Optimisation of FIA system for detection of organophosphorus and carbamate pesticides based on cholinesterase inhibition

The sensitivity of the bioanalytical FIA system containing different immobilised cholinesterases (AChEs from electric eel, human erythrocytes, bovine erythrocytes and BuChE from horse serum) for determination of organophosphorus and carbamate pesticides was tested. Responses to some frequently used organophosphorus (paraoxon, oxydemeton-methyl, triazophos) and carbamate (carbofuran, propoxur) pesticides were found to be dependent on the origin of cholinesterases. The highest sensitivity was obtained by bioanalytical columns prepared with electric eel AChE while the lowest sensitivity was shown by the bioanalytical columns prepared with horse serum BuChE. The differences in responses for different enzymes were found to be less pronounced when the contact time between the enzyme and the pesticide is long enough (low flow rates). The optimal flow rate was chosen as a compromise between the duration of analysis and reasonably low limits of detection.     

A Fluorometric Enzyme Inhibition Detector for Carbamate Pesticide Analysis by High Speed Liquid Chromatography

Abstract

A fluorometric enzyme inhibition detector has been developed for the detection of carbamate pesticides after separation using high speed liquid chromatography.

Housefly head cholinesterase was found to have the greatest sensitivity toward the carbamates, together with horse plasma and bovine erythrocyte cholinesterases. The substrate N-methyl indoxyl acetate is used for monitoring of enzyme activity.     

Analysis of Organophosphorous Pesticides Based on Housefly Acetylcholinesterase Using Sequential Injection Analysis

Organophosphorous pesticides were analyzed based on the inhibition of acetylcholinesterase (AChE) from housefly and the electric eel using a sequential injection analysis (SIA) system. This analytical system is fully computerized and is able to automatically monitor the pesticide residues in an assay cycle of 180 s including 30 s incubation time when the flow is stopped. The optimal operation conditions such as AChE, substrate, pH, flow rate, and stopping time were studied. The experimental setup gave good reproducibility with the linear ranges for paraoxon of 1–70 ppb and dichlorovos of 20–70 ppb. The detection limits of paraoxon and dichlorovos were 1 and 5 ppb, respectively. The AChE from housefly shows 10 times higher sensitivity than the enzyme from the eel for these pesticide determination in this system.     

Acetylcholinesterase sensor based on screen-printed carbon electrode modified with prussian blue

Acetylcholinesterase (ChE) sensor based on Prussian blue (PB) modified electrode was developed and tested for the detection of organophosphorus and carbamic pesticides. The signal of the sensor was generated in PB mediated oxidation of thiocholine recorded at+200 mv in DC mode. ChE from electric eel was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) on the surface of screen-printed carbon electrode covered with PB and Nafion. The content of the surface layer (specific enzyme activity, Nafion and BSA amounts) was optimized to establish high and reliable response toward the substrate and ChE inhibitors. The ChE/PB sensor makes it possible to detect Aldicarb, Paraoxon and Parathion-Methyl with limits of detection 30, 10 and 5 ppb, respectively (incubation 10 min). The feasibility of practical application of the ChE/PB sensor developed for the monitoring of degradation of the pesticides in wine fermentation was shown. To diminish matrix interferences, the electrolysis of the grape juice with Al anode and evaporation of ethanol were suggested, however the procedures decrease the sensitivity of pesticide detection and stability of the sample tested.     

Flow injection analysis of potassium using an all-solid-state potassium-selective electrode as a detector

The concentration of potassium was determined by a combination of flow injection analysis (FIA) with an all-solid-state potassium sensor detection. The all-solid-state potassium-selective electrode possessing long-term potential stability was fabricated by coating an electroactive polypyrrole/poly(4-styrenesulfonate) film electrode with a plasticized poly(vinyl chloride) membrane containing valinomycin. The simple FIA system developed in this laboratory demonstrated sensitivity identical to that in the batch system and achieved considerably rapid assay (150 samples h⁻¹). Analyses of soy sauce and control serum samples by this FIA system yielded results in good agreement with those obtained by conventional measurements.     

用于农药残留快速检测的两种酶的比较

以商品乙酰胆碱酯酶(C3389,Ⅵ-S,从电鳗中提取)和自制鸡肝酶为农药检测用酶,比较了不同浓度的敌敌畏、敌百虫、马拉硫磷和西维因对这两种酶的乙酰胆碱酯酶活力和总酯酶活力的抑制情况。结果表明,鸡肝酶的乙酰胆碱酯酶活力较低,而其总酯酶活力对4种农药的灵敏度与商品乙酰胆碱酯酶活力的灵敏度相近,且总酯酶活力对除马拉硫磷外的其余3种农药的检出限都较乙酰胆碱酯酶低。同时对总酯酶活力测定的pH进行了优化,发现pH6．5(40mmol／L柠檬酸盐缓冲液)时总酯酶活力较高。     

Disposable potentiometric sensors for monitoring cholinesterase activity

A highly sensitive disposable screen-printed butyrylcholine (BuCh) potentiometric sensor, based on heptakis (2,3,6-tri-o-methyl)-β-cyclodextrin (β-CD) as ionophore, was developed for butyrylcholinesterase (BuChE) activity monitoring. The proposed sensors have been characterized and optimized according to the constituents of homemade printing carbon ink including β-CD, anionic sites, and plasticizer. The fabricated sensor showed Nernstian responses from 10⁻⁶ to 10⁻² mol L⁻¹ with detection limit of 8 × 10⁻⁷ mol L⁻¹, fast response time (1.6 s) and adequate shelf-life (6 months). Improved selectivity towards BuCh with minimal interference from choline (Ch) was achieved and the sensor was used for determination of 0.06-1.25 U mL⁻¹ BuChE. The developed disposable sensors have been successfully applied for real-time intoxication monitoring through assaying cholinesterases (ChEs) activity in human serum. Determination of organophosphate pesticide was conducted by measuring their inhibition of BuChE with successful assaying of malathion in insecticide samples with high accuracy and precision.     

Flow-injection amperometric determination of pesticides on the basis of their inhibition of immobilized acetylcholinesterases of different origin

Determination of the organophosphorus pesticides paraoxon, chlorpyrifos oxon, and malaoxon has been performed by a method based on inhibition of acetylcholinesterase (AChE) and amperometric detection in a flow-injection system with enzymes obtained from the electric eel (eeAChE) and Drosophila melanogaster (dmAChE) and immobilized on the surface of platinum electrode within a layer of poly(vinyl alcohol) bearing styrylpyridinium groups. dmAChE is more sensitive than eeAChE to inhibition by chlorpyrifos oxon and paraoxon. The sensitivity difference was largest for chlorpyrifos oxon (detection limit approx. 17 times lower), and practically none for malaoxon. Determination of the analytes in spiked river water samples by use of the dmAChE biosensor resulted in recoveries from 50 to 90 % for chlorpyrifos oxon at levels of 20 to 40 nmol L(-1), 50 to 100 % for paraoxon at 0.6 to 0.8 micro mol L(-1), and 140 to 190 % for malaoxon at 0.6 to 1.2 micro mol L(-1).     

Cholinesterases in Biorecognition and Biosensors Construction: A Review

Acetylcholinesterase and butyrylcholinesterase are the only two known cholinesterases. Acetylcholinesterase plays an important part in cholinergic system. It terminates neurotransmission by hydrolysis of transmitter acetylcholine. The role of butyrylcholinesterase is not well understood. It is able to detoxify several compounds such as cocaine, succinylcholine, and so forth. The current review is focused on the application of cholinesterases in biorecognition. Cholinesterases are important markers in the body. Butyrylcholinesterase activity in plasma can serve as a liver function test or specific marker for sensitivity to myorelaxants or liver carcinoma. Both cholinesterases can serve as markers of poisoning by some neurotoxic compounds. Nerve agents (sarin, soman, tabun, VX), some Alzheimer disease drugs (galantamine, huperzine, donepezil, rivastigmine), pesticides (carbofuran, trichlorfon, paraoxon, malaoxon), and natural toxins (aflatoxin, pyridostigmine) can act as inhibitors of butyrylcholinesterase and/or acetylcholinesterase. Devices filled with immobilized cholinesterases can be used for the assay of the aforementioned toxins. In this review, methods for examination of cholinesterases activity in the body and in analytical devices are described. Applications, types of diagnosis, and assays are described as well.     

The use of Artificial Neural Networks for the selective detection of two organophosphate insecticides: Chlorpyrifos and chlorfenvinfos

Amperometric acetylcholinesterase (AChE) biosensors have been developed to resolve mixtures of chlorpyrifos oxon (CPO) and chlorfenvinfos (CFV) pesticides. Three different biosensors were built using the wild type from electric eel (EE), the genetically modified Drosophila melanogaster AChE B394 and B394 co-immobilized with a phosphotriesterase (PTE). Artificial Neural Networks (ANNs) were used to model the combined response of the two pesticides. Specifically two different ANNs were constructed. The first one was used to model the combined response of B394 + PTE and EE biosensors and was applied when the concentration of CPO was high and the other, modelling the combined response of B394 + PTE and B394 biosensors, was applied with low concentrations of CPO. In both cases, good prediction ability was obtained with correlation coefficients better than 0.986 when the obtained values were compared with those expected for a set of six external test samples not used for training.     

凝胶渗透色谱净化气相色谱-质谱法检测河豚鱼、鳗鱼和对虾中191种农药残留

建立了河豚鱼、鳗鱼和对虾中191种农药多残留的气相色谱-质谱分析方法。样品用乙酸乙酯-环己烷(体积比为1:1)均质提取,凝胶渗透色谱净化,收集26～44 min的流出液并进行在线浓缩,通过气相色谱柱(DB-1701)分离后在选择离子监测(SIM)模式下进行质谱检测。分别以最低定量限和4倍最低定量限为添加浓度对河豚鱼、鳗鱼和对虾样品进行了两个水平的添加回收率实验,方法的回收率范围为50.2%～120%,其中89.5%的农药的回收率为70%～120%,相对标准偏差范围为0.6%～21.6%。方法的最小检出限和最低定量限范围分别为0.002～0.3 mg/kg和0.007～1.2 mg/kg。该方法的灵敏度、准确度和精密度均符合农药多残留检测的技术要求,适用于河豚鱼、鳗鱼和对虾等动物源性水产品中多种农药残留的检测。     

Amperometric biosensors based on nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors

Screen-printed electrodes coated with the nafion layer have been investigated for cholinesterase biosensor design. The butyrylcholinesterase (ChE) from horse serum was immobilised onto the nafion layer by cross-linking with glutaraldehyde vapours. The biosensors obtained showed better long-term stability and lower working potential in comparison to those obtained with no nafion coating. The sensitivity of a biosensor toward organophosphate pesticides is not affected by the nafion coating. The detection limits were found to be 3.5x10(-7) M for trichlorfon and 1.5x10(-7) M for coumaphos.     

Acetylcholinesterase-based biosensors for quantification of carbofuran, carbaryl, methylparaoxon, and dichlorvos in 5% acetonitrile

Amperometric acetylcholinesterase biosensors have been developed for quantification of the pesticides carbofuran, carbaryl, methylparaoxon, and dichlorvos in phosphate buffer containing 5% acetonitrile. Three different biosensors were built using three different acetylcholinesterase (AChE) enzymes—AChE from electric eel, and genetically engineered (B394) and wild-type (B1) AChE from Drosophila melanogaster. Enzymes were immobilized on cobalt(II) phthalocyanine-modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). Each biosensor was tested against the four pesticides. Good operational stability, immobilisation reproducibility, and storage stability were obtained for each biosensor. The best detection limits were obtained with the B394 enzyme for dichlorvos and methylparaoxon (9.6 × 10⁻¹¹ and 2.7 × 10⁻⁹ mol L⁻¹, respectively), the B1 enzyme for carbofuran (4.5 × 10⁻⁹ mol L⁻¹), and both the B1 enzyme and the AChE from electric eel for carbaryl (1.6 × 10⁻⁷ mol L⁻¹). Finally, the biosensors were used for the direct detection of the pesticides in spiked apple samples.     

Electronic Tongue Using an Enzyme Inhibition Biosensor Array for the Resolution of Pesticide Mixtures

An electronic tongue has been developed, employing an array of inhibition biosensors and Artificial Neural Networks (ANNs). The array of biosensors was formed by three amperometric pesticide biosensors that used different acetylcholinesterase (AChE) enzymes: the wild type from electric eel (EE) and two different genetically modified enzymes (B1 and B394). In order to model the response to dichlorvos and carbofuran mixtures, a total amount of 22 solutions were prepared, with random concentrations. Chronoamperometric responses of the biosensor array were used in order to obtain the inhibition bioelectronic tongue. Mean values of concentration of pesticides evaluated were 0.79 nM for dichlorvos and 4.1 nM for carbofuran. Good prediction ability was obtained with correlation coefficients better than 0.918 when the obtained values were compared with those expected for a set of 6 external test samples not used for training.     

平头pH电极-流动注射联用技术快速滴定食醋中醋酸的方法研究

采用一种具有平头结构的pH电极作为流动注射分析(FIA)的检测器,构建了流动注射自动化酸度滴定系统.优化了样品进样量、流速、载液浓度和反应管长度等参数.用NaOH溶液作为载液,在4.639×10-4～0.212 mol·L-1范围内醋酸浓度的对数与FIA峰的峰面积成正比,该方法的相对标准偏差(RSD)小于0.5%.采用...     

Enzymatic determination of N-methylcarbamate pesticides at the nanomolar level by the stopped-flow technique

A sensitive and selective kinetic enzymatic method for the determination of N-methylcarbamate pesticides is presented. It is based on their inhibitory effect on electric eel acetylcholinesterase and the use of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as chromogenic reagent for the thiocholine released from the acetylthiocholine iodide substrate. The fast DTNB-thiocholine reaction is monitored photometrically by the stopped-flow technique. Carbaryl, propoxur and carbofuran can be determined at concentrations in the ranges 6.5-120, 2-15 and 0.1-5.0 ng/ml, respectively, by the proposed method. An interference study was also reported.     

Postcolumn formation of fluorophores from nitrogenous pesticides by UV photolysis

The ultraviolet (UV) photolysis of several classes of nitrogenous pesticides was examined with a view to photo-induced fluorescence detection in flow-injection analysis (FIA) and liquid chromatography. The solvents evaluated as typical reversed-phase mobile phases included water, methanol, and 1:1 mixtures of methanol/water and acetonitrile/water, and methanol/acetonitrilelwater mixtures. Acetone, acetophenone, the surfactant triton X-100, and the photocatalyst titanium dioxide were assessed as photosensitizers to enhance the UV photolysis and fluorescence responses. FIA and liquid chromatographic separations of several pesticides were followed by post-column UV photolysis for the fluorescence detection. Ultraviolet photolysis produces some fluorescent products. The type of photolytic solvent seems to play a significant role. The presence of photosensitizers also affects the fluorescence response of some pesticides. The photochemical transformation products of some of the pesticides are suggested. Analytical figures of merit were evaluated for determination of several pesticides in ground water. The post-column UV photolysis approach for fluorescence detection in liquid chromatography was assessed for several nitrogenous pesticides in ground water samples at ng/g concentrations.     

Spectroscopic Determination of Cholinesterase Activity and Inhibition in Sol-Gel Media

Biological activity of cholinesterases can be determined by optically monitoring the enzymatic reaction with indophenyl acetate, (N-4′-acetoxyphenyl)-4-quinone imine. At pH 8.0 cholinesterases hydrolyze this yellow dye to yield a blue reaction product. Cholinesterase inhibitors reduce the rate of this hydrolysis. Thus, by monitoring absorbance of the hydrolysis product at its maximum (630 nm) as a function of time, reaction rates of both cholinesterase activity and cholinesterase inhibition may be quantified spectroscopically. Using this technique, we measured the enzymatic activity of butyrylcholinesterase (BuChE) molecules encapsulated in tetramethyl orthosilicate (TMOS) silicate gel-glass prepared by hydrolysis and condensation. This activity is reduced, in a concentration-dependent manner, by the reversible cholinesterase inhibitors 1,5-bis(4-allyldimethyl-ammoniumphenyl) pentan 3-one dibromide (BADAPP) and 9-amino-1,2,3,4-tetrahydroacridine (THA; tacrine, Cognex). The gel-glasses are rigid, and compact, transparent and porous enough to allow reagents to diffuse in and out.     

Polymer nanoparticles as fluorescent labels in a fluoroimmunoassay for human chorionic gonadotropin

A sensitive fluoroimmunoassay (FIA) was developed for the determination of human chorionic gonadotropin (β-HCG). It is based on fluorescent polymer nanoparticles (PFNPs) coated with anti-β-HCG monoclonal antibodies in a sandwich type of fluoroimmunoassay. The PFNPs were synthesized by precipitation polymerization using methacrylic acid (MAA) as the monomer, trimethylolpropane trimethacrylate as the cross-linker, azobisisobutyronitrile as the radical initiator, and fluorescein as the fluorophore. Anti-β-HCG monoclonal antibody was labeled with the PFNPs and then used in a FIA of β-HCG in human serum samples using low-fluorescent transparent 96-well microtiter plates. The calibration graph for β-HCG is linear over the range from 1.25 to 300 mIU mL-¹ with a detection limit of 0.3 mIU mL-¹ (3σ). The relative standard deviation for seven parallel measurements of 10 mIU mL-¹ of β-HCG is 3.8%. The method has the specificity of an immunoassay and the sensitivity of fluorescent nanoparticle label technology.     

Fluoroimmunoassay for tumor necrosis factor-α in human serum using Ru(bpy)3Cl2-doped fluorescent silica nanoparticles as labels

A novel fluoroimmunoassay (FIA) method was developed for the determination of tumor necrosis factor-α (TNF-α) in this study. The proposed method has the advantage of showing the specificity of immunoassays and sensitivity of fluorescent nanoparticles label technology. With the well-established inverse microemulsion polymerisation process, the tris(2′,2-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy)-doped fluorescent silica nanoparticles (RuDFSNs) were prepared. Then a RuDFSNs-labeled anti-TNF-α monoclonal antibody was prepared and used for FIA of TNF-α in human serum samples with a sandwich FIA by using the low fluorescent 96-well transparent microtiter plates. The assay response was linear from 1.0 to about 250.0 pg/mL with a detection limit of 0.1 pg/mL for TNF-α. The intra- and inter-assay precision are 4.9%, 4.4%, 4.6%; 6.1%, 5.9%, 5.3% for five parallel measurements of 2.0, 20.0, 200.0 pg/mL TNF-α respectively, and the recoveries are in the range of 96-104% for human serum sample measurements by standard-addition method. We also explored the application of fluorescence microscopy imaging in the study of the FIA for TNF-α with the fluorescent nanoparticle labels. The results demonstrate that the method offers potential advantages of sensitivity, simplicity and good reproducibility for the determination of TNF-α, and is applicable to the determination of TNF-α in serum samples and being capable of fluorescence microscopy imaging for the determination of TNF-α.