Ethyl Paraoxon Research Articles

Altering the substrate specificity of methyl parathion hydrolase with directed evolution

Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1×106s−1M−1), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1×103s−1M−1). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9×105s−1M−1). These studies highlight the ‘plasticity’ of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system.

Carbon dot reduced Cu2O nanohybrid/hyperbranched epoxy nanocomposite: mechanical, thermal and photocatalytic activity

In the present study a highly tough thermostable hyperbranched epoxy nanocomposite was fabricated by the incorporation of carbon dot reduced Cu2O nanohybrid, which exhibited efficient reusable photocatalytic activity towards the degradation of pesticide under solar light (light intensity: 800–1000 lux). The catalytic efficacy of the above nanohybrid was compared with that of the parent carbon dot. Carbon dot reduced Cu2O nanohybrid particles were prepared by the reduction of cupric acetate solution with carbon dots at 70 °C for 6 h. The formation of nanohybrid (size: 3–4 nm), as well as its nanocomposites with hyperbranched epoxy, was confirmed by FTIR, XRD, Raman and TEM analyses. The significant improvement in the performance in terms of tensile strength (20%), elongation at break (2.5 fold), toughness (3.5 fold) and thermal stability (23 °C) of the pristine epoxy thermoset was observed by the formation of nanocomposite with 1.5 wt% of nanohybrid. The degradation of ethyl paraoxon pesticide was studied under ambient conditions using normal solar light and the changes of concentration with respect to initial were monitored by UV study. Thus, the high performance nanocomposite with photocatalytic attributes has strong potential to be used as a functional thin film material, as well as thermostable reusable photocatalyst.

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.

Detoxification of organophosphate residues using phosphotriesterase and their evaluation using flow based biosensor

Among known pesticide groups, organophosphates (OPs) have grasped attention due to their hazardous nature and their applications as pesticides and chemical weapons. This work presents the development of cost-effective column based biosensor for detoxification of OPs in water and milk. Enzyme phosphotriesterase (PTE) was immobilized on an activated Sepharose 4B via covalent coupling using an Omnifit glass column. Three different OPs, ethyl paraoxon (EPOx), malaoxon (MAO) and chlorpyriphos-oxon (CPO) were spiked in water and milk to test the detoxification of OPs. Mixtures of these pesticides were also tested to check the cumulative detoxification in the real samples. The efficiency of detoxification was evaluated using a highly sensitive acetylcholinesterase (AChE) B394 biosensor based flow system. The column conditions were optimized for the detoxification studied. The method was shown to be promising when we tested real milk samples spiked with OPs. Detoxification obtained in milk was up to 86% whereas in water, 100% detoxification was obtained.

Acetylcholinesterase based assay of eleven organophosphorus pesticides: finding of assay limitations

The study includes findings about limitations of acetylcholinesterase (AChE) based assay. Eleven organophosphorus pesticides: chlorpyrifos ethyl, chlorpyrifos methyl, DFP, dichlorvos, dimethoate, fenthion, paraoxon ethyl, paraoxon methyl, phosalone, pirimiphos methyl and pirimiphos ethyl were photometrically assayed using AChE as a recognition element. The study was carried out in order to find approachability of AChE based assay. In the first round, common organic solvents were tested for interfering in assay, since samples collection and extraction is a necessary part in samples processing. Isopropanol was found as the most convenient due to minimal inhibition not exceeding 5%. Though all analysed pesticides inhibit AChE in vivo, some of them are toxic after metabolisation. We found AChE based assay approachable for assay of DFP, paraoxons, and dichlorvos. These are oxoforms of organophosphorus pesticides. From thioforms of assayed pesticides, only fenthion was able significantly inhibit AChE in vitro. Electrochemical biosensor with AChE attached on platinum electrode was used for confirmation of interaction pesticide – AChE and complex stability estimation. DFP, paraoxons and dichlorvos were allowed to interact with AChE in biosensor. These pesticides were settled firmly in AChE active site as no spontaneous recovery of AChE activity was observed.

Flow-Injection System with Site-Specific Immobilization of Acetylcholinesterase Biosensor for Amperometric Detection of Organophosphate Pesticides

ABSTRACTA flow-injection system with integrated amperometric biosensor featuring an easily replaceable immobilzed acetylcholinesterase (AChE) membrane was studied. The amperometric biosensor was constructed on the basis of site-specific immobilization of AChE on a hybrid polymer membrane with integrated multi-walled carbon nanotubes. Multistage modification of the membrane and immobilization of the enzyme was proved by Fourier transform infrared spectroscopy. The optimum flow-rate of the flow-injection analysis (FIA) system was 0.5 mL/min. It gave a linear response to acetylthiocholine chloride from 2 μM to 100 μM, with an average RSD of 3.0% (n = 6). The sensitivity of the constructed biosensor was 0.093 μA/μM·cm2. The Kmapp value of the immobilized AChE was 1.15 mM and the linear correlation coefficient R2, 0.9949. The method had a low detection limit for three organophosphorus pesticides (OPs) in model pesticide solutions—paraoxon ethyl (0.9×10−12 M), monocroptophos (1.8×10−12 M) and dichlorvos (2.0×10−12 M). This indicated that the action of multi-walled nanotubes and controlled site-specific enzyme immobilization ensured high electrocatalytic activity and selectivity of the biosensor towards pesticides. It was found that the biosensor can be reused 15 operation cycles. After storage for 30 days the enzyme membrane retained over 80% of its initial response. The FIA system was used for detection of anti-cholinesterase activity of two binary OP mixtures. The results for paraoxon + monocroptophos and paraoxon + dichlorvos showed that the total inhibition activity was not simply additive, but was lower than the sum of the individual inhibition values. Moreover, the difference between the sum of the individual inhibition values and the real results for the mixture was bigger for the binary system paraoxon and dichlorvos (7-10%) compared with that for paraoxon and monocroptophos (5-7%). The developed biosensor system is an ideal tool for monitoring of organophosphate pesticides.

A novel automated flow-based biosensor for the determination of organophosphate pesticides in milk

This work describes the development of an automated flow-based biosensor that employs genetically modified acetylcholinesterase (AChE) enzymes B394, B4 and wild type B131. The biosensor was based on a screen printed carbon electrode (SPE) that was integrated into a flow cell. Enzymes were immobilised on cobalt (II) phthalocyanine (CoPC) modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). The automated flow-based biosensor was successfully used to quantify three organophosphate pesticides (OPs) in milk samples. The OPs used were chlorpyriphos-oxon (CPO), ethyl paraoxon (EPOx) and malaoxon (MOx). The total analysis time for the assay was less than 15 min. Initially, the biosensor performance was tested in phosphate buffer solution (PBS) using B394, B131 and B4 biosensors. The best detection limits were obtained with B394; therefore, this biosensor was used to produce calibration data in milk with three OPs in the concentration range of 5 × 10 −6 M to 5 × 10 −12 M. The limit of detection (LOD) obtained in milk for CPO, EPOx and MOx were 5 × 10 −12 M, 5 × 10 −9 M and 5 × 10 −10 M, respectively, with a correlation coefficient R 2 = 0.9910. The automated flow-based biosensor successfully quantified the OPs in different fat-containing milk samples. There were no false positives or false negatives observed for the analytical figures of merit for the constructed biosensors. This method is inexpensive, sensitive, portable, non-invasive and provides real-time results. This analytical system can provide rapid detection of highly toxic OPs in food matrices such as milk.

Core/shell molecular imprinting microparticles prepared using RAFT technology for degradation of paraoxon

We have developed a uniform core-shell structured surface hydrogel imprinted molecular imprinting polymer (MIP). This was achieved by surface grafting on a disulfide ester modified polystyrene core, using reversible addition-fragmentation transfer polymerization (RAFT) with N-methacryloyl-histidine-Cu2+ complex (MAHCu 2+) as the functional monomer and methyl paraoxon as the template to simulate phosphotriesterase (PTE). Subsequently, we investigated the catalytic (hydrolytic) activities of MIP to the template methyl paraoxon and the template analogue ethyl paraoxon. The results showed that the catalytic (hydrolytic) activity of MIP to the template methyl paraoxon was the highest, and the value of k was 8.67×10−5 mM·L−1·min−1, which was 3.89-fold higher than MIP to ethyl paraoxon and 2.79-fold higher than the non-imprinting polymer (NIP) to methyl paraoxon. The K m and r m of MIP were also determined: the K m was 3.95×10−4 M and the r m 2.12 μM/min. The MIP can be reused with only lose 7% of catalytic activity for four cycles.

Amperometric inhibition-based detection of organophosphorus pesticides in unary and binary mixtures employing flow-injection analysis

The present work is focused on the application of an acetylthiocholine (ATCh) biosensor in a flow-injection system for the detection of organophosphorus pesticides. The optimal operating conditions of the flow-injection system were determined: flow-rate – 0.5 mL min −1, substrate concentration – 100 μM, incubation and reactivation time–10 min. A calibration plot was obtained for ATCh concentration ranging from 20 to 200 μM. A linear interval was detected along the calibration curve from 20 to 100 μМ with a correlation coefficient R 2 = 0.996. The sensitivity of the constructed biosensor was calculated to be 0.083 μA μM −1 cm −2. The application of the flow-injection system for detection and quantification of three organophosphorus pesticides – paraoxon ethyl, monocrotophos and dichlorvos in unary solutions and in binary mixtures was investigated as well. The inhibition curves for each pesticide was plotted and the linear intervals were determined along with the corresponding equations and detection limits – 0.87 × 10 −11 M for paraoxon, 1.08 × 10 −11 M for monocrotophos and 1.22 × 10 −10 M for dichlorvos. The bimolecular inhibition constants k i were calculated by performing amperometric measurements of the residual enzyme activity after incubation for 10 min in a series of samples with varying pesticide concentrations (from 2 to 100 μM). The highest inhibition potency was observed for paraoxon (2.3 × 10 5 M −1 min −1), and the lowest – for dichlorvos (3.5 × 10 4 M −1 min −1). The flow-injection system was used in the detection of anti-cholinesterase activity of two binary mixtures – paraoxon + monocrotophos and paraoxon + dichlorvos. It was interesting to observe that the total anti-cholinesterase activity of the mixtures was lower than the anti-cholinesterase activity of paraoxon with the same concentration in the sample. The storage stability of the enzyme membrane was considerably improved with respect to our previous work. After storage for 30 days, the enzyme membrane retained over 90% of its initial response. The half-life storage time of the enzyme membrane (50% residual activity) was almost tripled – from 25 to 75 days.

Preparation of a PTE simulacrum based on surface molecular imprinting

Abstract Firstly, we synthesized N-methacryloyl-histidine monomer and N-methacryloyl-histidine-Cu2+ complex (MAH-Cu2+). Then the molecular imprinting polymers (MIP) has been prepared by surface grafting on uniform polystyrene (PS) core using reversible addition-fragmentation transfer polymerization (RAFT) with MAH-Cu2+ as the functional monomer, methyl paraoxon as the template to simulate phosphotriesterase (PTE). Finally, we have investigated the catalytic hydrolytic activities of MIP and non-imprinting polymers (NIP) to the template methyl paraoxon and the template analogue ethyl paraoxon respectively by UV spectrophotometry. The results showed that the catalytic hydrolytic activity of MIP to the template methyl paraoxon was highest and the value of k is 8.67 × 10−5 mmol L−1 min−1, 3.89-fold higher than MIP to the template analogue ethyl paraoxon, 2.79-fold higher than NIP to the template methyl paraoxon. The KM, rm of MIP are also determined, and KM = 3.95 × 10−4 mol/L, rm = 2.12 μmol/min. The MIP can be reused with only lose 7% of catalytic activity for four cycles.

Investigation on the Behavior of Pesticides in Atmosphere

Although pesticides are widely used in agriculture, they and in particular the relative residues in foodstuffs, water and atmosphere, may cause remarkable sanitary problems due to the harmful effects (carcinogenic and mutagenic effects) on the human health. In fact, their spread in waters and atmosphere can produce undesired effects on various organisms and/or water contamination. This paper shows an analytical approach based on XAD-2 adsorbent and GC analysis for evaluating the pesticide trend in atmosphere: in particular, the pesticides investigated are omethoate, dicrotofos, disulfoton, dimethoate, parathion methyl, formothion, paraoxon ethyl, malaoxon, parathion ethyl, iodofenfos and triazofos. For the analytical methodology a linearity response was obtained (r2 = 0.9988) in GC-NPD whereas the limits of detection range between 2 and 5 pg/μL in GC-NPD with a Relative Standard Deviation below 9.5. Finally, this approach has been successfully applied to real samples: the results show that dimethoate concentration decreases with increasing distance from the sampling site but it is still persistent in atmosphere after few days from the pesticide spraying.

Application of bimetallic nanoparticles modified screen printed electrode for the detection of organophosphate compounds using an enzyme inhibition approach

A novel, unique gold–platinum bimetallic nanoparticles modified screen printed electrode (SPE) has been developed for the sensitive detection of paraoxon ethyl, carbofuran and simulant of nerve agent diisopropyl fluorophosphate (DFP), based on the enzyme inhibition approach. The gold–platinum nanoparticles were electrochemically deposited on a 3-aminopropyl triethoxy silane modified electrode. Following this acetylcholineesterase (AChE)/choline oxidase (ChOx) bienzymes were immobilized through cross-linking with glutaraldehyde onto a modified electrode. Electrodes were characterized by scanning electron microscopy (SEM), EDX (energy dispersive X-ray analysis) and cyclic voltammetry (CV). The bimetallic alloy nanoparticles have an excellent high surface area and the unique effect of electrocatalytic activity for the oxidation of hydrogen peroxide (H2O2). The IC50 values were determined for the inhibitors using immobilized enzymes on the modified electrode. Paraoxon ethyl, carbofuran, and DFP could be detected up to 0.20 μM, 0.20–0.25 μM, and 0.20–0.25 μM respectively at a 25–30% inhibition level of AChE enzyme (residual enzyme activity) with an incubation time of 10 min.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for detection and identification of albumin phosphylation by organophosphorus pesticides and G- and V-type nerve agents

Toxic organophosphorus compounds (OPC), e.g., pesticides and nerve agents (NA), are known to phosphylate distinct endogenous proteins in vivo and in vitro. OPC adducts of butyrylcholinesterase and albumin are considered to be valuable biomarkers for retrospective verification of OPC exposure. Therefore, we have detected and identified novel adducts of human serum albumin (HSA) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Pure albumin and plasma were incubated with numerous pesticides and NA of the V- and G-type in different molar ratios. Samples were prepared either by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel enzymatic cleavage using endoproteinase Glu-C (Glu-C) or by combining highly albumin-selective affinity extraction with ultrafiltration followed by reduction, carbamidomethylation, and enzymatic cleavage (Glu-C) prior to MALDI-TOF MS analysis. Characteristic mass shifts for phosphylation revealed tyrosine adducts at Y(411) (Y(401)KFQNALLVRY(411)TKKVPQVSTPTLVE(425)), Y(148) and Y(150) (I(142)ARRHPY(148)FY(150)APE(153), single and double labeled), and Y(161) (L(154)LFFAKRY(161)KAAFTE(167)) produced by original NA (tabun, sarin, soman, cyclosarin, VX, Chinese VX, and Russian VX) as well as by chlorpyrifos-oxon, diisopropyl fluorophosphate (DFP), paraoxon-ethyl (POE), and profenofos. MALDI-MS/MS of the single-labeled I(142)-E(153) peptide demonstrated that Y(150) was phosphylated with preference to Y(148). Aged albumin adducts were not detected. The procedure described was reproducible and feasible for detection of adducts at the most reactive Y(411)-residue (S/N ≥ 3) when at least 1% of total albumin was labeled. This was achieved by incubating plasma with molar HSA/OPC ratios ranging from approximately 1:0.03 (all G-type NA, DFP, and POE) to 1:3 (V-type NA, profenofos). Relative signal intensity of the Y(411) adduct correlated well with the spotted relative molar amount underlining the usefulness for quantitative adduct determination. In conclusion, the current analytical design exhibits potential as a verification tool for high-dose exposure.

Synergism of resistance to phosalone and comparison of kinetic properties of acetylcholinesterase from four field populations and a susceptible strain of Colorado potato beetle

The susceptibility to phosalone and biochemical characteristics of acetylcholinesterase (AChE) were compared between susceptible (SS) strain and four field populations of Colorado potato beetle (CPB) collected from commercial potato fields of Hamedan Province in west of Iran. Bioassays involving topical application of phosalone to fourth instars revealed up to 252 fold resistance in field populations compared with the SS strain. Synergism studies showed that although esterase and/or glutathione S-transferase metabolic pathways were present and active against phosalone, they were not selected for and did not have a major role in resistance. It is likely that piperonyl butoxide (PBO) reduced phosalone toxicity by inhibiting bio-activation of phosalone. The affinity ( K m) and hydrolyzing efficiency ( V max) of AChE to selected substrates, namely, acetylthiocholine iodide (ATC), propionylthiocholine iodide (PTC), and butyrylthiocholine iodide (BTC) were examined. AChE inhibition by higher substrate concentration was evident only in the SS strain. In resistant field populations, Aliabad (Aa), Bahar (B) and Dehpiaz (Dp), substrate inhibition at higher concentrations was not seen. There was no definite optimal concentration found for any of the substrates examined. When ATC, PTC, and BTC were used as substrate, the reaction rates of AChE from Yengijeh (Yg) population increased as the concentration of all three substrates were increased, but were almost constant at concentration of ATC ⩾ 3.98, PTC ⩾ 2.8, and BTC ⩾ 5 mM. The susceptible form of AChE had the most efficient ATC hydrolysis but very low BTC hydrolysis activity. In contrast, AChEs from field populations elicited relatively reduced ATC hydrolysis, but relatively increased BTC hydrolysis. The in vitro inhibition potency of some organophosphates (OPs), on AChEs of the field populations and SS strain was determined. The rank order from the most potent inhibitor to the least as determined by their bimolecular reaction constants ( K i) was ethyl paraoxon > diazoxon > methyl paraoxon for AChE from Aa, B, Dp, and Yg populations, respectively, whereas the rank order for the susceptible strain was methyl paraoxon > ethyl paraoxon > diazoxon.

Immobilization of acetylcholineesterase–choline oxidase on a gold–platinum bimetallic nanoparticles modified glassy carbon electrode for the sensitive detection of organophosphate pesticides, carbamates and nerve agents

A novel, highly sensitive amperometric biosensor, based on electrodeposition of gold–platinum bimetallic nanoparticles onto 3-aminopropyltriethoxy silane modified glassy carbon electrode for the detection of paraoxon ethyl, aldicarb, and sarin has been developed. The biosensor consists of acetylcholineesterase (AChE)/choline oxidase (ChOx) immobilized by cross-linking with glutaraldehyde on a modified electrode. The properties of nanoparticles modified electrodes are characterized by scanning electron microscopy (SEM), energy dispersive X-ray (EDX), cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS). The synergistic action of Au and Pt nanoparticles showed excellent electrocatalytic activity with low applied potential for the detection of hydrogen peroxide (H2O2). The IC50 and inhibition rate constant (Ki) values were determined for the inhibitors using immobilized enzymes on modified electrode and the data were compared by spectrophotometric determination of these kinetic parameters using free enzymes in solution. Paraoxon ethyl, sarin, and aldicarb could be detected up to 150–200nM, 40–50nM, and 40–60μM respectively at 30–40% inhibition level of AChE enzyme and followed linearity in wide range concentration.

Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100 °C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60 °C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.

In vitro assessment of the genotoxicity of ethyl paraoxon in newborns and adults

This in vitro experiment measured the genotoxic effects of ethyl paraoxon, the active metabolite of ethyl parathion. To assess genotoxicity, we used the micronuclei (MN) technique by blocking cytokinesis, and the 'comet' assay. We cultured peripheral blood samples from healthy adults and umbilical cord blood samples from four clinically healthy newborns to identify the frequency of MN. After 48 hours, we added the following ethyl paraoxon concentrations to the cultures: 0.0, 0.075, 0.100, 0.160, and 0.200 microg/mL. For the comet assay, following Singh's technique, we treated the blood samples for 2 hours with similar doses of the metabolite. The comet assay results, at a concentration of 0.075 microg/mL, showed that ethyl paraoxon causes a greater DNA migration that followed a dose-response pattern, a greater intensity being observed in lymphocytes from newborns. A comparison of the treatment and control groups indicated that only the 0.200 microg/mL concentration produced a slight increase in MN. In conclusion, our study identified primary DNA damage due to ethyl paraoxon, with a major effect on newborn lymphocytes, as well as an effect on the frequency of MN in the study groups at high concentrations only.

Early-warning electrochemical biosensor system for environmental monitoring based on enzyme inhibition

Potentiometric and conductometric biosensors based on immobilised cholinesterases have been developed. Detection of some organophosphorous pesticides (methyl parathion, methyl paraoxon, ethyl paraoxon, trichlorphon, diisopropyl fluorophosphates) and carbamate pesticide (carbofuran) have been realised through enzyme inhibition mechanisms. Comparison of sensitivity of the biosensor systems developed with that of traditional analytical techniques (high performance liquid chromatography and Lumistox test based on Vibrio fischeri luminescent bacteria) has shown that biosensor measuring procedures are well suited for fast environmental control. Analytical characteristics of the systems created, their advantages and shortcomings are discussed.

Early-warning electrochemical biosensor system for environmental monitoring based on enzyme inhibition

Potentiometric and conductometric biosensors based on immobilised cholinesterases have been developed. Detection of some organophosphorous pesticides (methyl parathion, methyl paraoxon, ethyl paraoxon, trichlorphon, diisopropyl fluorophosphates) and carbamate pesticide (carbofuran) have been realised through enzyme inhibition mechanisms. Comparison of sensitivity of the biosensor systems developed with that of traditional analytical techniques (high performance liquid chromatography and Lumistox test based on Vibrio fischeri luminescent bacteria ) has shown that biosensor measuring procedures are well suited for fast environmental control. Analytical characteristics of the systems created, their advantages and shortcomings are discussed.

Coupled biosensor, biomimetic and chemometrics strategies for analysis of the metals in complex environmental matrices

An analytical strategy to determine metals in environmental samples contaminated with both organophosphate pesticides and metals is presented. In the first step, parathion and arsenate were determined separately. Inhibition of the enzyme butyrylcholinesterase was quantified employing an enzyme thermistor and also with a CCD camera. Subsequently, parathion mixed with arsenate was measured. A molecularly imprinted polymer catalyst was then introduced as a pre-column to degrade OPs. The catalytic MIP enabled the measurement of metal from the mixture containing OPs. Hydrolysis of parathion, ethyl paraoxon, O-Ethyl-O-(4-nitrophenyl)-phenylphosphonothioate EPN and dichlorvos were monitored by using catalytic MIPs in the enzyme thermistor. Finally, inhibition patterns of parathion and arsenate were obtained employing multivariate analysis. The patterns generated after eliminating the OPs can be used for monitoring and mapping of metals in various environmental matrices.