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1.
Acetylcholinesterase (AChE), an enzyme that degrades the neurotransmitter acetylcholine, when covalently inhibited by organophosphorus compounds (OPs), such as nerve agents and pesticides, can be reactivated by oximes. However, tabun remains among the most dangerous nerve agents due to the low reactivation efficacy of standard pyridinium aldoxime antidotes. Therefore, finding an optimal reactivator for prophylaxis against tabun toxicity and for post-exposure treatment is a continued challenge. In this study, we analyzed the reactivation potency of 111 novel nucleophilic oximes mostly synthesized using the CuAAC triazole ligation between alkyne and azide building blocks. We identified several oximes with significantly improved in vitro reactivating potential for tabun-inhibited human AChE, and in vivo antidotal efficacies in tabun-exposed mice. Our findings offer a significantly improved platform for further development of antidotes and scavengers directed against tabun and related phosphoramidate exposures, such as the Novichok compounds.  相似文献   

2.
将对氧磷(Paraoxon)与肟类重活化剂双复磷(Obidoxime)反应制得膦酰化肟(DEP-obidoxime);利用基于效应标志物的质谱定量技术研究了膦酰化肟对乙酰胆碱酯酶(AChE)的抑制作用及中毒酶的重活化特性.结果表明,膦酰化肟具有极强的AChE抑制毒性,但因膦酰化肟中毒酶与原有机磷毒物中毒酶结构相同,故其膦酰化AChE仍可被经典重活化剂[如氯解磷定(Pralidoxime)、双复磷(Obidoxime)及酰胺磷定(HI-6)]重活化,根据EC_(50)表征结果,这3种重活化剂的重活化效果强弱顺序依次为氯解磷定HI-6双复磷.  相似文献   

3.
Organophosphorus (OP) nerve agents and pesticides present significant threats to civilian and military populations. OP compounds include the nefarious G and V chemical nerve agents, but more commonly, civilians are exposed to less toxic OP pesticides, resulting in the same negative toxicological effects and thousands of deaths on an annual basis. After decades of research, no new therapeutics have been realized since the mid-1900s. Upon phosphylation of the catalytic serine residue, a process known as inhibition, there is an accumulation of acetylcholine (ACh) in the brain synapses and neuromuscular junctions, leading to a cholinergic crisis and eventually death. Oxime nucleophiles can reactivate select OP-inhibited acetylcholinesterase (AChE). Yet, the fields of reactivation of AChE and butyrylcholinesterase encounter additional challenges as broad-spectrum reactivation of either enzyme is difficult. Additional problems include the ability to cross the blood brain barrier (BBB) and to provide therapy in the central nervous system. Yet another complication arises in a competitive reaction, known as aging, whereby OP-inhibited AChE is converted to an inactive form, which until very recently, had been impossible to reverse to an active, functional form. Evaluations of uncharged oximes and other neutral nucleophiles have been made. Non-oxime reactivators, such as aromatic general bases and Mannich bases, have been developed. The issue of aging, which generates an anionic phosphylated serine residue, has been historically recalcitrant to recovery by any therapeutic approach—that is, until earlier this year. Mannich bases not only serve as reactivators of OP-inhibited AChE, but this class of compounds can also recover activity from the aged form of AChE, a process referred to as resurrection. This review covers the modern efforts to address all of these issues and notes the complexities of therapeutic development along these different lines of research.  相似文献   

4.
Here we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix‐assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI‐FTMS) for screening irreversible AChE inhibitors. Due to its good salt‐tolerance and low sample consumption, MALDI‐FTMS could facilitate rapid detection, especially detection in real application. AChE activity was determined through calculating abundance of substrate and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) concentrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibitions still kept good correlation with concentration of OPs. Finally, this AChE‐inhibited method was applied to screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of decedents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following detecting in GC‐MS/MS, the possible death reason of these three decedents was confirmed, and another decedent actually died for sumicidin, a non‐AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by detecting enzyme activity in MALDI‐FTMS provided fast and accurate analysis results and excluded another toxicants not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.  相似文献   

8.
《Electroanalysis》2006,18(22):2163-2167
Poly (N‐vinyl‐2‐pyrrolidone) (PVP)‐capped CdS quantum dots (QCdS‐PVP) was synthesized with CdCl2 and Na2S in the presence of PVP. QCdS‐PVP has been used for the immobilization and stabilization of the acetylcholinesterase (AChE). The electrocatalytic activity of QCdS‐PVP leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product, and higher sensitivity and stability. The GCE/QCdS‐PVP/AChE biosensor was used for the detection of organophosphate pesticides (OPs), such as trichlorfon. The sensor performance, including pH and inhibition time, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 12 ppb trichlorfon with a 5‐min inhibition time.  相似文献   

9.
《Electroanalysis》2003,15(14):1185-1191
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.  相似文献   

10.
《Electroanalysis》2017,29(2):339-344
In the electrochemical detection method for pesticides that measures their inhibitory effects on acetylcholinesterases (AChEs), the direct electrooxidation of the enzyme product (thiocholine, SCh) is slow at conventional electrodes. To overcome this limitation, an electron mediator is required to lower the applied potential and facilitate the transfer of electrons between the enzyme product and electrode. In this study, [Ru(NH3)5py]3+ is introduced as an electron mediator in inhibition‐based pesticide detection. To obtain a better signal‐to‐background ratio, [Ru(NH3)5py]3+, which undergoes a fast outer‐sphere reaction, is combined with low‐electrocatalytic indium‐tin‐oxide (ITO) electrodes at which many interfering species undergo slow redox reactions. AChE is immobilized onto an avidin‐modified ITO electrode via the direct adsorption of avidin onto ITO followed by the biospecific binding of biotinylated AChE to the avidin. SCh is generated from acetylthiocholine by AChE. Subsequently, SCh converts [Ru(NH3)5py]3+ to [Ru(NH3)5py]2+, which is then oxidized at the ITO electrode. This procedure allows the sensitive detection of carbaryl at a low applied potential of 0.15 V vs Ag/AgCl. The calculated detection limit for carbaryl is approximately 0.3 pM. This simple and sensitive pesticide sensor is thus very promising and should be extendable to the onsite environmental monitoring of other pesticides.  相似文献   

11.
A nanoparticle-based electrochemical immunosensor has been developed for the detection of phosphorylated acetylcholinesterase (AChE), which is a potential biomarker of exposure to organophosphate (OP) pesticides and chemical warfare nerve agents. Zirconia nanoparticles (ZrO(2) NPs) were used as selective sorbents to capture the phosphorylated AChE adduct, and quantum dots (ZnS@CdS, QDs) were used as tags to label monoclonal anti-AChE antibody to quantify the immunorecognition events. The sandwich-like immunoreactions were performed among the ZrO(2) NPs, which were pre-coated on a screen printed electrode (SPE) by electrodeposition, phosphorylated AChE and QD-anti-AChE. The captured QD tags were determined on the SPE by electrochemical stripping analysis of its metallic component (cadmium) after an acid-dissolution step. Paraoxon was used as the model OP insecticide to prepare the phosphorylated AChE adducts to demonstrate proof of principle for the sensor. The phosphorylated AChE adduct was characterized by Fourier transform infrared spectroscopy (FTIR) and mass spectroscopy. The binding affinity of anti-AChE to the phosphorylated AChE was validated with an enzyme-linked immunosorbent assay. The parameters (e.g., amount of ZrO(2) NP, QD-anti-AChE concentration,) that govern the electrochemical response of immunosensors were optimized. The voltammetric response of the immunosensor is highly linear over the range of 10 pM to 4 nM phosphorylated AChE, and the limit of detection is estimated to be 8.0 pM. The immunosensor also successfully detected phosphorylated AChE in human plasma. This new nanoparticle-based electrochemical immunosensor provides an opportunity to develop field-deployable, sensitive, and quantitative biosensors for monitoring exposure to a variety of OP pesticides and nerve agents.  相似文献   

12.
用于农药残留快速检测的两种酶的比较   总被引:12,自引:0,他引:12  
张慧君  许学勤  徐斐  华泽钊 《分析化学》2004,32(11):1517-1520
以商品乙酰胆碱酯酶(C3389,Ⅵ-S,从电鳗中提取)和自制鸡肝酶为农药检测用酶,比较了不同浓度的敌敌畏、敌百虫、马拉硫磷和西维因对这两种酶的乙酰胆碱酯酶活力和总酯酶活力的抑制情况。结果表明,鸡肝酶的乙酰胆碱酯酶活力较低,而其总酯酶活力对4种农药的灵敏度与商品乙酰胆碱酯酶活力的灵敏度相近,且总酯酶活力对除马拉硫磷外的其余3种农药的检出限都较乙酰胆碱酯酶低。同时对总酯酶活力测定的pH进行了优化,发现pH6.5(40mmol/L柠檬酸盐缓冲液)时总酯酶活力较高。  相似文献   

13.
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.  相似文献   

14.
《Analytical letters》2012,45(9):1419-1429
A new and simple method has been developed to allow the direct determination of pesticides in samples of vegetable oils via pH transduction. It is based on the inhibitory effect of the pesticides on the enzyme acetylcholinesterase (AChE). The strategy involves the use of a biphasic system composed of an organic phase (the oil) containing the pesticide (carbofuran) and an aqueous phase containing the AChE. The pesticide is extracted from the oil phase at the interface of the two phases which causes the inhibition of AChE and formation of acetic acid that is monitored with a pH meter. The degree of inhibition observed in the Tris-HCl solution is in the same order of magnitude as the one observed in the 2-phase system. This approach is sensitive, accurate, and does not require tedious extraction and sample clean-up. It is, therefore, well suited for rapid screening.  相似文献   

15.
A spectrophotometric method for simultaneous analysis of methamidophos and fenitrothion was proposed by application of chemometrics to the spectral kinetic data, which was based upon the difference in the inhibitory effect of the two pesticides on acetylcholinesterase (AChE) and the use of 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) as a chromogenic reagent for the thiocholine iodide (TChI) released from the acetylthiocholine iodide (ATChI) substrate. The absorbance of the chromogenic product was measured at 412 nm. The different experimental conditions affecting the development and stability of the chromogenic product were carefully studied and optimized. Linear calibration graphs were obtained in the concentration range of 0.5–7.5 ng·mL?1 and 5–75 ng·mL?1 for methamidophos and fenitrothion, respectively. Synthetic mixtures of the two pesticides were analysed, and the data obtained processed by chemometrics, such as partial least square (PLS), principal component regression (PCR), back propagation‐artificial neural network (BP‐ANN), radial basis function‐artificial neural network (RBF‐ANN) and principal component‐radial basis function‐artificial neural network (PC‐RBF‐ANN). The results show that the RBF‐ANN gives the lowest prediction errors of the five chemometric methods. Following the validation of the proposed method, it was applied to the determination of the pesticides in several commercial fruit and vegetable samples; and the standard addition method yielded satisfactory recoveries.  相似文献   

16.
Two-enzyme systems based on acetylcholinesterase (AChE) - a mono-enzyme system based on AChE, with p-aminophenyl acetate as substrate, and a bi-enzyme system based on AChE and tyrosinase, with phenyl acetate as substrate - have been studied for detection of organophosphate insecticides. The analytical performance and detection limits for determination of the pesticides were compared for the two AChE configurations. The enzyme loading, pH, and applied potential of the bi-enzyme system were optimised. When phenyl acetate was used as substrate for AChE activity the phenol generated by enzymatic hydrolysis was determined with a second enzyme, tyrosinase. Amperometric measurements were performed at 100 mV and -150 mV relative to the Ag/AgCl reference electrode for the mono-enzyme and bi-enzyme systems. Screen-printed sensors were used to detect the organophosphorus pesticides paraoxon and chlorpyrifos ethyl oxon; the detection limits achieved with phenyl acetate as substrate were 5.2x10(-3) mg L(-1) and 0.56x10(-3) mg L(-1), respectively.  相似文献   

17.
A highly sensitive sensor for determination of carbamate pesticides based rhodamine B (RB) modified silver nanoparticle (RB-AgNPs) was developed. Compared with the classical method, it combined colorimetric with fluorescence for detecting carbamate pesticides in complex solutions. Carbamate pesticides can inhibit the activity of acetylcholinesterase (AChE), thus preventing the generation of thiocholine. On the other hand, thioncholine can transform the yellow RB-AgNPs solutions gray color and unquenches the fluorescence of RB simultaneously. Once the activity of AChE was inhibited by the pesticide, the color of the RB-AgNPs solution remains yellow and the fluorescence of RB molecules remains quenched. Under optimized experimental conditions, carbaryl was detected in a concentration range from 0.1 ng/L to 8.0 ng/L with a detection limit of 0.023 ng/L (it was detected by fluorescence spectra). This simple method is suitable for determination of carbamate pesticides in complex samples, such as tomato, apple and river water.  相似文献   

18.
Oxidation of oximes via photosensitized electron transfer (PET) results in the formation of the corresponding ketones as the major product via oxime radical cations and iminoxyl radicals. The influence of electron-releasing and electron-accepting substituents on these reactions was studied. The observed substituent effect strongly supports formation of iminoxyl radicals from the oximes via an electron transfer-proton transfer sequence rather than direct hydrogen atom abstraction. Correlation of the relative conversion of the oximes with Hammett parameters shows that radical effects dominate for the meta-substituted acetophenone oximes (rho(rad)/rho(pol) = 5.4; r2 = 0.93), whereas the para-substituted oximes are influenced almost equally by radical and ionic effects (rho(rad)/rho(pol) = -1.1; r2 = 0.98). From these data sets we conclude that the follow-up reactions proceed through a number of intermediates with both radical and ionic character. This was confirmed by product studies with the use of an isotopically labeled nucleophile. In addition to the major oxidation product (ketone), a chlorine-containing product was often identified as well. Studies on the formation of this product show that the most likely pathway is either via a direct nucleophilic addition in a complex formed between the oxime radical cation and the chloranil radical anion or via a radical substitution (SH2) mechanism. These studies show that with the increasing use of oximes as drugs and pesticides, intake of these chemicals followed by enzymatic oxidation may result in the formation of a variety of reactive intermediates, which may lead to cell and tissue damage.  相似文献   

19.
《Electroanalysis》2018,30(8):1801-1810
Mesoporous bimetallic PtPd nanoflowers (MPtPdN) were synthesized by a surfactant‐directing method. The MPtPdN was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and wide‐angle X‐ray diffraction (XRD). The use of MPtPdN as a platform for the indirect electrochemical detection of organophosphate pesticides (OPs) via enzymatic inhibition pathway was demonstrated. Due to the nanostructure of MPtPdN and the synergy effect of the noble metal nanoparticles, a novel and sensitive acetylcholinesterase (AChE) biosensor for OPs detections based on MPtPdN and enzyme inhibition was prepared. The inhibition of omethoate exhibited a linear relationship from 4.7×10−11 to 4.7×10−8 M with a detection limit of 1.7×10−12 M (S/N=3). The proposed AChE biosensor was reliable and can be used to measure the concentration of omethoate in different spiked samples with high accuracy and satisfactory recovery. The preparation of biosensors based on the MPtPdN can be further extended to construct many other important enzyme biosensors.  相似文献   

20.
《化学:亚洲杂志》2017,12(7):775-784
Two different acetylcholinesterase (AChE)‐capped mesoporous silica nanoparticles (MSNs), S1‐AChE and S2‐AChE , were prepared and characterized. MSNs were loaded with rhodamine B and the external surface was functionalized with either pyridostigmine derivative P1 (to yield solid S1 ) or neostigmine derivative P2 (to obtain S2 ). The final capped materials were obtained by coordinating grafted P1 or P2 with AChE′s active sites (to give S1‐AChE and S2‐AChE , respectively). Both materials were able to release rhodamine B in the presence of diisopropylfluorophosphate (DFP) or neostigmine in a concentration‐dependent manner via the competitive displacement of AChE through DFP and neostigmine coordination with the AChE‘s active sites. The responses of S1‐AChE and S2‐AChE were also tested with other enzyme inhibitors and substrates. These studies suggest that S1‐AChE nanoparticles can be used for the selective detection of nerve agent simulant DFP and paraoxon.  相似文献   

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