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 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.

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.

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.

Control of tachycardia and hypertension following coronary artery bypass graft surgery: efficacy and haemodynamic effects of esmolol.

Hypertension following coronary artery bypass grafting is not uncommon, especially in patients having good left ventricular function. It is often accompanied by tachycardia. The purpose of this study is to determine the efficacy of esmolol in the treatment of tachycardia and hypertension immediately following cardiopulmonary bypass and to study other haemodynamic effects of esmolol. Thirty patients undergoing elective [corrected] coronary artery bypass grafting were included in this prospective study. Morphine-based anaesthetic technique along-with standard bypass techniques were used in all the patients. The study was performed in the operating room about 30-45 minutes after the termination of cardiopulmonary bypass. Patients having a heart rate of more than 90 bpm and systolic blood pressure of more than 130 mm Hg without any inotropic support were included and randomly assigned to esmolol or control group. Esmolol was administered in a bolus dose of 500 micrograms/kg followed by infusion of upto 100 micrograms/kg/min. The patients in the control group were administered comparable volumes of normal saline. Baseline haemodynamic measurements were obtained just before the administration of esmolol or normal saline and were repeated after 5, 10, 15, 30 and 45 min. The baseline measurement in both the groups showed that patients were maintaining a state of hyperdynamic circulation with high systolic blood pressure (esmolol group 148 +/- 15 mm Hg, control group 140 +/- 8 mm Hg; p = NS), heart rate (esmolol group 128 +/- 17 bpm, control group 127 +/- 17 bpm; p = NS) and cardiac index (esmolol group 3.1 +/- 1 L/min/m2, control group 3.3 +/- 0.5 L/min/m2; p = NS). Esmolol decreased systolic blood pressure (p < 0.001), heart rate (p < 0.01) and cardiac index (p < 0.05) at five minutes. These changes persisted throughout the study period. The left ventricular stroke work index decreased at five minutes (p < 0.05) and remained so till 30 minutes. The maximum fall in heart rate (15%) and systolic blood pressure (16%) was observed at 45 minutes. There were no haemodynamic changes in the control group except that cardiac index, stroke volume and left ventricular stroke work index increased at five minutes. We conclude that esmolol lowers the indices of cardiovascular work in patients who demonstrated hyperdynamic circulation. This was achieved by decreasing the heart rate and systolic blood pressure which was accompanied by decrease in cardiac index and left ventricular stroke work index.

Glutathione-mediated transfer of Cu(I) into phytochelatins.

Room temperature luminescence attributable to Cu(I)-thiolate clusters has been used to probe the transfer of Cu(I) from Cu(I)-glutathione complex to rabbit liver thionein-II and plant metal-binding peptides phytochelatins (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly and (gamma-Glu-Cys)4Gly. Reconstitutions were also performed using CuC1. The Cu(I)-binding stoichiometry of metallothionein or phytochelatins was generally independent of the Cu(I) donor. However, the luminescence of the reconstituted metallothionein or phytochelatins was higher when Cu(I)-GSH was the donor. This higher luminescence is presumably due to the stabilizing effect of GSH on Cu(I)-thiolate clusters. As expected, 12 Cu(I) ions were bound per molecule of metallothionein. The Cu(I) binding to phytochelatins depended on their chain length; the binding stoichiometries being 1.25, 2.0 and 2.5 for (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly and (gamma-Glu-Cys)4Gly respectively at neutral pH. A reduced stoichiometry for the longer phytochelatins was observed at alkaline pH. No GSH was found to associate with phytochelatins by a gel-filtration assay. The Cu(I) binding to (gamma-Glu-Cys)2Gly and (gamma-Glu-Cys)3Gly occurred in a biphasic manner in the sense that the relative luminescence increased approximately linearly with the amount of Cu(I) up to a certain molar ratio whereafter luminescence increased dramatically upon the binding of additional Cu(I). The luminescence intensity declined once the metal-binding sites were saturated. In analogy with the studies on metallothioneins, biphasic luminescence suggests the formation of two types of Cu(I) clusters in phytochelatins.

Flow injection amperometric enzyme biosensor for direct determination of organophosphate nerve agents.

A flow injection amperometric biosensor for the determination of organophosphate nerve agents was developed. The biosensor incorporated an immobilized enzyme reactor that contains the enzyme organophosphorus hydrolase covalently immobilized on activated aminopropyl controlled pore glass beads and an electrochemical flow-through detector containing carbon paste working electrode, a silver/silver chloride reference electrode, and stainless steel counter electrode. The organophosphorus hydrolase catalyzed the hydrolysis of organophosphate with nitrophenyl substituent to generate p-nitrophenol which is then detected downstream electrochemically at the carbon paste electrode poised at 0.9 V vs the reference electrode. The amperometric response of the biosensor was linear up to 120 microM and 140 microM, with lower detection limits of 20 nM and 20 nM, for paraoxon and methyl parathion, respectively. The response was very reproducible (RSD 2%, n = 35) and stable for over 1 month when the immobilized enzyme column was stored at 4 degrees C. Each assay took ca. 2 min giving a sample throughput of 30 h(-1). The applicability of the biosensor to monitor paraoxon and methyl parathion in distilled water and simulated well water was demonstrated.

Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 1. Potentiometric microbial electrode.

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.

Role of CdS quantum crystallites in cadmium resistance in Candida glabrata.

Glutathione-related peptides (gamma-Glu-Cys)nGly, trivially known as phytochelatins (PCs), sequester Cd(II) and other heavy metals in all plants and some yeasts. However, the metal resistance levels may depend on factors such as the PC concentrations, their chain length and ability to incorporate labile sulfide. We show here that a highly Cd(II)-resistant mutant of yeast Candida glabrata exhibited Cd(II)-dependent formation of extremely high levels of PC-coated CdS quantum crystallites. The CdS crystallites were formed in the cytosol but finally accumulated in the vacuoles. Cd(II)-stimulated sulfide production required sulfate and was inhibited by both cysteine and methionine. GSH synthesis inhibition sensitized the resistant strain to Cd(II) indicating that GSH still provided primary defense against the metal ion.

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.

Optical spectroscopic and reverse-phase HPLC analyses of Hg(II) binding to phytochelatins.

Optical spectroscopy and reverse-phase HPLC were used to investigate the binding of Hg(II) to plant metal-binding peptides (phytochelatins) with the structure (gammaGlu-Cys)2Gly, (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly. Glutathione-mediated transfer of Hg(II) into phytochelatins and the transfer of the metal ion from one phytochelatin to another was also studied using reverse-phase HPLC. The saturation of Hg(II)-induced bands in the UV/visible and CD spectra of (gammaGlu-Cys)2Gly suggested the formation of a single Hg(II)-binding species of this peptide with a stoichiometry of one metal ion per peptide molecule. The separation of apo-(gammaGlu-Cys)2Gly from its Hg(II) derivative on a C18 reverse-phase column also indicated the same metal-binding stoichiometry. The UV/visible spectra of both (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly at pH 7.4 showed distinct shoulders in the ligand-to-metal charge-transfer region at 280-290 mm. Two distinct Hg(II)-binding species, occurring at metal-binding stoichiometries of around 1.25 and 2.0 Hg(II) ions per peptide molecule, were observed for (gammaGlu-Cys)3Gly. These species exhibited specific spectral features in the charge-transfer region and were separable by HPLC. Similarly, two main Hg(II)-binding species of (gammaGlu-Cys)4Gly were observed by UV/visible and CD spectroscopy at metal-binding stoichiometries of around 1.25 and 2.5 respectively. Only a single peak of Hg(II)-(gammaGlu-Cys)4Gly complexes was resolved under the conditions used for HPLC. The overall Hg(II)-binding stoichiometries of phytochelatins were similar at pH 2.0 and at pH 7.4, indicating that pH did not influence the final Hg(II)-binding capacity of these peptides. The reverse-phase HPLC assays indicated a rapid transfer of Hg(II) from glutathione to phytochelatins. These assays also demonstrated a facile transfer of the metal ion from shorter- to longer-chain phytochelatins. The strength of Hg(II) binding to glutathione and phytochelatins followed the order: gammaGlu-Cys-Gly<(gammaGlu-Cys)2Gly<(gammaGlu-Cy s)3Gly<(gamma Glu-Cys)4Gly.

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 &mgr;M for paraoxon with a detection limit of 8 &mgr;M. 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.