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1.
J Hazard Mater ; 402: 123830, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33254811

RESUMO

Accurate screening of organophosphorus and carbamates pesticides from the complex real sample is crucial for water quality analysis and food safety control. Herein, a simple, low-cost and accurate pesticides screening method based on a high-throughput optical array system assembled by multiple cholinesterases (ChE) and thiocholine (TCh) sensors is described. The detection mechanism is that the inhibition of ChE activity by pesticides reduces the TCh produced by the hydrolysis of butyryl/acetylthiocholine iodide, thus changing the fluorescence intensity of TCh sensor. The diverse response of ChEs to pesticides and different affinity of sensors to TCh ensure the high-throughput and distinguishable signal output, which allow the establishment of high discrete pesticide database with intra-cluster agglomeration and inter-cluster dispersion. By using the database, the screening of unknown real contaminated samples were successfully operated, and the screened pesticide species and concentrations were consistent with high-performance liquid chromatography. This screening strategy demonstrates the feasibility of replacing existing complex mass spectrometry-based screening strategy with simple optical analysis, providing a new idea for the development of simple accurate screening technologies for widespread organic pollutants including pesticides.


Assuntos
Praguicidas , Tiocolina , Carbamatos , Colinesterases , Cromatografia Líquida de Alta Pressão , Praguicidas/análise
2.
J Mater Chem B ; 8(30): 6459-6468, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32597916

RESUMO

A self-templated strategy was adopted to design hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxides via the Mo or W doping of ZIF-67, and subsequent pyrolysis under an atmosphere of air at a low temperature of 450 °C. The hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxides displayed tunable oxidase-like and peroxidase-like activities able to efficiently catalyze the oxidation of TMB to generate a deep blue color in the absence or presence of H2O2. Relative to that of the un-doped Co3O4, the oxidase mimic activity of the Mo-doped Co3O4 increased to 1.3 to 2.1-fold, while its peroxidase mimic activity increased to 7.1 to 19.9-fold, depending on different Mo doping amounts. The oxidase mimic activity of the W-doped Co3O4 increased to 2.1 to 2.3-fold, while its peroxidase mimic activity increased to 4.8 to 5.9-fold, depending on the different W doping amounts. The Mo- and W-doped Co3O4 nanohybrid exhibited both higher O2 and H2O2 activating capability, and their H2O2 activating capacity was superior to the O2 activating capability. Furthermore, the Mo- and W-doped Co3O4 nanohybrids exhibited similar O2 activating abilities, while the Mo-doped one displayed a higher H2O2 activating capability than the W-doped one. The discrepant peroxidase-like nature of Mo- and W-doped Co3O4 nanohybrids is likely attributed to their different catalytic mechanisms. The peroxidase-like activity of Mo-doped Co3O4 is highly related to the ˙OH free radical, while that of W-doped Co3O4 is likely ascribed to the electron transfer between TMB and H2O2. The Km values of Co3O4/MoO3 for TMB and H2O2 were 0.0352 mM and 0.134 mM, which were 3.2- and 1.9-fold lower than that of pure Co3O4, respectively. A Co3O4/MoO3-based colorimetric platform was developed for the determination of H2O2 in the 0.1-200 µM range, with a limit of detection of 0.08 µM (3σ). Based on the thiocholine (TCh) inhibition of the excellent peroxidase-like activity of Co3O4/MoO3 and the TCh generation via acetylcholinesterase (AChE) catalyzed hydrolysis of acetylthiocholine chloride (ATCh), the colorimetric platform was extended to screen AChE activity and its inhibitor.


Assuntos
Acetilcolinesterase/metabolismo , Materiais Biomiméticos/química , Inibidores da Colinesterase/química , Cobalto/química , Peróxido de Hidrogênio/análise , Nanopartículas Metálicas/química , Molibdênio/química , Óxidos/química , Tiocolina/análise , Técnicas Biossensoriais , Catálise , Colorimetria , Temperatura Alta , Limite de Detecção , Oxirredutases/metabolismo , Tiocolina/metabolismo
3.
BMC Neurosci ; 20(1): 32, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286881

RESUMO

BACKGROUND: Three-dimensional (3D) reconstruction of human peripheral nerves, as a useful tool to understand the nerve internal information and functional basis, has become an important area of research in the peripheral nerve field. METHODS: In this study, we proposed a two-dimensional (2D) Karnovsky-Roots toluidine blue ponceau 2R (K-B-2R) staining method based upon conventional Karnovsky-Roots staining. It significantly improved the ability to display nerve fascicles, motor and sensory nerve fiber textures. In this method, Karnovsky-Roots staining was carried out, followed by toluidine blue counterstain and ponceau 2R counterstain. RESULTS: Comparisons were conducted between the three methods in staining of median nerve sections, which showed similar distribution characters in acetylcholinesterase-positive sites. The additional counterstaining did not change the basis of Karnovsky-Roots staining. However, the resulting images from this new method significantly facilitated the subsequent 3D nerve reconstruction and 3D printing. CONCLUSIONS: These results show that the new staining method significantly enhanced the display qualities of nerve fascicle edges and fiber textures of motor and sensory nerves and facilitated 3D nerve reconstruction.


Assuntos
Imageamento Tridimensional/métodos , Nervos Periféricos/anatomia & histologia , Coloração e Rotulagem/métodos , Acetilcolinesterase/metabolismo , Compostos Azo , Humanos , Indicadores e Reagentes , Impressão Tridimensional , Tiocolina , Cloreto de Tolônio
4.
Mikrochim Acta ; 186(6): 390, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31152243

RESUMO

A novel and highly sensitive enzyme inhibition assay was developed for the rapid detection of the organophosphate pesticide dichlorvos and the carbamate pesticide carbofuran. It achieves signal amplification by the secondary catalysis of platinum nanoparticles. Acetylcholinesterase (AChE) is capable of catalyzing the hydrolysis of acetylthiocholine to form thiocholine. Thiocholine causes the aggregation of citrate-capped platinum nanoparticles which then lose their peroxidase-mimicking properties. After addition of pesticides, the activity of AChE is inhibited, less thiocholine is produced, less aggregation occurs, and the peroxidase-mimetic properties are increasingly retained. In the presence of tetramethylbenzidine and H2O2, a deep blue coloration with an absorption maximum at 650 nm will be formed. The assay was applied to the determination of dichlorvos and carbofuran, and detection limits of 2.3 µg·L-1 and 1.4 µg·L-1 were obtained, respectively. Recovery experiments with spiked tap water and pears gave satisfactory relative standard deviations. Graphical abstract The blue product formed by platinum nanoparticle-catalyzed oxidation of 3,3'5,5'-tetramethylbenzidine (TMB) by H2O2 is reduced if acetylthiocholine (ATCh) is hydrolyzed by acetylcholinesterase (AChE) to form thiocholine. However, if AChE is inhibited by pesticides, color formation will recover.


Assuntos
Carbofurano/análise , Colorimetria/métodos , Diclorvós/análise , Nanopartículas Metálicas/química , Praguicidas/análise , Acetilcolinesterase/química , Acetiltiocolina/química , Benzidinas/química , Materiais Biomiméticos/química , Inibidores da Colinesterase/análise , Água Potável/análise , Peróxido de Hidrogênio/química , Limite de Detecção , Peroxidase/química , Platina/química , Tiocolina/química , Poluentes Químicos da Água/análise
5.
Int J Biol Macromol ; 137: 895-903, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31247229

RESUMO

Use of immobilized acetylcholine esterase (AChE) for detecting organophosphorus pesticides in water sources and body fluids can bring down the detection costs dramatically. In the present study, AChE was directly doped on multiwalled carbon nanotube (MWCNT) surface modified with carboxylic groups through amide bond and used for organophosphorus pesticide detection. Amide bond formation between MWCNTs and the enzyme molecules avoid use of any intermediate membranes, cross-linkers or binding materials. This strategy overcomes the hindrance to electron transfer posed by membranes or cross-linkers and increases the sensitivity of detection. MWCNTs carrying carboxyl groups were deposited on glassy carbon electrode and were subsequently immobilized with AChE. The activity of AChE was monitored by cyclic voltammetry after immobilization. Scanning electron microscopy and atomic force microscopy were used to characterize the electrode surface. FT-IR spectra were taken to characterize enzyme-MWCNT complex. Under optimized parameters, the electrode showed linear range between 10 and 50 nM, which is promising for detection of trace amounts of the pesticide. The lower and higher detection limits of the sensor are 0.1 nM and 500 nM respectively. The stability and reusability of the electrode were determined. Finally, successful detection of organophosphorus compounds in real samples established it as reliable for sensor applications.


Assuntos
Acetilcolinesterase/química , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Nanotubos de Carbono/química , Compostos Organofosforados/análise , Praguicidas/análise , Acetilcolinesterase/metabolismo , Animais , Técnicas Biossensoriais/instrumentação , Eletroquímica , Eletrodos , Electrophorus , Enzimas Imobilizadas/metabolismo , Vidro/química , Cinética , Limite de Detecção , Compostos Organofosforados/química , Oxirredução , Paraoxon/análise , Paraoxon/química , Praguicidas/química , Tiocolina/metabolismo
6.
Analyst ; 144(10): 3436-3441, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31020297

RESUMO

Organophosphorus pesticides (OPs) are widely used in agricultural fields, but exhibit high toxicity to human beings. A sensitive fluorescence assay for organophosphorus pesticides was developed using the inhibition of acetylcholinesterase (AChE) activity and the copper-catalyzed click chemical reaction. In the click reaction, two hybridized DNA probes can be ligated with copper ions, inducing a fluorescence quenching during the strand displacement reaction. AChE can hydrolyze acetylthiocholine (ATCh) to form thiocholine (TCh) which contains a thiol group. TCh will react with copper ions, blocking the click reaction and a high fluorescence signal is observed. But in the presence of OPs, the activity of AChE is inhibited, releasing a high concentration of copper ions that catalyze the click chemical reaction and resulting in decreased fluorescence signals. Taking advantage of the copper-mediated signal amplification effect, the sensitivity was improved. This assay has also been applied to detect OPs in river water samples with satisfactory results, which demonstrates that the method has great potential for practical applications in environmental protection and food safety fields.


Assuntos
Inibidores da Colinesterase/análise , Compostos Organofosforados/análise , Praguicidas/análise , Espectrometria de Fluorescência/métodos , Acetilcolinesterase/química , Acetiltiocolina/química , Catálise , Quelantes/química , Inibidores da Colinesterase/química , Química Click , Cobre/química , DNA/química , Sondas de DNA/química , Fluorescência , Corantes Fluorescentes/química , Limite de Detecção , Compostos Organofosforados/química , Praguicidas/química , Rios/química , Tiocolina/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química
7.
Anal Chim Acta ; 1060: 97-102, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-30902336

RESUMO

A portable and user-friendly method using personal glucose meters for on-site quantitative detection of organophosphorus pesticide (OP) was developed. The inhibition of organophosphorus compounds on acetylcholinesterase (AChE) leads to reduced yields of thiocholine formed by the enzymatic hydrolysis of acetylthiocholine chloride. Ferricyanide ([Fe(CN)6]3-), the mediator used in glucose test strips for electron transfer to the electrode, can be rapidly reduced to ferrocyanide ([Fe(CN)6]4-) by thiocholine. This reaction enables direct measurement of thiocholine by personal glucose meters in the same way as measuring the glucose in blood, offering an interesting choice to quantify OP. After incubation of AChE for 30 min and enzymatic reaction of 10 min, the yield of thiocholine was measured by a personal glucose meter, achieving detection limit of 5 µg L-1 for paraoxon. The proposed method was successfully applied to the detection in apples and cucumbers, presenting promising potential for on-site OP detection in food samples.


Assuntos
Técnicas Biossensoriais , Automonitorização da Glicemia , Compostos Organofosforados/análise , Praguicidas/análise , Tiocolina/química , Humanos
8.
Mikrochim Acta ; 185(10): 446, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-30187211

RESUMO

The authors describe a colorimetric method for the determination of the activity of acetylcholinesterase (AChE). Manganese dioxide (MnO2) nanosheets directly reacts with 3,3',5,5'-tetramethylbenzidine (TMB) in the absence of hydrogen peroxide (H2O2). This leads to the formation of a blue product (oxTMB) with an absorption peak at 652 nm. If AChE hydrolyzes its substrate acetylthiocholine chloride, thiocholine is formed which blocks the oxidative power of the MnO2 nanosheets. Hence, oxTMB will not be formed. The decreased absorbance is directly related to the AChE activity in the 0.01-1.0 mU·mL-1 range. The detection limit is 0.01 mU·mL-1 and the relative standard deviation is 1.2% (for n = 11 at 0.5 mU·mL-1). The method was also applied to screen for inhibitors of AChE. Graphical abstract Based on the oxidizing properties of manganese dioxide nanosheets (MnO2 nanosheets), we report a colorimetric method for determining acetylcholinesterase activity with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB).


Assuntos
Acetilcolinesterase/metabolismo , Benzidinas/química , Colorimetria/métodos , Ensaios Enzimáticos/métodos , Compostos de Manganês/química , Nanoestruturas/química , Óxidos/química , Tiocolina/farmacologia , Benzidinas/metabolismo , Inibidores da Colinesterase/farmacologia , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Compostos de Manganês/metabolismo , Oxirredução , Óxidos/metabolismo
9.
Talanta ; 178: 1017-1023, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29136790

RESUMO

A simple paper-based sensor with electrochemical detection was first developed for rapid, selective, and sensitive determination of acetylcholinesterase (AChE). The screen-printed graphene electrode was used as working electrode providing sensitivity for the sensor. The amperometric detection of AChE is based on the determination of thiocholine (TCh) produced from hydrolysis of acetylthiocholine chloride (ATCh) by AChE. To detect TCh, the ATCh immobilized sheet was stacked onto the detection sheet using double adhesive tape, then samples of AChE were dropped on the back side of an ATCh immobilized sheet with only 1min of incubation time. To avoid interference, glutathione (GSH), the potential of 0.5V vs. Ag/AgCl was applied onto a graphene electrode and the current, which depends on AChE concentration, was measured. Under optimized conditions, the limit of detection (LOD) from the experiment of AChE determination was 0.1U/mL with AChE concentration in range of 0.1-15U/mL. The data correlated well with the data obtained using spectrophotometric method. The developed sensor had been successfully applied to detect AChE in blood samples.


Assuntos
Acetilcolinesterase/metabolismo , Ensaios Enzimáticos/instrumentação , Grafite/química , Impressão , Acetiltiocolina/metabolismo , Animais , Eletroquímica , Eletrodos , Electrophorus , Concentração de Íons de Hidrogênio , Cinética , Tiocolina/metabolismo
10.
Biosens Bioelectron ; 96: 167-172, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28494368

RESUMO

The binding of organometallic osmium carbonyl clusters onto the surface of gold nanoparticles (10OsCO-Au NPs) greatly enhanced the CO stretching vibration signal at ~2100cm-1, which is relatively free from interference due to the absorbance of biomolecules. By utilizing the acetylcholinesterase (AChE) mediated hydrolysis of acetylthiocholine to thiocholine where the activity of AChE is inhibited by the presence of organophosphate pesticides (OPPs), the subsequent thiocholine-induced aggregation of 10OsCO-Au NPs can be monitored by the change in color of the NPs solution and the variation in intensity of the SERS CO signal. The change in color offers a fast pre-screening method, whereas monitoring via SERS is used for greater accuracy and lower limit of detection (0.1 ppb) for quantitative detection. Its potential as a quick and accurate method of OPPs monitoring in consumer products was demonstrated in the detection of OPPs in real spiked samples such as beer.


Assuntos
Análise de Alimentos/métodos , Ouro/química , Nanopartículas Metálicas/química , Compostos Organofosforados/análise , Praguicidas/análise , Ressonância de Plasmônio de Superfície/métodos , Acetilcolinesterase/química , Acetiltiocolina/química , Cerveja/análise , Glicina/análogos & derivados , Glicina/análise , Hidrólise , Nanopartículas Metálicas/ultraestrutura , Tiocolina/química
11.
Bull Environ Contam Toxicol ; 98(5): 662-671, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28299407

RESUMO

Acetylcholinesterase (AChE) enzyme has been predominantly used for the detection of pesticides and metal ions. But, these sensors respond to pesticides as well as metal ions at certain concentration, which results in poor selectivity. Hence in this work, the amount of thiocholine produced during AChE inhibition has been estimated to detect the residual activity of AChE enzyme in-turn to enhance the efficiency of the biosensor. In this context, Pt/ZnO-CeO2/AChE/Chitosan based biosensor has been developed for sensitive voltammetric quantification of thiocholine in AChE. The sensor exhibited enhanced electron transfer rate, good conductivity and biocompatibility. Both the intrinsic and extrinsic parameters were simultaneously optimized using second order polynomial regression to get the best conditions for ATCh determination. Under optimized experimental conditions, the redox peak current was linear over the concentration range of 0.1-1.5 mM with detection and quantification limit of 0.05 and 0.15 µM respectively and the sensitivity of 1.47 µA mM-1.


Assuntos
Técnicas Biossensoriais/instrumentação , Cério/química , Desenho de Equipamento , Nanoestruturas/química , Platina/química , Óxido de Zinco/química , Acetilcolinesterase/metabolismo , Acetiltiocolina/análise , Quitosana/química , Inibidores da Colinesterase/metabolismo , Eletrodos , Concentração de Íons de Hidrogênio , Limite de Detecção , Praguicidas/análise , Sensibilidade e Especificidade , Tiocolina/análise
12.
Food Chem ; 202: 81-7, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26920268

RESUMO

In this paper, a simple and sensitive fluorescent sensor for dichlorvos was first constructed based on carbon dots-Cu(II) system. These carbon dots were obtained by simple hydrothermal reaction of feather. The fluorescence of these carbon dots can be selectively quenched by Cu(2+) ion. When acetylcholinesterase and acetylthiocholine were introduced into the system, thiocholine came into being, which can react with Cu(2+) ion and restore the fluorescence of the system. The reaction mechanism between Cu(2+) ion and thiocholine was confirmed by X-ray photoelectron spectroscopy. As one kind of acetylcholinesterase inhibitor, organophosphorus pesticides can be detected based on this sensing system. As an example of organophosphorus pesticides, dichlorvos was detected with a linear range of 6.0×10(-9)-6.0×10(-8)M. This sensing system has been successfully used for the analysis of cabbage and fruit juice samples.


Assuntos
Diclorvós/análise , Praguicidas/análise , Espectroscopia Fotoeletrônica/métodos , Acetilcolinesterase/química , Acetiltiocolina/química , Brassica/química , Carbono/química , Inibidores da Colinesterase/análise , Cobre/química , Fluorescência , Análise de Alimentos , Contaminação de Alimentos/análise , Sucos de Frutas e Vegetais/análise , Sensibilidade e Especificidade , Tiocolina/química
13.
Bioorg Chem ; 65: 57-60, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26874343

RESUMO

Heavy atom kinetic isotope effects (KIEs) were determined for the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine (FTC). The leaving-S, carbonyl-C, and carbonyl-O KIEs are (34)k=0.994±0.004, (13)k=1.0148±0.0007, and (18)k=0.999±0.002, respectively. The observed KIEs support a mechanism for both acylation and deacylation where the steps up to and including the formation of the tetrahedral intermediate are at least partially rate determining. These results, in contrast to previous studies with acetylthiocholine, suggest that the decomposition of a tetrahedral intermediate is not rate-determining for FTC hydrolysis. Structural differences between the two substrates are likely responsible for the observed mechanism change with FTC.


Assuntos
Biocatálise , Butirilcolinesterase/metabolismo , Isótopos/metabolismo , Tiocolina/análogos & derivados , Humanos , Hidrólise , Isótopos/química , Cinética , Estrutura Molecular , Tiocolina/química , Tiocolina/metabolismo
14.
Rev. toxicol ; 33(1): 39-43, 2016. tab, graf
Artigo em Espanhol | IBECS | ID: ibc-153972

RESUMO

Los plaguicidas organofosforados (POF) poseen una acción anticolinesterásica utilizada como indicador de intoxicación crónica del trabajador agrícola por POF. En esta población, los POF pueden actuar como pro-oxidantes, afectando la actividad de las enzimas antioxidantes y probablemente generar daños crónicos. Objetivo. Determinar la influencia de la exposición ocupacional a POF sobre el daño oxidativo y actividad de acetilcolinesterasa en trabajadores agrícolas. Metodología. Se realizó un estudio observacional, analítico, retrospectivo y comparativo en 45 trabajadores del Comité de Sanidad Vegetal de Durango A.C. (CESAVEDAC). Se evaluó la actividad de acetilcolinesterasa lipoperoxidación y capacidad antioxidante total en plasma, además de evaluar las concentraciones de colesterol, triglicéridos, glucosa. Resultados. El daño oxidativo se asocia a la exposición a POF (p=0.003) siendo 3.21 veces mayor el riesgo de desarrollar daño oxidativo en el grupo expuesto a POF, además existe una asociación entre la inhibición de acetilcolinesterasa y la exposición a POF (p=0.01) siendo 2.92 veces mayor el riesgo de presentar dicha inhibición con respecto al grupo no expuesto. Conclusiones. Existe una influencia negativa de la exposición ocupacional a POF sobre el daño oxidativo y la actividad de acetilcolinesterasa en la población de trabajadores agrícolas estudiada (AU)


Organophosphate pesticides (OPs) are considered as negative risk factors for the health POF farm workers. The anticholinesterase action these compounds have been used as an indicator POF chronic OP poisoning. Some authors have suggested that agricultural workers in the Ops can act as pro-oxidants, affecting the activity POF antioxidant enzymes and generating chronic damage likely. Objective. This study was conducted to determine the influence POF occupational exposure to OPs in oxidative damage and acetylcholinesterase activity in farmworkers Plant Health Committee POF Durango A.C (CESAVEDAC). Methodology. An observational, analytical, retrospective comparative study was conducted on 45 workers. The activity POF acetylcholinesterase as a biomarker POF toxicity assessed, indicator POF oxidative damage to lipid peroxidation antioxidant capability. Results. Was found that there is an association between oxidative damage and exposure to OPs (p = 0.003) being 3.21 times greater risk POF developing oxidative damage when it is exposed to OPs, moreover, there is an association between inhibition POF acetylcholinesterase and exposure to OPs (p = 0.01), 2.92 times higher risk POF show inhibition activity acetylcholinesterase when it is exposed to OPs Conclusions. There is a negative influence POF occupational exposure to OPs on oxidative damage and acetylcholinesterase activity in farmworkers (AU)


Assuntos
Humanos , Masculino , Feminino , Exposição Ocupacional/análise , Exposição Ocupacional/prevenção & controle , Exposição Ocupacional/normas , Praguicidas/toxicidade , Acetilcolinesterase/toxicidade , Glucose/análise , Compostos Organofosforados/toxicidade , Inseticidas Organofosforados/efeitos adversos , Inseticidas Organofosforados/métodos , Inibidores da Colinesterase/toxicidade , Estudos Retrospectivos , Estudos Longitudinais , Tiocolina/toxicidade
15.
Anal Sci ; 31(7): 591-5, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26165279

RESUMO

A microdevice for coulometric detection of organophosphate pesticides (OPs) was fabricated based on the measurement of the inhibition of an enzyme, acetylcholinesterase (AChE), by OPs. Thiocholine (TCh) produced in the enzymatic reaction of AChE with acetylthiocholine (ATCh) as a substrate was oxidized on a microelectrode array formed in a main flow channel. Volumes of plugs of necessary solutions were measured using a structure consisting of a row of rhombuses formed in an auxiliary flow channel. The plugs were merged and solution components were mixed at a T-junction formed with the main and auxiliary flow channels. A linear relationship was confirmed between the generated charge and the logarithm of the OP (malathion) concentration in a concentration range between 10(-6) and 10(-3) M with a correlation coefficient of 0.951. The lower limit of detection was 412 nM.


Assuntos
Técnicas Biossensoriais/instrumentação , Inibidores da Colinesterase/análise , Dispositivos Lab-On-A-Chip , Malation/análise , Acetilcolinesterase/metabolismo , Acetiltiocolina/metabolismo , Animais , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Electrophorus , Malation/química , Malation/farmacologia , Microeletrodos , Oxirredução , Tiocolina/metabolismo
16.
Biosens Bioelectron ; 74: 177-82, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26141104

RESUMO

Based on the specific binding of Cu(2+) ions to the 11-mercaptoundecanoic acid (11-MUA)-protected AuNCs with intense orange-red emission, we have proposed and constructed a novel fluorescent nanomaterials-metal ions ensemble at a nonfluorescence off-state. Subsequently, an AuNCs@11-MUA-Cu(2+) ensemble-based fluorescent chemosensor, which is amenable to convenient, sensitive, selective, turn-on and real-time assay of acetylcholinesterase (AChE), could be developed by using acetylthiocholine (ATCh) as the substrate. Herein, the sensing ensemble solution exhibits a marvelous fluorescent enhancement in the presence of AChE and ATCh, where AChE hydrolyzes its active substrate ATCh into thiocholine (TCh), and then TCh captures Cu(2+) from the ensemble, accompanied by the conversion from fluorescence off-state to on-state of the AuNCs. The AChE activity could be detected less than 0.05 mU/mL within a good linear range from 0.05 to 2.5 mU/mL. Our proposed fluorescence assay can be utilized to evaluate the AChE activity quantitatively in real biological sample, and furthermore to screen the inhibitor of AChE. As far as we know, the present study has reported the first analytical proposal for sensing AChE activity in real time by using a fluorescent nanomaterials-Cu(2+) ensemble or focusing on the Cu(2+)-triggered fluorescence quenching/recovery. This strategy paves a new avenue for exploring the biosensing applications of fluorescent AuNCs, and presents the prospect of AuNCs@11-MUA-Cu(2+) ensemble as versatile enzyme activity assay platforms by means of other appropriate substrates/analytes.


Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Cobre/química , Ensaios Enzimáticos/métodos , Ácidos Graxos/química , Ouro/química , Nanopartículas Metálicas/química , Compostos de Sulfidrila/química , Acetilcolinesterase/análise , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Fluorescência , Humanos , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Tiocolina/química
17.
J Phys Chem B ; 119(13): 4749-57, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25782026

RESUMO

In this work, the diffusion of thiocholine ion into an enzyme-loaded polypyrrole film was evaluated by different methods, and the results were compared to identify the most suitable method. The enzyme-loaded polypyrrole film was coated with a thin layer of gelatin and gluteraldehyde so as to prevent enzyme leaching. Diffusion coefficients under normal and prepolarized conditions were calculated by five different methods, namely, the Cottrell method, the method of Peerce and Bard, the theoretical impedance model, the electrochemically stimulated conformational relaxation (ESCR) method, and the direct impedance measurement method. The theoretical model of Vortynstev was used to calculate the parameters from the impedance spectra using simplex technique in MATLAB. The results indicate that under normal unpolarized condition the ESCR method gives a diffusion coefficient close to that given by Vortynstev method, but under polarized conditions the Cottrell method can provide a better value of diffusion coefficient than ESCR. The diffusion coefficient of thiocholine in PPy composite film from an electrolytic background of phosphate buffer of pH 7.4 was found to be 1.00 × 10(-8) cm(2) s(-1) based on Vortynstev method. The mechanism of thiocholine diffusion into the positively charged/polarized matrix is attributed to be through the formation of a dinegative ion between thiocholine and phosphate anion via electrostatic attraction.


Assuntos
Eletrodos , Polímeros/química , Pirróis/química , Tiocolina/química , Difusão , Impedância Elétrica , Eletrólitos/química , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Modelos Químicos , Software , Análise Espectral , Eletricidade Estática
18.
Biosens Bioelectron ; 68: 168-174, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25569873

RESUMO

This paper reports a novel nanosensor for organophosphorus pesticides based on the fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The detection mechanism is based on the facts that AuNPs quench the fluorescence of UCNPs and organophosphorus pesticides (OPs) inhibit the activity of acetylcholinesterase (AChE) which catalyzes the hydrolysis of acetylthiocholine (ATC) into thiocholine. Under the optimized conditions, the logarithm of the pesticides concentration was proportional to the inhibition efficiency. The detection limits of parathion-methyl, monocrotophos and dimethoate reached 0.67, 23, and 67 ng/L, respectively. Meanwhile, the biosensor shows good sensitivity, stability, and could be successfully applied to detection of OPs in real food samples, suggesting the biosensor has potentially extensive application clinic diagnoses assays.


Assuntos
Acetilcolinesterase/química , Técnicas Biossensoriais , Praguicidas/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Ouro/química , Nanopartículas/química , Praguicidas/química , Tiocolina/química
19.
J Org Chem ; 80(3): 1905-8, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25545007

RESUMO

The carbonyl-C, carbonyl-O, and leaving-S kinetic isotope effects (KIEs) were determined for the hydrolysis of formylthiocholine. Under acidic conditions, (13)k(obs) = 1.0312, (18)k(obs) = 0.997, and (34)k(obs) = 0.995; for neutral conditions, (13)k(obs) = 1.022, (18)k(obs) = 1.010, and (34)k(obs) = 0.996; and for alkaline conditions, (13)k(obs) = 1.0263, (18)k(obs) = 0.992, and (34)k(obs) = 1.000. The observed KIEs provided helpful insights into a qualitative description of the bond orders in the transition state structure.


Assuntos
Isótopos/química , Compostos de Enxofre/química , Tiocolina/química , Hidrólise , Cinética , Estrutura Molecular , Tiocolina/análogos & derivados
20.
J Insect Sci ; 14: 18, 2014 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-25373165

RESUMO

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (K(M)) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively correlated sensitivity, being insensitive to phosphamidon and monocrotophos and sensitive to N-methyl carbamates.


Assuntos
Acetilcolinesterase/metabolismo , Hemípteros/efeitos dos fármacos , Resistência a Inseticidas , Inseticidas/farmacologia , Compostos Organotiofosforados/farmacologia , Acetiltiocolina/análogos & derivados , Acetiltiocolina/metabolismo , Animais , Butiriltiocolina/metabolismo , Hemípteros/enzimologia , Hemípteros/genética , Irã (Geográfico) , Tiocolina/análogos & derivados , Tiocolina/metabolismo
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