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...     

基于氧化锌纳米棒的乙酰胆碱酯酶生物传感器用于辛硫磷农药测定

以喷金的聚碳酸酯模板为工作电极,采用电沉积法从氯化锌和氯化钾溶液中制得氧化锌纳米棒.将沉积了氧化锌纳米棒的模板固定在打磨后的玻碳电极表面,并将模板溶解.再通过在氧化锌纳米棒修饰电极的表面直接固定乙酰胆碱酯酶,制备出乙酰胆碱酯酶生物传感器.自然晾干后,所得乙酰胆碱酯争氧化锌生物传感器用于辛硫磷农药的测定.试验结果表明:在含有0.5 mmol·L-1的巯基乙酰胆碱的PH 7.38磷酸盐缓冲溶液中,乙酰胆碱酯酶-氧化锌修饰电极的氧化峰电流显著提高,而再向其中加入酶抑制剂辛硫磷后,电流明显减小,在此基础上提出了一种高灵敏度的测定辛硫磷农药的方法.在优化的条件下,辛硫磷浓度在9.85 × 10-6～4.95×10-4mol·L-1之间,其浓度的对数与抑制率呈线性关系,检出限(3S/N)为5.99×10-6mol·L-1.     

Kinetic-Turbidimetric Determination of Stachyose Based on Its Inhibitory Action on Sucrose Crystallization.

Abstract

The effects of 9 saccharides on the crystallization of sucrose have been comparatively studied with analytical purposes. A sensitive (detection limit 0.1 μg mL^?1) and simple kinetic turbidimetric procedure is used for determination of stachyose, based on its inhibitory action on the crystallization of sucrose. The supersaturated solutions of sucrose were prepared by addition of acetone to stable water sucrose solutions. The induction period of the crystallization process was turbidimetrically measured. Controlled constant stirring and temperature are convenient in order to obtain reproducible results. The method was applied to the determination of stachyose in synthetic mixtures.     

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.     

Study of Inhibition,Reactivation and Aging Processes of Pesticides Using Graphene Nanosheets/Gold Nanoparticles‐Based Acetylcholinesterase Biosensor

Organophosphate (OP) and carbamate pesticides exert their toxicity via attacking the hydroxyl moiety of serine in the ‘active site’ of acetylcholinesterase (AChE). In this paper we developed a stable AChE biosensor based on self‐assembling AChE to graphene nanosheet (GN)‐gold nanoparticles (AuNPs) nanocomposite electrode for investigation of inhibition, reactivation and aging processes of different pesticides. It is confirmed that pesticides can inhibit AChE in a short time. OPs poisoning is treatable with oximes while carbarmates exposure is insensitive to oximes. The proposed electrochemical approach thus provides a new simple tool for comparison of pesticide sensitivity and guide of therapeutic intervention.     

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.     

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.     

Determination of pesticides in vegetable samples using an acetylcholinesterase biosensor based on nanoparticles ZrO2/chitosan composite film

An amperometric pesticides inhibition biosensor has been developed and used for determination of pesticides in vegetable samples. To eliminate the interference of ascorbic acid, multilayer films of polyelectrolyte (chitosan/polystyrensulfonate) were coated on the glass carbon electrode. Then, acetylcholinesterase was immobilized on the electrode based on surface-treated nanoporous ZrO₂/chitosan composite film as immobilization matrix. As a modified substrate, acetylthiocholine was hydrolysed by acetylcholinesterase and produced thiocholine which can be oxidized at +700?mV vs. SCE. Pesticides inhibit the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to acetylthiocholine within 9.90?×?10^?6 to 2.03?×?10^?3?M. The electrode provided a linear response over a concentration range of 6.6?×?10^?6 to 4.4?×?10^?4?M for phoxim with a detection limit of 1.3?×?10^?6?M, over a range of 1.0?×?10^?8 to 5.9?×?10^?7?M for malathion, and over a range of 8.6?×?10^?6 to 5.2?×?10^?4?M for dimethoate. This biosensor has been used to determine pesticides in a real vegetable sample.     

基于聚多巴胺纳米粒子的荧光增强型探针检测乙酰胆碱酶

以多巴胺盐酸盐为原料,在碱性条件下通过氧化反应制备聚多巴胺荧光纳米粒子(F-PDA),再与二氧化锰(MnO_2)纳米片进行复合,构建了用于检测乙酰胆碱酶(AChE)的F-PDA@MnO_2复合物荧光探针。MnO_2纳米片和F-PDA复合,体系的荧光被猝灭。在底物乙酰硫代胆碱(ATCh)存在下,加入AChE后,体系荧光恢复,恢复程度与AChE浓度在5.0～100 mU/mL范围内呈良好的线性关系,检出限为0.14 mU/mL(S/N=3)。该方法用于缓冲溶液中AChE的检测,加标回收率为89.5%～120%,相对标准偏差为1.6%～2.5%,且具有较高的选择性。可为基于F-PDA传感体系构建提供新的方法学模型。     

Simple and Sensitive Electrochemiluminescence Method for Determination of Bisphenol a Based on its Inhibitory Effect on the Electrochemiluminescence of Luminol

An electrochemiluminescence inhibition method has been developed for the determination of bisphenol A based on studying the inhibition phenomena of bisphenol A to the electrochemiluminescence of luminol. There is a linear relationship between the inhibitive intensity of electrochemiluminescence and the concentration of bisphenol A in the range of 4.4 × 10^?7 to 2.2 × 10^?5 mol/l. The method was successfully applied to the determination of bisphenol A in hot water in contact with commercially available table‐water bottle samples.     

亚临界水萃取-酶抑制法快速检测蔬菜中的灭多威农药

本文研究了蔬菜中灭多威农药的亚临界水萃取-酶抑制法快速检测技术.研究表明,在萃取温度70℃,萃取时间5 min,提取液的pH为8.0,提取液体积为5 mL的条件下,灭多威有最高萃取效率.将本法分别应用于测定模拟样品和实际样品,结果表明亚临界水萃取的萃取效率和精密度都要高于国家标准方法中的手摇振荡萃取.     

基于固定化酶的乙酰胆碱酯酶抑制剂体外筛选模型的建立

以可重复使用的固定化酶代替游离态酶, 建立一种基于比色分析的乙酰胆碱酯酶(AChE)抑制剂体外筛选新模型. 采用以氨基化硅胶为载体固定的AChE优化了实验条件, 用AChE抑制剂阳性对照物他克林和毒扁豆碱对该模型进行验证, 还对模型技术参数进行评价, 并将新模型用于单体化合物及天然产物粗提物AChE抑制活性评价. 结果表明, 最佳实验条件为: 固定化酶用量55 μL, 底物浓度5 mmol/L, 甲醇、 乙醇及体积分数不高于6%的二甲基亚砜水溶液均可作为样品溶剂; 模型验证及模型技术参数评价结果良好, 该模型对AChE抑制剂筛选有较好的特异性和灵敏度, 可用于筛选AChE抑制剂. 该模型具有适用性强、 固定化酶可重复使用及结果可靠等优点, 是单体化合物及天然产物粗提物AChE抑制剂活性评价的有效方法.     

Superficially Bound Acetylcholinesterase Based on a Chitosan Matrix for Neurotoxic Compound Assay by a Photographic Technique

Smartphones have become popular in the last decade and they have been used in analytical chemistry as easy detection systems with comparable parameters to standard laboratory equipment. This work focuses on the attachment of enzyme acetylcholinesterase on the previously activated chitosan. Acetylcholine splits the neurotransmitter acetylcholine, as a natural substrate, into choline and acetic acid. However, this compound can cleave alternative substrates, e.g., indoxyl acetate, which was used in this work. The conversion of the substrate is blocked or slowed down by inhibitors from which galantamine and tacrine were tested as model inhibitors with detection limits of 1.1 and 0.18?µM, respectively. The measurement procedure was performed on a three-dimensional printed holder and red–green–blue channels were used for digital photography evaluation. The method was validated using a standard Ellman’s spectrophotometric assay. We successfully attached acetylcholinesterase on the chitosan surface that was used for the inhibitor assay. The long-term stability of the immobilized enzyme as well as the sensitivity to organic solvents were also tested. The proposed method appeared to be suitable for the rapid and inexpensive assay of neurotoxic compounds.     

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.     

便携式检测仪检测呋喃丹和对氧磷的研究

基于胆碱酯酶被抑制前后反应速率不同的原理,研制了便携式荧光探针农药残留检测仪,并对检测条件进行了优化,获得呋喃丹和对氧磷的检出限分别为3．0和12μg／L,线性范围分别为3．0—37．8μg／L和12-350μg／L,该检测仪具有操作简便,检测怏速,可用于现场检测等优点,在对蔬菜和水果样品检测中,获得呋喃丹的回收率在87％～114％之间。     

新型2H-噻唑[3,2-b][1,2,4]三嗪-3,7-二酮的超声辅助一锅法合成及乙酰胆碱酯酶抑制活性研究

以取代苯甲醛、溴乙腈和6-苄基-3-硫代-3,4-二氢-1,2,4-三嗪-5(2H)-酮为起始原料,在超声辅助下,一锅合成了9个新型6-苄基-2-亚苄基-2H-噻唑[3,2-b][1,2,4]三嗪-3,7-二酮类化合物,结构经_¹H NMR,_¹³C NMR和MS（ESI）表征并对其合成机理进行了初步研究。测试了目标化合物(4a~4i)的体外乙酰胆碱酯酶的抑制活性,其中4a和4d抑制活性最好,有进一步研究的价值。     

Acetylcholinesterase Based Dipsticks with Indoxylacetate as a Substrate for Assay of Organophosphates and Carbamates

Enzyme acetylcholinesterase (AChE) is an important part of cholinergic neurotransmission. It is targeted by many toxins such as nerve agents, organophosphates, and carbamate pesticides. Several drugs for treatment of Alzheimer's disease and Myasthenia gravis are also AChE inhibitors. The inhibitory effect can be used for assay purposes. The presented experiment is devoted to the construction of a colorimetric dipstick with immobilized AChE and using indoxylacetate as a chromogenic substrate. Standard qualitative cellulose filter papers, high-performance TLC plates, cotton gauze, and parafilm were chosen as matrices for the testing. The constructed dipsticks were created for assays of paraoxon as a model organophosphate pesticide and neostigmine as a model carbamate. The assessed limit of detection was 10^?7 mol/l for both inhibitors. It responds absolutely by detecting 4 pmol of inhibitor when a sample volume of 40 µL is considered. Long term stability and optimization of immobilization were also done and practical importance is discussed. The prepared dipsticks were also used for assays of paraoxon spiked tap and rain water. The suitability of the dipsticks for practical performance was approved. Intensive color changes from white to blue are suitable for scoring by a naked eye.     

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.     

阻抑茜素红S褪色动力学光度法测定痕量汞

在HAc—NaAc介质中,以邻菲啰啉作活化剂,痕量Cr（Ⅵ）对H2O2氧化茜素红S褪色反应存在强烈的催化作用,而Hg（Ⅱ）对此褪色反应具有明显的阻抑作用,由此建立了测定痕量Hg（Ⅱ）的新阻抑动力学光度法。该方法线性范围为0～1．6μg／mL,检出限为2．6×10^-8g／mL,应用于污水中Hg（Ⅱ）的测定,结果满意。     

Kinetic Spectrophotometric Determination of Vanadium (V) Based on its Inhibitory Effect on the Thionine – Ascorbic Acid Reaction

ABSTRACT

A simple and sensitive kinetic method for the determination of vanadium(V) based on its inhibitory effect on the reduction of thionine by ascorbic acid at pH=5 is described. The reaction rate is monitored spectrophotometrically by measuring the decrease in absorbance of thionine at 598 nm after a fixed time (10 min). The calibration graph is linear in the range of 10 ? 120 ng ml^?1 of vanadium(V) and the detection limit is 6 ng ml^?1. The relative standard deviation (RSD) for 80 ng ml^?1 of V(V) was 0.96% (n=10). The method was successfully applied to the determination of vanadium in a certified reference sample.