Studies on acetylcholine sensor and its analytical application based on the inhibition of cholinesterase (original) (raw)
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This study was aimed at the development of a conductometric biosensor based on acetylcholinesterase considering the feasibility of its application for the inhibitory analysis of various toxicants. In this paper, the optimum conditions for enzyme immobilization on the transducer surface are selected as well as the optimum concentration of substrate for inhibitory analysis. Sensitivity of the developed biosensor to different classes of toxic compounds (organophosphorus pesticides, heavy metal ions, surfactants, aflatoxin, glycoalkaloids) was tested. It is shown that the developed biosensor can be successfully used for the analysis of pesticides and mycotoxins, as well as for determination of total toxicity of the samples. A new method of biosensor analysis of toxic substances of different classes in complex multicomponent aqueous samples is proposed.
Sensors, 2013
Numerous amperometric biosensors have been developed for the fast analysis of neurotoxic insecticides based on inhibition of cholinesterase (AChE). The analytical signal is quantified by the oxidation of the thiocholine that is produced enzymatically by the hydrolysis of the acetylthiocholine pseudosubstrate. The pseudosubstrate is a cation and it is associated with chloride or iodide as corresponding anion to form a salt. The iodide salt is cheaper, but it is electrochemically active and consequently more difficult to use in electrochemical analytical devices. We investigate the possibility of using acetylthiocholine iodide as pseudosubstrate for amperometric detection. Our investigation demonstrates that operational conditions for any amperometric biosensor that use acetylthiocholine iodide must be thoroughly optimized to avoid false analytical signals or a reduced sensitivity. The working overpotential determined for different screen-printed electrodes was: carbon-nanotubes (360 mV), platinum (560 mV), gold (370 mV, based on a catalytic effect of iodide) or cobalt phthalocyanine (110 mV, but with a significant reduced sensitivity in the presence of iodide anions).
Amperometric acetylcholine and choline sensors with immobilized enzymes
Analytica Chimica Acta, 1986
Acetylcholine and choline sensors are prepared by immobilizing enzymes on nylon net attached to a hydrogen peroxide sensor. Choline oxidase is used for the choline sensor, acetylcholinesterase and choline oxidase are used for acetylcholine. The platinum/ silver electrode pair is polarized at +0.6 V. The assembly is protected with an acetate cellulose membrane to enhance selectivity. The ranges measured are l-10 pmol 1-l in 0.1-l ml of sample. The response times are l-2 min.
Biosensors and Bioelectronics, 2014
The work presented here describes a novel, easy and low-cost method of fabrication of a highly sensitive acetylcholinesterase biosensor and its application to detect organophosphate and organocarbamate pesticides. Acetylcholinesterase was electro-immobilized into a thick conducting layer of polypyrrole. Porcine skin gelatin and gluteraldehyde mixture was used for stabilizing the system. Acetylthiocholine chloride was used as the substrate. Polypyrrole catalyzed the electrochemical oxidation of thiocholine and promoted the electron transfer, thus lowering the oxidation potential and increasing the detection sensitivity. Electro oxidation of thiocholine in polypyrrole matrix occurred at 0.1 V under low potential scan rate. The thiocholine sensitivity of the electrode was found to be 143 mA/M. The sensor was applied to detect the sample organophosphate pesticide ethylparaoxon and organocarbamate pesticide carbofuran. The detection limit for paraoxon was found to be 1.1 ppb and that for carbofuran is 0.12 ppb. The sensor showed good intra and inter state precision with relative standard deviation (RSD) 0.742% and 6.56% respectively. Both dry and wet storage stability were studied. The sensor stored at 0 1C in dry condition had a good storage stability retaining 70% of its original activity for 4 months. During wet storage, the activity decrease followed the same trend, however, the operational stability at the end of the storage period was found to be less compared to the dry storage case. The developed biosensor is as a promising new tool for analysis of cholinesterase inhibitors.
2004
Potentiometric and amperometric enzyme sensors based on modified gold electrodes have been developed and compared in pesticide determination. PAMAM dendrimer (generation G4) stabilized with 1-hexadecanethiol was used for the immobilization of acetylcholinesterase from electric eel and choline oxidase from Alcaligenes species in the assembly of amperometric sensor. Polyaniline-doped with camphorsulfonic acid was used to obtain potentiometric response. Trichlorfon, carbofuran and eserine suppress the biosensor response due to their inhibitory effect. The detection limits of 0.003 and 200 nmol l −1 (trichlorfon), 0.04 and 6 nmol l −1 (carbofuran) and 0.1 and 700 nmol l −1 were obtained for amperometric and potentiometric sensors, respectively. The difference in the biosensor behavior and the high sensitivity of the dendrimer modified sensor to the inhibitors is due to the specific organization of protein layer at charged surface of the modifier macromolecules.
Acetylcholinesterase sensors based on gold electrodes modified with dendrimer and polyaniline
Analytica Chimica Acta, 2004
Potentiometric and amperometric enzyme sensors based on modified gold electrodes have been developed and compared in pesticide determination. PAMAM dendrimer (generation G4) stabilized with 1-hexadecanethiol was used for the immobilization of acetylcholinesterase from electric eel and choline oxidase from Alcaligenes species in the assembly of amperometric sensor. Polyaniline-doped with camphorsulfonic acid was used to obtain potentiometric response. Trichlorfon, carbofuran and eserine suppress the biosensor response due to their inhibitory effect. The detection limits of 0.003 and 200 nmol l −1 (trichlorfon), 0.04 and 6 nmol l −1 (carbofuran) and 0.1 and 700 nmol l −1 were obtained for amperometric and potentiometric sensors, respectively. The difference in the biosensor behavior and the high sensitivity of the dendrimer modified sensor to the inhibitors is due to the specific organization of protein layer at charged surface of the modifier macromolecules.
An acetylcholinesterase biosensor for determination of low concentrations of Paraoxon and Dichlorvos
Journal of Biotechnology, 2010
The characterization of an economic and ease-to-use carbon paste acetylcholinesterase (AChE) based biosensor to determine the concentration of pesticides Paraoxon and Dichlorvos is discussed. AChE hydrolyses acetylthiocholine (ATCh) in thiocoline (TC) and acetic acid (AA). When AChE is immobilized into a paste carbon working electrode kept at +410 mV vs. Ag/AgCl electrode, the enzyme reaction rate using acetylthiocholine chloride (ATCl) as substrate is monitored as a current intensity. Because Paraoxon and Dichlorvos inhibit the AChE reaction, the decrease of the current intensity, at fixed ATCl concentration, is a measure of their concentration. Linear calibration curves for Paraoxon and Dichlorvos determination have been obtained. The detection limits resulted to be 0.86 ppb and 4.2 ppb for Paraoxon and Dichlorvos, respectively, while the extension of the linear range was up 23 ppb for the former pesticide and up to 33 ppb for the latter. Because the inhibited enzyme can be reactivated when immediately treated with an oxime, the biosensor reactivation has been studied when 1,1 0 -trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were used. TMB-4 resulted more effective. The comparison with the behavior of similar AChE based biosensors is also presented.
Determination of binary pesticide mixtures by an acetylcholinesterase–choline oxidase biosensor
Biosensors and Bioelectronics, 2004
In this study, acetylcholinesterase (AChE) and choline oxidase (ChO) were co-immobilized on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes to construct a biosensor for the detection of anti-cholinesterase compounds. pHEMA membranes were prepared with the addition of SnCl 4 to achieve the desired porosity. Immobilization of the enzymes was done by surface attachment via epichlorohydrin (Epi) and Cibacron Blue F3G-A (CB) activation. Enzyme immobilized membrane was used in the detection of anti-cholinesterase activity of aldicarb (AS), carbofuran (CF) and carbaryl (CL), as well as two mixtures, (AS + CF) and (AS + CL). The total anti-cholinesterase activity of binary pesticide mixtures was found to be lower than the sum of the individual inhibition values.
An enzyme electrode for acetylcholine
Biochimica et Biophysica Acta (BBA) - Enzymology, 1978
A new enzyme electrode is described to measure continuously acetylcholine concentration. A coating containing active acetylcholinesterase is produced on a pH-glass electrode. The mean thickness of the coating is 50 ~zm. Optimal operational conditions with respect to buffer concentration, ionic strength, linearity, stability, sensitivity, pH of the bulk solution, and response time are studied and discussed. The use of acetylcholinesterase-containing membranes as sensors could offer several novel advantages.