An optical fiber biosensor for chlorpyrifos using a single sol-gel film containing acetylcholinesterase and bromothymol blue

An optical fiber biosensor consisting of acetylcholinesterase (AChE) and bromothymol blue (BTB) doped sol-gel film was employed to detect organophosphate pesticide chlorpyrifos. The main advantage of this optical biosensor is the use of a single sol-gel film with immobilized AChE and BTB. The compatibility of this mixture (AChE and BTB) with the sol-gel matrix has prevented leaching of the film. The immobilization of the enzyme and indicator was simple without chemical modification. The biosensing element on single sol-gel film has been placed inside the flow-cell for flow system. In the presence of a constant AChE, a color change of the BTB and the measured reflected signal at wavelength 622nm could be related to the pesticide concentration in the sample solutions. The performance of optical biosensor in the flow system has been optimized, including chemical and physical parameters. The response time of the biosensor is 8min. A linear calibration curve of chlorpyrifos against the percentage inhibition of AChE was obtained from 0.05 to 2.0mg/L of chlorpyrifos (18-80% inhibition, R(2)=0.9869, n=6). The detection limit for chlorpyrifos was 0.04mg/L. The results of the analysis of 0.5-1.5mg/L of chlorpyrifos using this optical biosensor agreed well with chromatographic method.     

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。     

An amperometric biosensor based on acetylcholinesterase immobilized onto iron oxide nanoparticles/multi-walled carbon nanotubes modified gold electrode for measurement of organophosphorus insecticides

An acetylcholinesterase (AChE) purified from maize seedlings was immobilized covalently onto iron oxide nanoparticles (Fe₃O₄NP) and carboxylated multi walled carbon nanotubes (c-MWCNT) modified Au electrode. An organophosphorus (OP) biosensor was fabricated using this AChE/Fe₃O₄/c-MWCNT/Au electrode as a working electrode, Ag/AgCl as standard and Pt wire as an auxiliary electrode connected through a potentiostat. The biosensor was based on inhibition of AChE by OP compounds/insecticides. The properties of nanoparticles modified electrodes were studied by scanning electron microscopy (SEM), Fourier transform infrared (FTIR), cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS). The synergistic action of Fe₃O₄NP and c-MWCNT showed excellent electrocatalytic activity at low potential (+0.4 V). The optimum working conditions for the sensor were pH 7.5, 35 °C, 600 μM substrate concentration and 10 min for inhibition by pesticide. Under optimum conditions, the inhibition rates of OP pesticides were proportional to their concentrations in the range of 0.1–40 nM, 0.1–50 nM, 1–50 nM and 10–100 nM for malathion, chlorpyrifos, monocrotophos and endosulfan respectively. The detection limits were 0.1 nM for malathion and chlorpyrifos, 1 nM for monocrotophos and 10 nM for endosulfan. The biosensor exhibited good sensitivity (0.475 mA μM⁻¹), reusability (more than 50 times) and stability (2 months). The sensor was suitable for trace detection of OP pesticide residues in milk and water.     

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.     

基于乙酰胆碱酯酶生物传感平台的西维因检测

为给农药西维因检测提供一种新方法,根据西维因抑制乙酰胆碱酯酶活性的原理,以黑珍珠2000（BP2000）为乙酰胆碱酯酶的固定化材料,采用滴凃电极法构建了基于乙酰胆碱酯酶的西维因生物传感平台. 结果表明,固定在BP2000 上的乙酰胆碱酯酶保持了对氯化乙酰胆碱的催化活性,并且由于BP2000 材料的引入,提升了电极有效的电化学活性表面积,而且电极上物质的电化学氧化拥有较低氧化电位（0.630 V）并伴随质子传输. 由BP2000 搭建成功的乙酰胆碱酯酶生物传感平台对西维因检测的线性响应范围为2.0 ng·mL^-1 ~ 12.5 ng·mL^-1,检测限为3.15 ng·mL^-1. 本研究对酶生物传感平台和酶生物燃料电池体系中酶电极的构建提供了一种简单方法及高效载体.     

基于功能化石墨烯-聚乙烯醇复合膜固定乙酰胆碱酯酶的甲拌磷传感器

将功能化离子液体修饰石墨烯(IL-GR)分散在聚乙烯醇(PVA)中,制得IL-GR-PVA分散液,与乙酰胆碱酯酶(ACh E)溶液混匀后滴涂在电极表面,利用PVA良好的成膜特性,制得新型有机磷检测酶电极ACh E/IL-GR-PVA/GCE,并用于有机磷农药的检测。采用透射电镜(TEM)表征了IL-GR的形貌,采用循环伏安法(CV)和差示脉冲伏安法(DPV)研究了酶电极的电化学性质。结果表明,IL-GR-PVA复合膜具有良好的导电性和生物相容性,能很好地保持ACh E的生物活性,并显著促进了其电化学过程。在优化实验条件下,抑制率(I%)与甲拌磷浓度的负对数在1.0×10-14~1.0×10-9mol/L和1.0×10-9~1.0×10-6mol/L范围内呈良好的线性关系,检出限(S/N=3)为8.0×10-15mol/L。该传感器制备简单,稳定性好,灵敏度高,为有机磷农药的测定提供了新方法。     

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.     

Highly selective NIR fluorescent probe for acetylcholinesterase and its application in pesticide residues detection

A series of near-infrared (NIR) fluorescent substrates (NDRO-1～8) derived from fluorophore NDRH with different volumes of ester bond as the recognition group were designed and synthesized for the detection of acetylcholinesterase (AChE), among which NDRO-1 with the smallest acetate group displayed the highest activity toward AChE. The detection limit of NDRO-1 for sensing AChE was 0.32 µg/mL, and K_m was 6.40 µmol/L, indicating ultra-sensitivity and good affinity of NDRO-1 toward AChE. NDRO-1 was used to detect the inhibitory of four kinds of pesticides including methamidophos, dichlorvos, and the detection limit was lower than 50 µg/L, which was further used in pesticide residues detection.     

Assisted inhibition effect of acetylcholinesterase with n-octylphosphonic acid and application in high sensitive detection of organophosphorous pesticides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

A simple and practical approach to improve the sensitivity of acetylcholinesterase (AChE)-inhibited method has been developed for monitoring organophosphorous (OP) pesticide residues. In this work, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to detect AChE activity. Due to its good salt-tolerance and low sample consumption, MALDI-FTMS facilitates rapid and high-throughput screening of OP pesticides. Here we describe a new method to obtain low detection limits via employing external reagents. Among candidate compounds, n-octylphosphonic acid (n-Octyl-PA) displays assistant effect to enhance AChE inhibition by OP pesticides. In presence of n-Octyl-PA, the percentages of AChE inhibition still kept correlation with OP pesticide concentrations. The detection limits were improved significantly even by 10²–10³ folds in comparison with conventional enzyme-inhibited methods. Different detection limits of OP pesticides with different toxicities were as low as 0.005 μg L⁻¹ for high toxic pesticides and 0.05 μg L⁻¹ for low toxic pesticides. Besides, the reliability of results from this method to analyze cowpea samples had been demonstrated by liquid-chromatography tandem mass spectrometry (LC–MS/MS). The application of this commercial available assistant agent shows great promise to detect OP compounds in complicated biological matrix and broadens the mind for high sensitivity detection of OP pesticide residues in agricultural products.     

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.     

纳米SnSe2空心球上乙酰胆碱酯酶的固定及辛硫磷传感器的构建

描述了一种将乙酰胆碱酯酶固定在SnSe2空心球上检测辛硫磷的简单方法,用水热法合成了SnSe2空心球,并用透射电镜对其表征.固定的乙酰胆碱酯酶能保持其生物学活性,催化乙酰胆碱为胆碱,胆碱被氧化产生可检测的信号.基于辛硫磷对乙酰胆碱酯酶活性有抑制作用这个机理,在理想条件下,这种传感器对辛硫磷检测的线性范围是0.008～56μg/mL,检测限为0.004μg/mL.这种新型的传感器有很好的稳定性和重现性.这项工作表明SnSe2空心球可以作为固定乙酰胆碱酯酶的理想载体并用于构建相应的传感器.     

Fabricating of Acetylcholine Biosensor by a Layer‐by‐layer Deposition Technique for Determining Trichlorfon

An acetylcholine (ACh) biosensor has been fabricated with bienzymes/poly(diallyldimethylammonium chloride) (PDDA) multilayer film‐modified platinum (Pt) electrodes by a layer‐by‐layer technique (LBL). The ACh biosensor was optimized and the properties are described. This ACh biosensor was used for the detection of organophosphate pesticide trichlorfon. The detection limits (found 0.001 μg/mL for trichlorfon) make it possible to detect the pollutants. This simple protocol of biosensor preparation, high sensitivity and stability are very promising for the determination of environmental pollutants in field conditions.     

Electrochemical Biosensor for Organophosphate Pesticides and Huperzine‐A Detection Based on Pd Wormlike Nanochains/Graphitic Carbon Nitride Nanocomposites and Acetylcholinesterase

A new electrochemical biosensor was developed for organophosphorus pesticides (OPs) and huperzine‐A (hupA) detection based on Pd wormlike nanochains/graphitic carbon nitride (Pd WLNCs/g‐C₃N₄) nanocomposites and acetylcholinesterase (AChE). The morphologies and components of the nanocomposites were analyzed by transmission electron microscopy (TEM) and X‐ray photoelectron spectroscopy (XPS). The Pd WLNCs/g‐C₃N₄ nanocomposites could effectively immobilize enzymes and promote the signal amplification. Under the optimum condition, the proposed biosensor displayed well performance. The linear response ranges for the determination of OPs and hupA were 1.00 nM to 14.96 µM and 3.89 nM to 20.80 µM, respectively. The corresponding detection limits were 0.33 nM and 1.30 nM (S/N=3). Meanwhile, the biosensor owned good reproducibility and stability, and could also be applied to analyze practical samples, which would be a new hopeful method for pesticide analysis.     

基于金属纳米粒子增敏的SPR生物传感器检测吲哚乙酸

本实验建立了表面等离子体共振(SPR)生物传感器检测3-吲哚乙酸(IAA)的方法。制备了两种SPR生物传感器检测IAA:传统模式的SPR生物传感器1和Au/Ag合金纳米粒子增敏的SPR生物传感器2。结果发现:传感器1在IAA浓度范围为175~350μg/L时,浓度与其波数位移值呈线性关系,检出限为25μg/L(S/N=3);传感器2在IAA浓度范围为17.5~250μg/L时,浓度与其波数位移值呈线性关系,检出限为2.2μg/L(S/N=3)。说明基于Au/Ag合金纳米粒子的传感器2比传感器1有较高的灵敏度和较低的检出限。加标回收实验测得加标回收率范围为96%~100.2%,平均值为98.4%。本实验制备的SPR生物传感器具有较好的精密度、稳定性、重现性和特异性。     

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.     

Acetylcholinesterase Sensor with Patterned Structure for Detecting Organophosphorus Pesticides Based on Titanium Dioxide Sol‐gel Carrier

In this paper, novel and stable acetylcholinesterase (AChE) sensor with patterned structure for detecting organophosphorus pesticides (OPs) based on titanium dioxide sol‐gel carrier was proposed and prepared. Biosensor was assembled by dropping titanium oxide, chitosan (CS) and enzyme to the surface of the glass carbon step by step. The concentration range of the sensor detection for dichlorvos (DDVP) is 1.13 nM to 22.6 μM, and the limit of detection (LOD) is 0.23 nM. It can also detect dichlorvos in cabbage juice samples accurately. The preparation of biosensor adopted a patterned novel structure for the first time, which opens a new way for the structure optimization of organophosphorus pesticide sensor.     

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.     

Disposable screen-printed electrode coupled with recombinant Drosophila melanogaster acetylcholinesterase and multiwalled carbon nanotubes for rapid detection of pesticides

Recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE), multiwalled carbon nanotubes (MWCNTs), and Prussian blue have been combined for development of a three-electrode biosensor with more rapid responses and higher stability than in our previous study. A new disposable screen-printed electrode (SPE) was developed for rapid detection of organophosphate and carbamate pesticides. After optimization, 10 microg MWCNT and 5 microL enzyme immobilization solution consisting of 0.2% glutaraldehyde, 0.1% Nafion, 0.2% bovine serum albumin, 0.1 g/L MWCNT, and 1.5 mU R-DmAChE were fixed on each of the R-DmAChE/MWCNT SPEs. The LOD of this biosensor was 0.5 microg/L for pesticide standards of dichlorvos (DDV) and carbofuran. The performance of this biosensor was tested for vegetable and water samples at various spiked levels, and good stability and sensitivity were found. The obtained recoveries were from 82.6 to 110.5% for DDV at levels of 0.5-5 microg/L and 73.4 to 118.4% for carbofuran at 1-10 microg/L in lake and sea water samples, demonstrating that the proposed approach is an alternative means for rapid detection of pesticide residues and contaminants in food safety and environmental monitoring.     

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.     

Au掺杂Fe3O4纳米粒子酶传感器的制备及其应用于有机磷农药检测的研究

合成了金掺杂的四氧化三铁纳米粒子(Au-Fe₃O₄), 以壳聚糖为交联剂, 制备了电流型乙酰胆碱酯酶(AChE)生物传感器, 并将其应用于有机磷农药(OPs)的检测. 实验表明, Au-Fe₃O₄纳米粒子具有良好的生物兼容性, 能够有效地促进电子传递, 修饰了Au-Fe₃O₄纳米粒子的酶传感器, 响应速度快, 检测灵敏度高, 稳定性好; 固定在传感器上的乙酰胆碱酯酶有良好的酶动力学响应, 其表观米氏常数( )为10.3 mmol/L. 利用有机磷农药对乙酰胆碱酯酶的抑制作用, 以硫代乙酰胆碱(ATCh)为底物, 对有机磷农药敌敌畏进行了检测, 检测限达到4.0×10^－13 mol/L.