pesticides have been studied.