Stabilization of the Closed-Ring Isomer of Spiropyran by Amide Naphthotube in Water and Its Application in Naked-Eye Detection of Toxic Paraoxon

The thermodynamically unstable, colourless closed-ring isomer of spiropyran can be stabilized in water by the anti-configurational isomer of amide naphthotube. The influence of the binding on the thermodynamics and kinetics of spiropyran have been studied. The complex was further used to prepare a test paper that allows naked-eye detection of toxic paraoxon.     

Molecular dynamics simulations of the detoxification of paraoxon catalyzed by phosphotriesterase

Combined QM(PM3)/MM molecular dynamics simulations together with QM(DFT)/MM optimizations for key configurations have been performed to elucidate the enzymatic catalysis mechanism on the detoxification of paraoxon by phosphotriesterase (PTE). In the simulations, the PM3 parameters for the phosphorous atom were reoptimized. The equilibrated configuration of the enzyme/substrate complex showed that paraoxon can strongly bind to the more solvent‐exposed metal ion Zn_β, but the free energy profile along the binding path demonstrated that the binding is thermodynamically unfavorable. This explains why the crystal structures of PTE with substrate analogues often exhibit long distances between the phosphoral oxygen and Zn_β. The subsequent S_N2 reaction plays the key role in the whole process, but controversies exist over the identity of the nucleophilic species, which could be either a hydroxide ion terminally coordinated to Zn_α or the μ‐hydroxo bridge between the α‐ and β‐metals. Our simulations supported the latter and showed that the rate‐limiting step is the distortion of the bound paraoxon to approach the bridging hydroxide. After this preparation step, the bridging hydroxide ion attacks the phosphorous center and replaces the diethyl phosphate with a low barrier. Thus, a plausible way to engineer PTE with enhanced catalytic activity is to stabilize the deformed paraoxon. Conformational analyses indicate that Trp131 is the closest residue to the phosphoryl oxygen, and mutations to Arg or Gln or even Lys, which can shorten the hydrogen bond distance with the phosphoryl oxygen, could potentially lead to a mutant with enhanced activity for the detoxification of organophosphates. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

基于SERS与PCA-SLR实现乙基对氧磷定量检测

利用表面增强拉曼光谱(SERS),结合主成分分析(PCA)与分段线性回归(SLR)算法实现乙基对氧磷的定量检测。首先采集820～1 630 cm-1乙基对氧磷溶液SERS,并对820～1 630 cm-1(全范围)与845～875 cm-1(特征范围)光谱分别进行标准正态变换(SNV)、多元散射校正(MSC)、一阶导数绝对值、二阶导数等预处理;然后经PCA降维后利用SLR建立乙基对氧磷溶液浓度预测模型。通过对比不同模型的预测准确度,发现特征范围光谱采用MSC预处理后所建立的模型为最优,总体预测均方误差值(RMSEP)为0.33,满足乙基对氧磷定量检测的需要。     

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.     

A label-free electrochemical immunosensor based on gold nanoparticles for detection of paraoxon

An electrochemical immunosensor for the direct determination of paraoxon has been developed based on the biocomposites of gold nanoparticles loaded with paraoxon antibodies. The biocomposites are immobilized on the glassy carbon electrode (GCE) using Nafion membrane. On the immunosensor prepared paraoxon shows well-shaped CV with reduction and oxidation peaks located −0.08 and −0.03 mV versus SCE, respectively. The detection of paraoxon performed at −0.03 mV is beneficial for guaranteeing sufficient selectivity. The amount of the biocomposite consisting gold nanoparticles loaded with antibodies and the volume of Nafion solution used for fabricating the immunosensor have been studied to ensure sensitivity and conductivity of the immunosensor. The immunosensor has been employed for monitoring the concentrations of paraoxon in aqueous samples up to 1920 μg l⁻¹ with a detection limit of 12 μg l⁻¹.     

基于Fe_3O_4-Au磁性纳米粒子及碳纳米管修饰电极的对氧磷生物传感器研究

采用化学共沉淀技术制备磁性Fe3O4-Au纳米粒子复合物(Fe3O4-AuNPs),并以此磁性纳米复合物和碳纳米管(CNTs)构建用于快速检测对氧磷的乙酰胆碱酯酶(AChE)生物传感器。通过磁力作用将Fe3O4-AuNPs纳米粒子固定在自制的磁铁/玻碳电极(MGCE)上,并以此作为AChE的载体。分别通过X射线衍射、振动样品磁强和透射电镜表征了磁性纳米粒子复合物Fe3O4-AuNPs的成分、磁性及其形貌特征。利用电化学交流阻抗(EIS)、循环伏安法和微分脉冲伏安法(DPV)表征了自制的MGCE修饰电极以及生物传感器(AChE/Fe3O4-AuNPs/CNTs/MGCE)的电化学特征,建立了用该生物传感器微分脉冲伏安法检测对氧磷的方法。在最佳实验条件下,酶抑制率与对氧磷浓度的对数在3.6×10-6~2.9×10-2mol/L范围内呈线性关系,检出限为1.6×10-7mol/L。用提出的方法对实际水样中的对氧磷进行加标回收实验,回收率为98.0%~107%。     

Biophysical aspects of cyclodextrin interaction with paraoxon

Cyclodextrins are torus‐shaped polymers of glucose that can bind organophosphorous compounds such as nerve agents and pesticides. We demonstrate here that cyclodextrin can bind up to two paraoxon molecules with a K_av of 6775 M^‐1. Molecular modeling shows that the paraoxon appears to bind in polar opposite orientation and have an average binding energy of ?89 Kcals/mol. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

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.     

Immobilization of Acetylcholinesterase onto Carbon Nanotubes Utilizing Streptavidin-biotin Interaction for the Construction of Amperometric Biosensors for Pesticides

Abstract

Carbon nanotubes (CNTs) are very promising materials onto which bioactive molecules can be immobilized in the construction of biosensors. Streptavidin was used as a molecular linker to immobilize biotinylated acetylcholinesterase (AChE) on CNTs in a gentle and controllable fashion for pesticide biosensors. Glassy carbon electrodes coated with the CNT-enzyme complex had high affinity for the substrate acetylthiocholine and produced strong peak oxidation currents in electrochemical assays. We also propose a new method, i.e., the use of relative net slope rather than the percentage of inhibition, in the calculation of pesticide concentrations. The biosensors could detect low levels of the pesticide methyl paraoxon.     

Preparation of Polyclonal Antibodies with Application for an Organophosphorus Pesticide Immunoassay

Haptens of dichlorvos and paraoxon were conjugated to the carrier proteins of bovine serum albumin. The obtained conjugates were characterized by infrared and ultraviolet–visible spectroscopy. The binding ratios of dichlorvos and paraoxon-to-carrier proteins were also evaluated. The number of hapten molecules per protein molecule of dichlorvos–cationized bovine serum albumin conjugate was higher than for paraoxon–bovine serum albumin conjugate. The sheep polyclonal antibodies were produced against the dichlorvos and paraoxon. New multipolyclonal antibodies were obtained and characterized following the immunization of a 1:1 mixture of the immunogens for the simultaneous determination of dichlorvos and paraoxon by the immunoassay. An indirect enzyme-linked immunosorbent assay was used to characterize the reactivity of the antibodies to hapten conjugates. The multiantibodies showed lower affinities than the separate antibodies, but their affinities were sufficient for an immunoassay for the simultaneous determination of the analytes. The detection limit and linear range for the determination of dichlorvos and paraoxon alone and together were determined. The recovery was characterized to determine dichlorvos and paraoxon fortified in model solutions and milk. These results demonstrate the potential of this immunoassay for the quantitative screening of dichlorvos and paraoxon.