An enzyme electrode for acetylcholine

Biosensors and Bioelectronics, 1990

Acetylcholine esterase electrodes, based on glass, Pd/pdO and Ir/IrOz electrodes as pH sensor, using the immobilized acetylcholine esterase in acrylamide-methacrylamide hydrazides prepolymer are reported and compared. New data on the analysis of nicotine, fluoride ion, and some otganophosphorus compounds are reported using the present AChE sensor based on the inhibition of the immobilized acetylcholine estemse. Reactivation of immobilized AChE afrer inhibition with reversible inhibitor, i.e. nicotine and fluoride ion is carried out using a mixture of working bu#er and acetylcholine, whereas reactivation afrer inhibition with inwersible inhibitor, i.e. organophosphorus compounds is carried out using a mixture of acetylcholine and pyridine-2-aldoxime methiodide (PAM). The detection limits for the nicotine and fluoride ion are found to be IO-'M whereas for paraoxon, methyl parathion and malathion are found to be 10m9~ and 10-'"M.

European Chemical Bulletin, 2016

A solid contact biosensor for Acetylcholine (ACh) based on host-guest interactions and potentiometric transduction has been designed and characterized. The biomimetic man-tailored host was synthesized using methacrylic acid as a functional monomer, ethylene glycol di-methacrylate as a crosslinker in the presence of benzoyl peroxide as an initiator. The imprinted beads were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. Slopes and detection limits are 55.2-59.6 mV decade -1 and 0.65-1.31 μg mL -1 , respectively. Significantly, improved accuracy, precision, good reproducibility, long-term stability, selectivity and sensitivity were offered by these simple and cost-effective potentiometric biosensors. A tubular version was further developed and coupled to a flow injection system for acetylcholine determination. This simple and inexpensive flow injection analysis manifold, with a good potentiometric detector, enabled the analysis of ∼30 samples h -...

Biosensors

An electrochemical biosensor was fabricated using nanoparticles of acetylcholinesterase (AChE) and choline oxidase (ChO)/Pt nanoparticles (PtNPs)/porous graphene oxide nanosheet (GONS) composite. A pencil graphite electrode (PGE) was used for the electrodeposition of nanocomposite and the determination of acetylcholine (ACh), a neurotransmitter. Various techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectra and cyclic voltammetry (CV) were used for characterization. This biosensor (AChENPs-ChONPs/GONS/PtNPs/PGE) indicated a very short response time (3 s), a lower limit of detection (0.001 µM), good linearity (0.001–200 µM), longer storage stability (6 months) and better reproducibility. The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 µM) were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor. The coefficients of variation were obtaine...

Electrochimica Acta, 1998

AbstractÐAn amperometric three-enzyme based biosensor for determination of acetylcholine has been developed with possible use for monitoring of brain microdialysates by co-immobilising acetylcholinesterase (AchE), choline oxidase (ChOx) and horseradish peroxidase (HRP) in an Os-based redox polymer on solid graphite electrodes. The redox hydrogel was formed by crosslinking the appropriate enzymes and the Ospolymer (PVI 13 -dmeOs) working as a non-diusing mediator between the electrode and HRP. The sensor was used in a¯ow injection system at an applied potential of À50 mV vs Ag/AgCl. A detection limit of 0.3 mM (twice the S/N ratio) for acetylcholine was obtained, thus representing a sensitive detection system. By adapting the electrode into a microsystem, the release of acetylcholine in real samples (rat brain dialysates) could be shown. Electrode design, optimisation steps and characteristics for the optimised electrode con®guration are presented. #

Talanta, 2019

Reversible inhibitors of acetylcholinesterase are increasingly applied in the Alzheimer's disease therapy. Their determination with biosensors requires special attention to the enzyme immobilization due to importance of electrostatic and steric factors affecting accessibility of the active site to the substrate and inhibitor present simultaneously. In this work, polyelectrolyte complexes have been assembled from electrochemically inactive components with implementation of acetylcholinesterase from electric eel. The influence of the complex components and enzyme position on the assembling efficiency was explored using surface plasmon resonance and kinetic parameters of the enzyme with amperometry with Co phtalocyanine mediator. The biosensor developed showed robust response toward donepezil and berberine with limit of detection of 0.46 and 70 nM, respectively, within 1 minute after inhibitor addition. The sensitivity of determination coincides or is better than characteristics of biosensors based on covalently attached AChE described in the literature. The biosensor was tested on the example of drug determination in spiked samples of artificial urine (recovery 103% for 0.6 nM donepezil and 110% for 0.1 μM berberine).

Analytica Chimica Acta, 2009

In this work two Zr(IV)-porphyrins were tested as potential acetate-selective ionophores. It is shown that these compounds show increased selectivity towards acetate ion, with selectivity sequence: Cl − < NO 3 − < SCN − < ClO 4 − < acetate − < F − , when incorporated into polymeric ion-selective membranes. Among tested ionophores, Zr(IV)-tetra(tert-butylphenyl)porphyrin was found to be the best in terms of response time (20 s) and lower detection limit (2 × 10 −4 M acetate − ).

Analytical and Bioanalytical Chemistry, 2005

Acetylcholinesterase (ChE) sensor based on Prussian blue (PB) modified electrode was developed and tested for the detection of organophosphorus and carbamic pesticides. The signal of the sensor was generated in PB mediated oxidation of thiocholine recorded at+200 mv in DC mode. ChE from electric eel was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) on the surface of screen-printed carbon electrode covered with PB and Nafion. The content of the surface layer (specific enzyme activity, Nafion and BSA amounts) was optimized to establish high and reliable response toward the substrate and ChE inhibitors. The ChE/PB sensor makes it possible to detect Aldicarb, Paraoxon and Parathion-Methyl with limits of detection 30, 10 and 5 ppb, respectively (incubation 10 min). The feasibility of practical application of the ChE/PB sensor developed for the monitoring of degradation of the pesticides in wine fermentation was shown. To diminish matrix interferences, the electrolysis of the grape juice with Al anode and evaporation of ethanol were suggested, however the procedures decrease the sensitivity of pesticide detection and stability of the sample tested.

Biosensors and Bioelectronics, 2003

In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme's natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using sidedirected mutagenesis and employing the enzyme in different assay formats.

Biosensors & Bioelectronics, 2004

A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25 °C. The steady-state current of these enzyme electrodes were reproducible within ±5.0% of the relative error. Response time was found to be 30–50 s and upper limit of the linear working portions was found to be 1.2 mM choline and acetylcholine concentrations in which produced detectable currents were 1.0×10−6 M substrate concentrations. The apparent Michaelis–Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.92 kJ mol−1, respectively. The effects of interferents and stability of the enzyme electrodes were also investigated.

Analytica Chimica Acta, 2003

An amperometric biosensor based on acetylcholinesterase was constructed by simple adsorption of the enzyme on screenprinted electrodes (SPEs). This sensor was used to detect the inhibitory effects of organophosphorus and carbamate insecticides on acetylcholinesterase, and more particularly of chlorpyrifos ethyl oxon (CP-o). We demonstrate that enzyme adsorption on SPEs allows to obtain stable sensors that present good characteristics and are as efficient as other screen-printed biosensors based on covalent binding or entrapment of acetylcholinesterase (AChE).