Amperometric determination of organophosphate pesticides using a acetylcholinesterase based biosensor made from nitrogen-doped porous carbon deposited on a boron-doped diamond electrode

Microchimica Acta - The authors describe an amperometric biosensor for the determination of organophosphate pesticides (OPs) via inhibition of the enzyme acetylcholinesterase (AChE). The enzyme was...     

Ionic liquid-functionalized graphene for fabricating an amperometric acetylcholinesterase biosensor

This work reports a sensitive amperometric biosensor for organophosphate pesticides (OPs) fabricated by modifying a glassy carbon electrode with acetylcholinesterase (AChE) immobilized on ionic liquid-functionalized graphene (IL-G). The functionalized graphene sheets had good dispersibility and long-term stability in various solvents. The as-prepared biosensor showed high affinity to acetylthiocholine (ATCl) with a Michaelis-Menten constant (K(m)) value of 0.77 mM. Furthermore, based on the inhibition by OPs of the enzymatic activity of the immobilized AChE, and using carbaryl as a model compound, the inhibition of carbaryl was proportional to its concentration ranging from 0.0025 to 0.48 and 0.48 to 1.42 μg mL(-1) with a detection limit of 0.8 ng mL(-1) (S/N = 3). The developed biosensor exhibited a good performance for OPs detection, including good reproducibility and acceptable stability, which provided a new and promising tool for the analysis of enzyme inhibitors.     

Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents

A screen-printed biosensor for the detection of pesticides in water miscible organic solvents is described based on the use of p-aminophenyl acetate as acetylcholinesterase substrate. The oxidation of p-aminophenol, product of the enzymatic reaction was monitored at 100 mV vs. Ag/AgCl screen-printed reference electrode. Miscible organic solvents as ethanol and acetonitrile were tested. The acetylcholinesterase (AChE) was immobilised on a screen-printed electrode surface by entrapment in a PVA-SbQ polymer and the catalytic activity of immobilised AChE was studied in the presence of different percentages of organic solvents in buffer solution. The sensor shows good characteristics when experiments were performed in concentrations of organic solvents below 10%. No significant differences were observed when working with 1 and 5% acetonitrile in the reaction media. Detection limits as low as 1.91x10(-8) M paraoxon and 1.24x10(-9) M chlorpyrifos ethyl oxon were obtained when experiments are carried out in 5% acetonitrile.     

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。     

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.     

Acetylcholine Biosensor Based on Dendrimer Layers for Pesticides Detection

We tested a new design of an enzyme biosensor based on acetylcholinesterase (AChE) and choline oxidase (ChO) immobilized on the supported monomolecular layer composed of poly(amidoamine) (PAMAM) dendrimers of the fourth generation (G4) mixed with 1‐hexadecanethiol (HDT). The resulting enzymatic activity, measured amperometrically, was substantially depressed in the presence of the organophosphate pesticide dimethyl‐2,2‐dichlorovinylphosphate (DDVP, Dichlorvos), carbamate pesticides carbofuran and carbamate drug eserine. The detection limits (1.3×10^?3 ppb for DDVP, 0.01 ppb for carbofuran and 0.03 for eserine) were considerably lower than so far reported for AChE based amperometric and potentiometric sensors. The relative simple protocol of biosensor preparation, high sensitivity and stability is very promising for determination of environmental pollutants in field conditions.     

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.     

AChE biosensor based on zinc oxide sol-gel for the detection of pesticides

Zinc oxide has been used as a matrix for immobilization of acetylcholinesterase (AChE) and detection of the pesticide paraoxon. The immobilized enzyme retained its enzymatic activity up to three months when stored in phosphate buffered saline (pH 7.4) at 4 °C. An amperometric biosensor for the detection of paraoxon was designed. The biosensor detected paraoxon in the range 0.035-1.38 ppm and can be used to detect other AChE inhibiting organophosphate pesticides.     

A rational design of the multiwalled carbon nanotube–7,7,8,8-tetracyanoquinodimethan sensor for sensitive detection of acetylcholinesterase inhibitors

A new, simple and effective amperometric acetylcholinesterase biosensor was developed using screen-printed carbon electrodes modified with carbon nanotubes (MWCNTs)–7,7,8,8-tetracyanoquinodimethane (TCNQ). The design of the biosensor was based on the supramolecular arrangement resulted from the interaction of MWCNTs and TCNQ. This arrangement was confirmed by spectroscopic and electrochemical techniques. Two different supramolecular arrangements were proposed based on different MWCNTs:TCNQ ratios. The synergistic effect of MWCNTs and TCNQ was, for the first time, exploited for detection of thiocholine at low potential with high sensitivity. The biosensor developed by immobilization of acetylcholinesterase (AChE) in sol–gel allowed the detection of two reference AChE inhibitors, paraoxon-methyl and chlorpyrifos with detection limits of 30 pM (7 ppt) and 0.4 nM (0.1 ppb), respectively. Efficient enzyme reactivation was obtained by using obidoxime.     

Adsorption: an easy and efficient immobilisation of acetylcholinesterase on screen-printed electrodes

An amperometric biosensor based on acetylcholinesterase was constructed by simple adsorption of the enzyme on screen-printed electrodes (SPEs). This sensor was used to detect the inhibitory effects of organophosphorus and carbamate insecticides on acetylcholinesterase, and more particularly of chlorpyrifos ethyl oxon (CP-o). We demonstrate that enzyme adsorption on SPEs allows to obtain stable sensors that present good characteristics and are as efficient as other screen-printed biosensors based on covalent binding or entrapment of acetylcholinesterase (AChE).     

Nerve Agents Assay Using Cholinesterase Based Biosensor

Electrochemical biosensor based on electric eel acetylcholinesterase (AChE) (EC 3.1.1.7) was performed for assay of nerve agents – tabun, sarin, soman, cyclosarin, and VX. The biosensor used AChE as biorecognition element. The presence of nerve agents was accompanied by a strong inhibition of AChE activity. Enzyme activity is easily measurable by electrochemical oxidation of thiocholine created from acetylthiocholine (ATChCl) by AChE‐catalyzed hydrolysis. The tested nerve agents were successfully assayed. The best limits of detection were achieved for sarin (5.88×10^?10 M) and VX (8.51×10^?10 M) after one‐minute assay. The biosensor was found long term stable at low as well as laboratory temperature.     

A mediator-free screen-printed amperometric biosensor for screening of organophosphorus pesticides with flow-injection analysis (FIA) system

A mediator-free amperometric biosensor for screening organophosphorus pesticides (OPs) in flow-injection analysis (FIA) system based on anticholinesterase activity of OPs to immobilized acetylcholinesterase enzyme (AChE) has been developed. The enzyme biosensor is prepared by entrapping AChE in Al₂O₃ sol-gel matrix screen-printed on an integrated 3-electrode plastic chip. This strategy is found not only increase the stability of the embedded AChE, but also effectively catalyze the oxidative reaction of thiocholine, making the Al₂O₃-AChE biosensor detects the substrate at 0.25 V (versus Ag/AgCl), hundreds mini-volt lower than other reported mediator-free ones. The Al₂O₃-AChE biosensor is thus coupled to FIA system to build up a simple and low-cost FIA-EC system for screening OPs in real samples. A wide linear inhibition response for dichlorvos, typical OP, is observed in the range of 0.1-80 μM, corresponding to 7.91-84.94% inhibition for AChE. The detection limit for dichlorvos is achieved at 10 nM in the simulated seawater for 15 min inhibiting time, which allows the biosensor quantitatively detects the ecotoxicological effect of the real samples from the seaports in eastern China, where the OPs pollution is confirmed by GC-MS.     

AuNPs/Sol-gel复合膜法固定乙酰胆碱酯酶生物传感器检测有机磷农药

基于有机磷农药对乙酰胆碱酯酶(Acetylcholinesterase,AChE)的抑制作用,用金纳米粒子(Au nanoparticles,AuNPs)与壳聚糖/SiO2杂化溶胶-凝胶构成复合固酶基质,将AChE固定于玻碳电极表面,制备了电流型AChE生物传感器选用久效磷进行实验,以氯化硫代乙酰胆碱为底物,建立了电化...     

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.     

An Electrochemical Acetylcholinesterase Biosensor Based on Fluorene(bisthiophene) Comprising Polymer for Paraoxon Detection

In this study, a novel conductive polymer comprising biosensor based on poly-2,2′-(9,9-dioctyl-9 h-fluorene-2,7-diyl)bistiophene (Poly(BT)) and acetylcholinesterase (AChE) was reported for the determination of paraoxon. This practical biosensor allowed to catalyze electrochemical oxidation of acetylthiocholine (+0.6 V vs. Ag reference). The detection range for acetylcholine chloride (AThCl) with Poly(BT)/AChE was found to be 0.025–4 mM. In pesticide analysis, wide linear ranges from 0.5 to 1 μg/L and 1 to 14 μg/L, and a low detection limit of 0.033 μg/L were estimated. Under optimum operating conditions, the developed biosensor was used for pesticide detection in milk and tap water samples, effectively.     

TiO2-decorated graphene nanohybrids for fabricating an amperometric acetylcholinesterase biosensor

This work describes a highly sensitive and rapid amperometric biosensor for organophosphate compounds (OPs) based on immobilization of acetylcholinesterase (AChE) on a novel TiO(2)-decorated graphene (TiO(2)-G) nanohybrid, which was constructed by in situ growth of TiO(2) nanoparticles (NPs) on the graphene sheet. The well-dispersed TiO(2) NPs eliminated the restacking of TiO(2)-G nanohybrids. Due to the integrating of TiO(2)-G nanohybrids, the as-prepared biosensor showed high affinity to acetylthiocholine (ATCl) with a Michaelis-Menten constant (K(m)) value of 0.22 mM, and rapid inhibition time (3 min). Further, based on the inhibition of OPs on the enzymatic activity of the immobilized AChE, and using carbaryl as a model compound, the inhibition of carbaryl was proportional to its concentration ranging from 0.001 to 0.015 and 0.015 to 2 μg mL(-1) with a detection limit of 0.3 ng mL(-1) (S/N = 3). The developed biosensor exhibited a good performance for organophosphate pesticide detection, including good reproducibility and acceptable stability, which provided a new and promising tool for the analysis of enzyme inhibitors.     

Acetylcholinesterase Sensor Based on Polyelectrolyte Complexes with DNA Inclusion for the Determination of Reversible Inhibitors

The acetylcholinesterase (AChE) biosensor has been developed for the determination of reversible inhibitors applied in the Alzheimer's disease therapy, i. e., Huperzine A (HupA) and galantamine (Gal). For this purpose, glassy carbon electrode (GCE) was first modified with carbon black (CB) and Co phthalocyanine and then polyelectrolyte complex was self‐assembled on its surface by drop casting of reactants and washing. To extend the stability and improve biosensor performance, it was proposed for the first time to use DNA as polyanion in the complex assembly. The DNA showed higher charge density than conventional polyelectrolytes and stabilized the surface coating by adsorption of higher enzyme amount and prevention of its leaching during the biosensor operation. Complex formation and the influence of structural factors were monitored with surface plasmon resonance. Kinetic study showed mixed inhibition of the enzyme within micro‐ and nanomolar range of inhibitor concentrations. The AChE biosensor showed limit of detection of HupA equal to 0.9 and that of Gal to 70 nM. The sensitivity of drug determination was found to be close or better than that of the AChE biosensors previously reported in the literature. The biosensor was tested on the sample of artificial urine and showed 102 % recovery of the drugs determination.     

Spectrophotomeric Assay of Aflatoxin B1 Using Acetylcholinesterase Immobilized on Standard Microplates

Aflatoxins are a group of hepatotoxic secondary metabolites produced by molds Aspergillus. During inappropriate storage or processing of food and beverages, aflatoxins can be distributed and become a serious health problem. Disparate analytical techniques were prepared for the assay in the past. Here, a method based on inhibition of enzyme acetylcholinesterase (AChE) is performed allowing fast and simple prove of aflatoxins presence. For the assay purposes, AChE was immobilized on standard polystyrene microplates using gelatin as a stabilizing substance. Both free and immobilized AChEs were used and compared one to each other. Immobilization protocol was optimized and interference of organic solvents such as methanol was tested. For the immobilized AChE, limit of detection 2.75 ppb for aflatoxin B1 was received. The immobilized AChE kept full activity at least one month. Suitability of the assay for a practical performance is considered.     

Sensor System Based on Acetylcholinesterase in Homogenous Phase for Analysis of Paraoxon

Abstract

An amperometric sensor system based on acetylcholinesterase (AChE) was used for a paraoxon assay. Paraoxon-inhibited AChE resulted in the decrease of acetylthiocholine hydrolysis and the further oxidation of thiocholine on a platinum electrode. Thus described, the system allows for the detection of 1.2 ppb of paraoxon responding to absolute 12 pg. The sensor system functionality was verified on spiked beverages. The practical impact of system was summarized and future implementation is expected.     

Acetylcholinesterase immobilised eggshell membrane-based optical biosensor for organophosphate detection

The development of an optical biosensor for the determination of malathion based on acetylcholinesterase (AChE) inhibition using Ellman’s reagent is reported. The AChE has been immobilised onto the eggshell membrane (ESM) using glutaraldehyde as a cross-linking agent. Scanning Electron Microscopic (SEM) studies and Fourier Transformed Infra-Red (FTIR) characterisations have been carried out to affirm the successful immobilisation of AChE onto the ESM. Under optimum conditions, the developed biosensor estimated the pesticide concentration in the range of 0.1–50 ng/mL with a limit of detection (LOD) of 0.1 ng/mL within 30 min. Parameters affecting the biosensor response such as concentration of enzyme, substrate and inhibition time were optimised. The stability and reusability of the AChE/ESM sensor have been observed as 31 days at 4°C and two times, respectively.