层层自组装纳米金与乙酰胆碱酯酶电化学生物传感器检测有机磷农药

采用层层自组装技术制备了快速检测有机磷农药的生物传感器,利用带正电荷的高分子聚电解质聚二烯丙基二甲基氯化铵(PDDA)将乙酰胆碱酯酶(AChE)和金纳米粒子(AuNPs)通过静电力逐层固定到玻碳电极(GCE)表面,并采用交流阻抗和微分脉冲伏安法研究了此生物传感器的电化学行为。由于金纳米粒子优异的电催化性能和良好的生物相容性,使固定化的乙酰胆碱酯酶对其底物具有更高的亲和力和更快的响应速度。实验结果表明:修饰金纳米粒子后,传感器的氧化电流明显增大,在4.6×10-5～5.3×10-3mol/L范围内,固定化酶的抑制率与甲基对硫磷浓度的对数成正比,检出限为7.6×10-6mol/L。该生物传感器具有制备方法简便、成本低、灵敏度高等优点,已成功用于蔬菜样品中甲基对硫磷含量的测定。     

Can immobilized enzymes Be applicable for homogeneous reaction? A novel technique of acetylcholinesterase immobilization for assaying carbaryl pesticide

A novel technique of enzyme immobilization is developed for making the immobilized enzymes capable of further releasing for homogeneous reaction. Water-soluble poly(vinyl alcohol) was used to prepare enzyme-loaded membranes with immobilized acetylcholinesterase (AChE). When used, a piece of enzymecontaining membrane is put into the solution and dissolves quickly. The released AChE is mixed and interacts with substrates and carbaryl inhibitors. The catalytical activity and inhibition sensitivity of released enzyme are comparable to those of free AChE. The values of Michaelis constant and maximum reaction rate for released AChE are also very close to those for free AChE. The experimental conditions such as the concentrations of PVA and acetone, the time of enzymatic reaction and that of AChE inhibition by carbaryl pesticide were optimized. The relative inhibition of AChE activity increased with the carbaryl concentration ranging from 0.1 μg/L to 100 mg/L. When compared to free AChE in solution or solid powder, the prepared PVA-AChE membranes are advantageous with respect to storage and handling. The suggested technique of enzyme immobilization is suitable for the variety of applications, when the enzyme catalysed reactions allows for single-using of the active material and does not require further enzyme recovering.     

Gold nanoparticle-coated multiwall carbon nanotube-modified electrode for electrochemical determination of methyl parathion

We report on an electrochemical sensor for the determination of methyl parathion. It is based on an electrode modified with multi-walled carbon nanotubes that were covered with gold nanoparticles (Au-NPs). The vertically aligned array of MWCNTs on a tantalum substrate was coated with Au-NPs by overhead magnetron sputtering deposition. Scanning and transmission electron microscopy and XRD were used to characterize the Au-NP-MWCNTs composite. Cyclic voltammetry and differential pulse voltammetry were employed to evaluate the suitability of the new electrode for the determination of methyl parathion. Under the optimal conditions, the current response of the electrode to methyl parathion is linear in the range from 0.50 to 16.0 mg mL^-1, with a detection limit of 50 ??g mL^-1 (signal/noise = 3), and the sensitivity is 4.5 times better than that of the plain MWCNTs electrode. We conclude that this method represents a simple, rapid, effective and sensitive approach for the detection of methyl parathion.

基于聚谷氨酸修饰玻碳电极的甲基对硫磷电化学传感器

制备了一种简单的聚谷氨酸修饰玻碳电极的用于检测甲基对硫磷的电化学传感器。 并应用循环伏安法研究了甲基对硫磷在该修饰电极上的氧化还原行为;甲基对硫磷的浓度检测采用差分脉冲伏安法,结果表明,甲基对硫磷在5.0×10-7～7.5×10-4 mol/L浓度范围与响应电流有良好的线性关系。 甲基对硫磷检测限（S/N=3）可达1.0×10-9 mol/L。 该法制备的传感器有望应用于实际样品中的甲基对硫磷的检测。     

Structure—activity relationships for <Emphasis Type="Italic">in vitro</Emphasis> oxime reactivation of chlorpyrifos-inhibited acetylcholinesterase

Organophosphorus pesticides parathion, chlorpyrifos, and malathion inhibit the enzyme acetylcholinesterase (AChE; EC 3.1.1.7) via phosphorylation of its active site. AChE reactivators and anticholinergics are compounds used as antidotes in the case of intoxication by these AChE inhibitors. In this work, chlorpyrifos, a representative member of this pesticide family, was used to inhibit the AChE activity of rat brain. The effect of twenty-one structurally different AChE reactivators was tested in vitro and subsequently, the relationship between their chemical structure and biological activity was outlined.     

Development of a stable biosensor based on a SiO2 nanosheet–Nafion–modified glassy carbon electrode for sensitive detection of pesticides

SiO₂ nanosheets (SNS) have been prepared by a chemical method using montmorillonite as raw material and were characterized by scanning electron microscopy and X-ray diffraction. SiO₂ nanosheet–Nafion nanocomposites with excellent conductivity, catalytic activity, and biocompatibility provided an extremely hydrophilic surface for biomolecule adhesion. Chitosan was used as a cross-linker to immobilize acetylcholinesterase (AChE), and Nafion was used as a protective membrane to efficiently improve the stability of the AChE biosensor. The AChE biosensor showed favorable affinity for acetylthiocholine chloride and catalyzed the hydrolysis of acetylthiocholine chloride with an apparent Michaelis–Menten constant of 134 μM to form thiocholine, which was then oxidized to produce a detectable and fast response. Based on the inhibition by pesticides of the enzymatic activity of AChE, detection of the amperometric response from thiocholine on the biosensor is a simple and effective way to biomonitor exposure to pesticides. Under optimum conditions, the biosensor detected methyl parathion, chlorpyrifos, and carbofuran at concentrations ranging from 1.0?×?10^?12 to 1?×?10^?10?M and from 1.0?×?10^?10 to 1?×?10^?8?M. The detection limits for methyl parathion, chlorpyrifos, and carbofuran were 5?×?10^?13?M. The biosensor developed exhibited good sensitivity, stability, reproducibility, and low cost, thus providing a new promising tool for analysis of enzyme inhibitors.

One-step fabrication of three-dimensional porous calcium carbonate–chitosan composite film as the immobilization matrix of acetylcholinesterase and its biosensing on pesticide

This work reports on a novel nanosized calcium carbonate–chitosan (nanoCaCO₃–chi) composite film fabricated by a one-step co-electrodeposition method. The generated nanoCaCO₃-based matrix possessed a three-dimensional (3D) porous, network-like structure, providing a favorable and biocompatible microenvironment to immobilize enzyme. By using such a composite film as enzyme immobilization matrix, a highly sensitive and stable acetylcholinesterase (AChE) sensor was achieved for determination of methyl parathion as a model of organophosphate pesticides (OPs) compounds. The inhibition of methyl parathion was proportional to its concentration ranging from 0.005–0.2 to 0.75–3.75 μg mL⁻¹. The detection limit was found to be as low as 1 ng mL⁻¹ (S/N = 3). The designed biosensor exhibited good reproducibility and acceptable stability.     

Ionic silsesquioxane film immobilized on silica applied in the development of carbon paste electrode for determination of methyl parathion

A mesoporous silica-based hybrid material composed of silica xerogel modified with an ionic silsesquioxane, which contains the 1,4-diazoniabicyclo[2.2.2]octane chloride group, was obtained. The silsesquioxane film is highly dispersed on the surface. This hybrid material was utilized to develop a carbon paste electrode (CPE) for determination of methyl parathion. Transmission FTIR, elemental analysis and N₂ adsorption–desorption isotherms were used for characterization of the material. The electrochemical behavior of methyl parathion was evaluated by cyclic voltammetry and differential pulse voltammetry. It was observed a linear response to methyl parathion in the concentration range from 1.25 × 10^?7 to 2.56 × 10^?6 mol L^?1 by employing the carbon paste electrode, in Britton–Robinson buffer solution (pH 6). The achieved detection limit (3 SD of the blank divided by the slope of calibration curve) was 0.013 µmol L^?1 and sensitivity was 6.3 µA µmol L^?1. This result shows the potentiality of this electrode for application as electrochemical sensor for methyl parathion.     

Organophosphorus pesticides detection using acetylcholinesterase biosensor based on gold nanoparticles constructed by electroless plating on vertical nitrogen-doped single-walled carbon nanotubes

An acetylcholinesterase (AChE) biosensor was constructed based on gold nanoparticles (AuNPs) using electroless plating on vertical nitrogen-doped single-walled carbon nanotubes (VNSWCNTs) for detecting organophosphorus pesticides (OPs). AChE was immobilised on AuNPs via Au–S bonding, and VNSWCNTs were produced by spontaneous chemical adsorption of NSWCNTs on gold electrode, also via Au–S bonding. This modified electrode exhibited excellent electron transfer capacity due to the synergy between AuNPs and VNSWCNTs. The developed biosensor showed good linear relations at concentrations of 10^?5 – 1 ppb, and the detection limits were 3.04 × 10^?6 ppb for methyl parathion, 1.96 × 10^?6 ppb for malathion and 2.06 × 10^?6 ppb for chlorpyrifos, respectively. The AChE biosensor revealed satisfactory stability, excellent sensitivity and good repeatability. These results suggest that this biosensor has good application prospects and can function as a sensitive device in OPs analysis.     

Photolithographically patterned enzyme membranes for the detection of pesticides and copper(II) based on enzyme inhibition

Summary A non-aqueous and an aqueous photopolymer system with an enzyme are used to prepare photolithographically patterned enzyme membranes for amperometric (thinfilm platinum electrode) and potentiometric (ISFET) sensors based on enzyme inhibition. Flow methods for enzyme inhibition tests are described. The decrease in enzyme (AChE) activity after incubation in a solution of dichlorvos as inhibitor is detected amperometrically. The enzyme urease is immobilized onto the pH-sensitive gate area of an ISFET. Such a biosensor is able to detect copper-(II) in water in the ppm-range without preconcentration.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Determination of Organophosphorus Pesticides Based on BDD Electrode Modified with Au/chitosan Fiber

The Au/chitosan fiber was firstly prepared by electrospinning and chemical reduction method and used to modify BDD electrode for the detection of methyl parathion. The results indicated that Au/chitosan fiber‐modified BDD electrode could improve the electrocatalytic activity, accelerate response, enhance the sensitivity, and reduce the detection limit, as compared with the bare BDD electrode. The repeatability and stability of Au/chitosan fiber‐modified BDD electrode was also studied. For the detection of methyl parathion in the apple juice samples, Au/chitosan fiber‐modified BDD electrode obtained favorable results and ideal recovery rate.     

A novel protocol for ultra-trace detection of pesticides: Combined electrochemical reduction of Ellman's reagent with acetylcholinesterase inhibition

This paper proposed a novel method for ultra-trace detection of pesticides combining electrochemical reduction of Ellman's reagent with acetylcholinesterase (AChE) inhibition. The amperometric biosensor, fabricated by immobilizing AChE on multi-walled carbon nanotubes-chitosan (MWCNTs-Chi) nanocomposites modified glassy carbon electrode, enjoyed high sensitivity owing to the excellent conductivity and favourable biocompatibility of MWCNTs-Chi nanocomposites. Meanwhile, the sensitivity of the biosensor was further enhanced using the electrochemical reduction signal of DTNB for determination. Under optimum conditions, methyl parathion was detected based on its inhibition effect on AChE activity and the subsequent change in electrochemical reduction response of DTNB. Good relationship was obtained between the reduction current and pesticide concentration in the ranges of 5.0 × 10⁻⁷ to 1.0 × 10⁻¹² M with a detection limit of 7.5 × 10⁻¹³ M (S/N = 3). Moreover, the proposed protocol was successfully employed for the determination of methyl parathion in water and soil samples.     

Electrochemical pesticide sensitivity test using acetylcholinesterase biosensor based on colloidal gold nanoparticle modified sol-gel interface

Based on the change in electrochemical behavior of enzymatic activity induced by pesticide, a novel electrochemical method for investigation of pesticide sensitivity using acetylcholinesterase (AChE) biosensor was developed. The sol-gel-derived silicate network assembling gold nanoparticles (AuNPs-SiSG) provided a biocompatible microenvironment around the enzyme molecule to stabilize its biological activity and prevented them from leaking out of the interface. The composite was characterized using atomic force microscopy and proved to be chemically clean, porous and homogeneous. AuNPs promoted a conductive pathway for electron transfer and improved electrochemical reactions at a lower potential. Typical pesticides such as monocrotophos, methyl parathion and carbaryl were selected for pesticide sensitivity tests. Due to the inhibitions of pesticides, the electrochemical responses of substrate on AChE-sensors decreased greatly. The inhibition curves showed good correspondence with the results by UV spectrophotometry assay. The proposed electrochemical pesticide sensitivity test exhibited high sensitivity, desirable accuracy, low cost and simplified procedures. This method could be developed as a conventional method to select efficient enzyme inhibitors and investigate toxic compounds against to enzyme.     

Acetylcholinesterase Immobilization on 3‐Mercaptopropionic Acid Self Assembled Monolayer for Determination of Pesticides

Studies on the immobilization of acetylcholinesterase onto a SAM gold electrode and the use of the fabricated biosensor for the determination of carbaryl and parathion are presented. The influence of pH, ionic strength, enzyme loading and concentration of glutaraldehyde on the response of the biosensor was investigated . The amperometric biosensor developed in this study provided linearity to parathion and carbaryl in the 2.0 a 30.0×10^?6 mol L^?1 concentration range. The detection limits under the optimum working conditions were found to be 9.3 μg L^?1 for parathion and 9.0 μg L^?1 for carbaryl. The enzyme electrode was found to be stable for 7 days.     

Electrochemical preparation of a novel type of C-dots/ZrO2 nanocomposite onto glassy carbon electrode for detection of organophosphorus pesticide

A selective, sensitive novel electrochemical sensor for detection of methyl parathion on the preparation of a carbon dots (C-dots)/ZrO₂ nanocomposite was developed. The C-dots/ZrO₂ nanocomposite was fabricated using electrochemical deposition onto a glassy carbon electrode and characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy and cyclic voltammetry. The optimum parameters such as effect of pH, accumulation time, accumulation potential, scan rate, effect of amount of C-dots and effect of amount of ZrO₂ were investigated. The C-dots/ZrO₂ modified glassy carbon electrode allowed rapid, selective determination of methyl parathion in rice samples by adsorptive stripping voltammetry. The stripping response was highly linear over the methyl parathion concentrations ranging from 0.2 ng mL^?1 to 48 ng mL^?1, with a detection limit of 0.056 ng mL^?1. This novel electrochemical nanocomposite-based electrochemical sensor was successfully applied for the detection of methyl parathion in rice samples.     

Electrochemical determination of methyl parathion at a Pd/MWCNTs-modified electrode

Palladium nanoparticles supported on MWCNTs (Pd/MWCNTs) were successfully prepared by a simple ethylene glycol reduction method in an oil bath. An electrochemical sensor based on Pd/MWCNTs nanocomposite-modified glassy carbon electrode was fabricated for the determination of methyl parathion by differential pulse voltammetry measurement. A highly linear response to methyl parathion in the concentration ranging from 0.10 μg mL^?1 to 14 μg mL^?1 was observed, and a detection limit of 0.05 μg mL^?1 was obtained with the calculation based on signal/noise?=?3. The present work provides a simple and rapid approach to the detection of methyl parathion.     

Cholinesterase-based dipstick assay for the detection of organophosphate and carbamate pesticides

A cholinesterase (ChE)-based dipstick-type assay for the class-specific detection of organophosphate (OP) and carbamate (CM) pesticides was developed. The principle of the assay is based on inhibition of the activity of a ChE by these two families of pesticides, which is dependent on the concentration of pesticides. The proposed assay system is composed of a test strip with an acetylcholinesterase (AChE)-coated membrane and an enzyme substrate solution. The assay protocol involves incubation of the enzyme-coated strip in the pesticide-containing sample solution followed by incubation of the sample-treated strip in a chromogenic enzyme substrate solution. The color intensity is estimated by the naked eye or a reflectometer. Of the membranes tested as the enzyme support, Hybond N⁺ was the most suitable. Among the compounds tested as the enzyme substrate, indophenyl acetate was the best. The detectable concentration range of the dipstick assay for the OP and CM pesticides was 10⁻⁶-10² and 10⁻⁶-10⁰ μg mL⁻¹, respectively. The sensitivity of the dipstick assay to the oxidized form of parathion (paraoxon) was higher than to parathion. The strip showed a large matrix effect with pesticide-spiked lettuce samples, whereas it showed a small matrix effect with pesticide-spiked rice samples.     

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.     

Nanocomposites composed of layered molybdenum disulfide and graphene for highly sensitive amperometric determination of methyl parathion

The authors describe an inexpensive electrode for the sensitive amperometric determination of the pesticide methyl parathion. A glassy carbon electrode was modified with a nanocomposite consisting of molybdenum disulfide nanosheets (MoS2) and graphene that was prepared via a hydrothermal process. Its morphology, elemental composition, diffraction, impedance and voltammetric characteristics were studied. The modified electrode displays excellent electrocatalytic ability towards methyl parathion, and the reduction peak current, measured typically at ?0.60 V (vs. Ag/AgCl) is related to the concentration of methyl parathion. The effect of concentration, scan rate and solution pH value were optimized. The calibration plot is linear in the 10 nM to 1.9 mM concentration range, with a 3.2 nM detection limit (at a signal-to-noise ratio of 3). The electrode is selective, stable, adequately repeatable and reproducible. The method was successfully applied to the determination of methyl parathion in spiked samples of homogenized apple, kiwi, tomato and cabbage.

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Graphical Abstract A reliable and robust methyl parathion sensor has been developed using heterostructured MoS₂/graphene. The linear range is 10 nM–1.9 nM and detection limit is 3.2 (±0.8) nM. The method was successful in real sample determination of spiked methyl parathion in food samples such as apple, kiwi, tomato and cabbage.

     

Electrochemical deposition of silicate–cetyltrimethylammonium bromide nanocomposite film on glassy carbon electrode for sensing of methyl parathion

A novel electrochemical sensor for methyl parathion based on silicate– cetyltrimethylammonium bromide nanocomposite film has been fabricated by electro-assisted deposition onto glassy carbon electrode in one-step via an electrochemical modulation of pH at the electrode/solution interface to promote controlled gelification of tetraethylorthosilicate sol, and was characterized with scanning electron microscopy, X-ray diffraction, and electrochemical impedance spectroscopy. The electrochemical sensing of methyl parathion on the film-modified electrode was investigated applying cyclic voltammetry and square wave voltammetry. Compared to the unmodified electrode, the shapes of the redox peaks were improved and the peak currents significantly increased. Experimental parameters such as deposition time, pH value, and accumulation conditions have been optimized. A linear relationship between the peak current and methyl parathion concentration was obtained in the range from 1.0 × 10⁻⁷ to 1.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 1.04 × 10 ⁻⁸ mol L⁻¹ (S/N = 3) after accumulation at 0 V for 120 s. The film electrode shows great promise for determination of methyl parathion in real samples.