Polyaniline-modified cholinesterase sensor for pesticide determination.

Cholinesterase sensors based on glassy carbon and planar epoxy graphite electrodes modified with processed polyaniline have been developed and examined for pesticide detection. The modification of electrode surface with polyaniline provides high operational stability and sensitivity towards the pesticides investigated. The detection limits found (coumaphos, 0.002, trichlorfon, 0.04, aldicarb, 0.03, methiocarb, 0.08 mg l(-1)) make it possible to detect the pollutants in the waters on the level of limited threshold levels without sample preconcentration.     

乙酰胆碱酯酶/Nafion/普鲁士蓝修饰玻碳电极的制备及其在有机农药检测中的应用

制备了乙酰胆碱酯酶/Nafion/普鲁士蓝修饰的玻碳电极,测试了该修饰电极检测有机农药西维因(carbaryl)和敌百虫(trichlorfon)的性能指标。 利用原子力显微镜和电化学技术研究了电极的构造及其对于有机农药检测性能指标的影响。 结果表明,乙酰胆碱酯酶均匀地分散到Nafion/普鲁士蓝修饰的玻碳电极上。 在最优的实验条件下,构筑的修饰电极检测西维因和敌百虫的线性范围分别为0.01~0.5 μmol/L及2.0~10.0 μmol/L和0.02~1.0 μmol/L及2.0~8.0 μmol/L,检出限分别为5.0和10.0 nmol/L。 并对模拟的实际样品进行了检测,发现该方法有较高的检测灵敏度、较好的重复性和抗干扰性。     

Enzyme-octadecylamine Langmuir-Blodgett membranes for ENFET biosensors

Langmuir-Blodgett (LB) films containing butyrylcholinesterase (BuChE) are fabricated to realise an enzymatic field effect transistor (ENFET) for the detection of organophosphorus pesticides in water. Trichlorfon as a common pesticide is examined in our work. The BuChE-immobilised LB films are formed by adsorbing the enzyme molecules onto a stearylamine monolayer using the electrostatic force. Enzyme/stearylamine mixed LB films are immobilized onto a pH-ISFET surface and treated by glutaraldehyde vapour to improve the LB film's stability. The ENFET thus obtained worked as a potentiometric biosensor for trichlorfon detection on the basis of enzyme inhibition. The detection limit for trichlorfon can reach 10(-7) M (26 ppb). The surface characteristics of BuChE/stearylamine LB films obtained under various conditions of the dipping surface pressures are analysed qualitatively by atomic force microscopy (AFM) and analysed quantitatively by FTIR spectroscopy.     

Rapid detection of organophosphorus pesticide residue on Prussian blue modified dual-channel screen-printed electrodes combing with portable potentiostat

A new method consisted of a dual-channel screen-printed electrode (DSPE) efficient modified with Prussian blue and acetylcholinesterase was developed for the rapid detection of organophosphorus pesticide residues.     

Amperometric Biosensor Based on Immobilization Acetylcholinesterase on Manganese Porphyrin Nanoparticles for Detection of Trichlorfon with Flow‐Injection Analysis System

A highly sensitive amperometric biosensor for the detection of organophosphate pesticides (OPs) is developed. The biosensor was fabricated by immobilized acetylcholinesterase (AChE) on manganese (III) meso‐tetraphenylporphyrin (MnTPP) nanoparticles (NPs)‐modified glassy carbon (GC) electrode. The MnTPP NPs used in this article were synthesized by mixing solvent techniques. AChE enzyme was immobilized on the MnTPP NPs surface by conjugated with chitosan (CHIT). The electrocatalytic activity of MnTPP NPs led to a greatly improved performance for thiocholine (TCh) product detection. The developed AChE‐CHIT/MnTPPNP/GC biosensor integrated with a flow‐injection analysis (FIA) system was used to monitor trichlorfon (typical OP). A wide linear inhibition response for trichlorfon is observed in the range of 1.0 nM–1.0 mM, corresponding to 10–83% inhibition for AChE with a detection limit of 0.5 nM.     

Improvement of acetylcholinesterase-based assay for organophosphates in way of identification by reactivators

Organophosphates present serious fulmination in several aspects of human life. Detection of organophosphates is frequently based on following acetylcholinesterase (AChE) inhibition. Although limit of detection and sensitivity for AChE-based assays seem to be intriguing, the identification of organophosphates is not currently efficient in this way. We introduce an improvement of AChE-based assay by reactivators using a selective come-back of AChE activity after previous inhibition. We have chosen four organophosphates: paraoxon-ethyl, paraoxon-methyl, trichlorfon, methamidophos as representative pesticides and the three most available reactivators: HI-6, obidoxime, pralidoxime. Reactivation was realized in the 96-wells photometric microplates and activity of human recombinant AChE was followed by reaction of Ellman's reagent with one of enzyme digestion product: thiocholine. Distinguishing of reactivation efficacy was judged by the independent two population t-test. The most significant identification was based on methamidophos inhibited AChE reactivation by HI-6 or pralidoxime and paraoxon-ethyl inhibited AChE by obidoxime; moreover, identification of trichlorfon and paraoxon-methyl was possible, too. Practical impact of described method is discussed.     

Enzyme and microbial sensors for phosphate, phenols, pesticides and peroxides

Biosensors employing a biocatalyst on a different level of integration have been developed for monitoring environmental pollution. These probes range from laboratory specimen to commercial detectors applied to analyzers. Recent developments on amperometric enzyme and microbial biosensors are presented here. A monoenzymatic bulk-type carbon electrode is described for biosensing organic hydroperoxides in aqueous solutions; peroxidase is immobilized within the electrode body and the direct electron transfer between electrode and enzyme is measured. Both, reversible and irreversible inhibitors of acetylcholinesterase have been quantified by using a kinetically controlled acetylcholine enzyme sequence electrode. The inhibitory effect of pesticides such as butoxycarboxime, dimethoate, and trichlorfon could be quantified within 6 min in molar concentrations. Different multi-enzyme electrodes have been developed for the determination of inorganic phosphate. These sensors represent examples of sequentially acting enzymes in combination with enzymatic analyte recycling. Using this type of amplification nanomolar concentrations can be measured.     

Amperometric Detection of Catecholamine Neurotransmitters Using Electrocatalytic Substrate Recycling at a Laccase Electrode

An enzyme electrode based on the coimmobilization of an osmium redox polymer and laccase on glassy carbon electrodes has been applied to ultra sensitive amperometric detection of the catecholamine neurotransmitters dopamine, epinephrine and norepinephrine, resulting in nanomolar detection limits, as low as 4 nM for dopamine. The sensitivity of the electrode is due to signal amplification via oxidation of the catecholamine by the immobilized laccase, which is regenerated by concomitant reduction of oxygen to water, coupled to the electrocatalytic re‐reduction of the oxidized catecholamine by the osmium redox complex: electrocatalytic substrate recycling. In addition because the sensor can be operated in reductive mode at ?0.2 V (vs. Ag/AgCl), noise and interferences are diminished. Combined with its high sensitivity this enzyme electrode also exhibited excellent selectivity allowing the detection of catecholamines in the presence of ascorbic acid. However, differentiation between the current responses achieved for the three catecholamines is not possible. The effective mode of constant recycling, resulting in amplification of the current response, of the laccase enzyme electrode sensor combined with the inherent advantages of using electrochemical techniques holds great promise for the future of catecholamine detection and monitoring.     

Structural characteristics and cyclic voltammetric behaviour of Sonogel-Carbon tyrosinase biosensors. A detailed comparative study of three immobilization matrixes

Structural characteristics an cyclic voltammetry of three amperommetric biosensors based on immobilization of tyrosinase on a Sonogel-Carbon electrode for detection of phenols are described. Cyclic voltammetry was applied to study the electrochemical behaviour of the electrode and the electrochemical reaction on the electrode surface. Scanning electron microscopy, X-ray energy dispersive spectroscopy and atomic force microscopy were used for the structure characterization of the electrode surface, enzyme film and polymers coatings. The influence of additive-protective polymers, such as polyethylene glycol and perfluorinated-Nafion ion-exchanger on the surface of the biosensor were explored.     

膜富集结合膜上图像比色法快速检测微量有机磷农药

建立了基于手机图像比色法结合多通道膜富集技术用于微量有机磷农药的快速检测方法. 根据乙酰胆碱酯酶活性受有机磷农药特异性抑制而降低的特点, 以吲哚乙酸酯为底物, 经酶水解生成靛蓝, 利用多通道膜富集装置将有色生成物富集到尼龙微孔滤膜上, 将标准溶液和样品溶液富集在同一张膜的不同位置. 用智能手机拍摄微孔滤膜的数码照片, 利用编制的手机APP软件对照片进行处理, 消除光照、 噪点等的影响, 可实现自动识别标准和未知样品. 通过提取样品颜色信号, 建立了颜色信号与浓度的定量分析模型, 用其检测敌百虫的检出限可达0.030 mg/L. 利用该方法对黄瓜中农药敌百虫的含量进行分析, 加标回收率在95.8%~106.9%之间, 相对标准偏差RSD(n=5)为3.8%~5.4%. 本方法用手机代替光谱仪, 简单实用, 与相关国标方法相比, 灵敏度和准确度均得到显著提高, 具有良好的应用前景.     

Enzyme-based biosensor as a selective detection unit in column liquid chromatography

A reagentiess enzyme electrode based on co-immobilized alcohol oxidase and horseradish peroxidase was used as the working electrode in an amperometric flow-through cell connected to a column liquid chromatographic (CLC) system for the selective detection of methanol and ethanol. The enzymes were covalently immobilized in carbon paste (graphite-phenylmethylsilicone oil) in the presence of polyethylenimine. Electrodes prepared from the enzyme-modified carbon paste were optimized with respect to their sensitivity and selectivity. Different membranes were cast or electropolymerized directly on the surface of the electrode to increase the long-term stability of the biosensor. The compatibility with the reversed-phase chromatographic system was established. A PLRP-S polymer-based separation column was used with phosphate buffer as the mobile phase. The selectivity of the enzyme electrode was also determined by injecting some easily oxidizable and possibly interfering species normally present in biological samples. The enzyme electrode was also used in an on-line system, consisting of a microdialysis probe as the sampling unit, the CLC system and the biosensor detection device, for the selective following of the ethanol produced when a paper pulp industrial waste water was ferinented with Saccharomyces cerevisiae.     

An electrochemical amplification immunoassay using bi-electrode signal transduction system

An electrochemical immunoassay technique has been developed based on the sensitive detection of the enzyme-generated product with a bi-electrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the redox reactions on two different electrodes, that is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system device is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30 min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity.     

Determination of trace trichlorfon by high performance liquid chromatography with UV detection based on its catalytic effect on sodium perborate oxidizing benzidine

Trichlorfon has the capacity to catalyze the oxidation of benzidine (4,4′-diamino-biphenyl) to 4-amino-4′-nitro biphenyl in the presence of sodium perborate. The product of the catalyzed reaction was validated by LC-MS method. Reversed-phase high performance liquid chromatography with 365 nm UV detection was used for separation and quantification of 4-amino-4′-nitro biphenyl. It can be proven there is a linear relationship between the peak areas of 4-amino-4′-nitro biphenyl and trichlorfon in the concentration range of 0.02-0.5 mg L⁻¹ (r = 0.9988). Limit of detection was 2.0 μg L⁻¹. A method for the indirect determination of trichlorfon using HPLC was developed based on catalytic effect of trichlorfon. Method validation was performed on samples spiked at three levels (0.5, 1.0, 1.5 mg kg⁻¹), the recoveries ranged from 67.5 to 82.1%, with relative standard deviations between 4.5 and 7.3%.0.01 mol L⁻¹ sodium dodecyl sulphate (SDS) solution was used to extract trichlorfon from samples and solid-phase extraction was used to isolate and concentrate trichlorfon in SDS solution. The recoveries of trichlorfon obtained with percolating the extraction through a SPE system were essentially in agreement with those obtained by liquid-liquid extraction. This new isolation technique decreases the use of toxic solvents and satisfies the requirements of Green Analytical Chemistry.     

Silicone-grease-based immobilisation method for the preparation of enzyme electrodes

An approach to the construction of amperometric biosensors based on the incorporation of an enzyme in silicone grease and using the grease to fill micropores on a graphite surface is described. The enzyme-grease electrode concept, illustrated with the enzyme tyrosinase, offers a very simple, rapid and inexpensive approach to the fabrication of enzyme electrodes. The tyrosinase electrode responds very rapidly to dynamic changes in the concentration of phenolic compounds. A response time (t95%) as low as 5 s has been determined. With flow injection, 120 samples per hour can be processed with a relative standard deviation of 2.4%. The electrode remains active for about 12 d. The detection limit for dopamine is 6 x 10(-6) M. This method of biosensor construction should be applicable to other enzyme-substrate systems.     

基于多孔金结构的三相界面酶电极的制备及高效电化学酶传感性能

界面微环境是影响酶催化反应及酶传感性能的关键因素. 本研究基于三维微纳米结构多孔金基底, 通过调控电极表面的亲水和疏水浸润性, 制备了具有固-液-气三相界面微环境的氧化酶电极, 并研究了界面微环境对酶催化反应动力学的影响规律. 基于所制备的三相界面多孔金结构酶电极, 反应物氧气能够从气相直接快速地传输到酶催化反应界面, 极大地提升了界面氧气浓度及其稳定性, 从而大幅度提高了氧化酶活性及酶电极响应的稳定性. 以葡萄糖为模型待测物, 基于该三相界面酶电极的电化学酶生物传感器拥有宽的线性范围、 高的灵敏度、 低的检出限以及良好的稳定性. 这类独特的三相反应界面设计为高效酶生物传感器的建构以及生物分子的精准检测提供了新思路.     

Hybridization detection of enzyme-labeled DNA at electrically heated electrodes

In this report we describe an electrochemical DNA hybridization sensor approach, in which signal amplification is achieved using heated electrodes together with an enzyme as DNA-label. On the surface of the heatable low temperature co-fired ceramic (LTCC) gold electrode, an immobilized thiolated capture probe was hybridized with a biotinylated target using alkaline phosphatase (SA-ALP) as reporter molecule. The enzyme label converted the redox-inactive substrate 1-naphthyl phosphate (NAP) into the redox-active 1-naphthol voltammetrically determined at the modified gold LTCC electrode. During the measurement only the electrode was heated leaving the bulk solution at ambient temperature. Elevated temperature during detection led to increased enzyme activity and enhanced analytical signals for DNA hybridization detection. The limit of detection at 53 °C electrode temperature was 1.2 nmol/L.     

基于纳米金的分子印迹传感器检测果蔬中的氧化乐果和乐果

以甲基丙烯酸为功能单体,氧化乐果为印迹分子,构建了一种可用于检测果蔬中氧化乐果和乐果的分子印迹传感器.在金电极上电沉积金纳米粒子,然后将修饰电极浸入10 mL含有氧化乐果和甲基丙烯酸的聚合物溶液中进行9次循环电聚合(-0.3～0.3 V),无水甲醇/乙酸洗涤除去模板分子.循环伏安法和电化学阻抗谱表征传感器,差分脉冲伏安...     

尸体脏器中敌百虫及其主要代谢物DDVP和水合氯醛的毛细柱GC/MS测定

本文应用Finnigan Mat 1020 GC/MS仪,以SE-54熔融硅毛细柱作分离柱,建立了尸体脏器中敌百虫及其主要代谢物DDVP和水合氯醛的分析方法。样品用CH_2Cl_2提取。敌百虫和DDVP的检测限均是15 ng,方法已成功用于刑事案件鉴定。     

A flow-injection system for the amperometric determination of xylose and xylulose with co-immobilized enzymes and a modified electrode

A flow-injection system is described for the detection of xylose and xylulose. The detection is based on three successive reactions taking place in an enzyme reactor containing xylose isomerase, mutarotase and glucose dehydrogenase co-immobilized on controlled-pore glass. The final product, NADH, is electrocatalytically oxidized at 0 mV vs. SCE at a chemically modified electrode. Straight calibration graphs for the two pentoses were obtained between 50 μM and 2 mM. The maximal sample throughput was 30 h^?1.     

Amperometric Enzyme Based Biosensor For the Detection of Xanthine Via Superoxide

Abstract

An amperometric enzyme modified carbon paste electrode for the detection of xanthinc by the xanthine oxidase catalyzed reaction is described. the product of the enzyme reaction monitored is the highly unstable radical superoxide, detected at O mV (vs SCE) poised potential. A dual working electrode configuration consisting of an enzyme modified carboa paste electrode and an unmodified glassy carbon electrode are utilized for this purpose.