Enzyme electrodes choline

Acetylcholineesterase and choline oxidase 300 pL 0.1 M phosphate buffer (pH 6.5) containing 16 mg BSA and 1 mg each of ChO and AChE were mixed with 30 pL 25% glutaral-dehyde diluted 10 fold with phosphate buffer. The solution was used to coat the surface of a Pt electrode. This enzyme electrode was used for the amperometric measurement of ACh and Ch. Calibration graphs were linear upto 0.09 and 0.08 mM Ch and ACh, respectively. Detection limits were 0.1 pM of both Ch and ACh. Response time was 1 s for both Ch and ACh. The use of the sensor as detector for HPLC analysis for both Ch and ACh was demonstrated. [91]... 

Acetylcholineesterase and choline oxidase Coated wire enzyme electrode was prepared by coating a Ag wire with a homogenized solution of CH2C12 containing 0.28 gm of polyvinyl bytyral, 0.15 gm of di-n-amylphthalate, 10 mg of tridodecy-lamine and 2 mg of sodium tetraphenylborate. Three different methods for immobilizing the enzyme on the coated electrode are described with use of AChE. The model electrode was used to determine ACh from 0.1 to 10 mM. Response time was 3-6 min. [94]... 

Yao et al. reported a flow injection analytical system for the simultaneous determination of acetylcholine and choline that made use of immobilized enzyme reactors and enzyme electrodes [25]. Acetylcholineesterase-choline oxidase and choline oxidase were separately immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, and incorporated in parallel as the enzyme reactors in a flow injection system. The sample containing acetylcholine and choline in 0.1 M phosphate buffer (pH 8.3) carrier solution was injected into the system. The flow was split to pass through the two reactors, recombined, and mixed with 0.3 mM K4Fe(CN)6 reagent solution before reaching a peroxidase immobilized electrode. Because each channel had a different residence time, two peaks were obtained for choline and total acetylcholine and choline. Response was linear for 5 pM-0.5 mM choline, and for 5 pM 1 mM acetylcholine plus choline. The detection limits were 0.4 pM for choline and 2 pM for acetylcholine. 

Mascini and Guilbault presented a review covering the methods used for immobilization of the enzymes, enzyme electrodes, and sensors for choline and acetylcholine [62]. A number of reports have appeared in the literature [21, 22, 63-124] describing biosensors used for the detection and the analysis of acetylcholine. The important characteristics of some of these reports are summarized in Table 1. 

Enzyme electrodes for choline and acetylcholine (300, 301) and for the analysis of choline-containing phospholipids (303, 304) is obtained by immobilizing choline oxidase or choline oxidase and acetylcholinesterase on membranes at the tip of platinum electrodes. The formation of hydrogen peroxide is monitored ... 

The above authors coimmobilized choline oxidase and AChE on a nylon net which was fixed to a hydrogen peroxide probe so that the esterase was adjacent to the solution. The apparent activities were 200-400 mU/cm2 for choline oxidase and 50-100 mU/cm2 for AChE. The sensitivity of the sequence electrode for ACh was about 90% of that for choline, resulting in a detection limit of 1 pmol/l ACh. The response time was 1-2 min. The parameters of this amperometric sensor surpass those of potentiometric enzyme electrodes for ACh (see Section 3.1.25). Application to brain extract analysis has been announced. 

Between 15 and 20 analyzers based on enzyme electrodes are on the market worldwide. They are one-parameter instruments for the measurement of glucose, galactose, uric add, choline, ethanol, lysine, lactate, pesti-ddes, sucrose, lactose, and the activity of a-amylase (Table 23). They provide for a negligible enzyme consumption of less than 1 pg per sample. 

Organophosphorus compounds are significant major environmental pollutants due to their intensive use as pesticides. The modem techniques based on inhibition of cholinesterase enzyme activity are discussed. Potentiometric electrodes based on detection of cholinesterase inhibition by analytes have been developed. The detection of cholinesterase activity is based on the novel pindple of molecular transduction. Immobilized peroxidase acting as the molecular transducer, catalyzes the electroreduction of hydrogen peroxide by direct (mediatorless) electron transfer. The sensing element consists of a carbon based electrode containing an assembly of co-immobilized enzymes cholinesterase, choline oxidase and peroxidase. 

Fortier [6] found that AQ polymer from Eastman was not deleterious for the activity of a variety of enzymes such as L-amino acid oxidase, choline oxidase, galactose oxidase, and GOD. Following mixing of the enzyme with the AQ polymer, the mixture was cast and dried onto the surface of a platinum electrode. The film was then coated with a thin layer of Nafion to avoid dissolution of the AQ polymer film in the aqueous solution when the electrode was used as a biosensor. These easy-to-make amperometric biosensors, which were based on the amperometric detection of H202, showed high catalytic activity. 

Figure 27.18 Common configuration for postcolumn reactors with electrochemical analysis. (A) LC-chemical reaction-EC. Postcolumn addition of a chemical reagent (for example, Cu2+ or an enzyme). (B) LC-enzyme-LC. Electrochemical detection following postcolumn reaction with an immobilized enzyme or other catalyst (for example, dehydrogenase or choline esterase). (C) LC-EC-EC. Electrochemical generation of a derivatizing reagent. The response at the second electrode is proportional to analyte concentration (for example, production of Br2 for detection of thioethers). (D) LC-EC-EC. Electrochemical derivatization of an analyte. In this case a compound of a more favorable redox potential is produced and detected at the second electrode (for example, detection of reduced disulfides by the catalytic oxidation of Hg). (E) LC-hv-EC. Photochemical reaction of an analyte to produce a species that is electrochemically active (for example, detection of nitro compounds and phenylalanine). Various combinations of these five arrangements have also been used. [Reprinted with permission from Bioanalytical Systems, Inc.]...

In this paper, we have evaluated three different protocols for the preparation of AQ-enzyme film using choline and glucose oxidases and mixture of AQ 29D and AQ 55D (1 1). This AQ mixture is recommended by Eastman Kodak to increase the adherency of the film to a surface such as a platinum electrode (17). The values 29 and 55 represent the glass transition temperature of each polymer. Also, the main structural difference between the two polymers is that, in the case of AQ 55D, an aliphatic glycol moiety replaces the cycloaliphatic glycol moiety found in the AQ29 (17,18). 

Figure 3. Calibration curve for AQ-choline oxidase electrode, prepared by casting 25 il of 1% AQ solution and 3 U of enzyme. The film was dried at 50°C during 30 min in an oven.

Acetylcholine Sensors. The general scheme for determination of the neurotransmitter acetylcholine is outlined in Figure 11. In this scheme, acetylcholine is first converted catalytically to choline by the enzyme acetylcholinesterase. The choline produced reduces the FAD redox centers of choline oxidase, and electron transfer from these centers to the electrode is facilitated by the polymeric relay system. 

Hale et al. reported the use of an enzyme-modified carbon paste for the determination of acetylcholine [21], The sensor was constructed from a carbon paste electrode containing acetylcholineesterase and choline oxidase, and the electron transfer mediator tetrathiafulvalene. The electrode was used for the cyclic voltammetric determination of acetylcholine in 0.1 M phosphate buffer at +200 mV versus saturated calomel electrode. Tetrathiafulvalene efficiently re-oxidized the reduced flavin adenine dinucleotide centers of choline oxidase. The calibration graph was linear up to 400 pM acetylcholine, and the detection limit was 0.5 pM. 

Acetylcholineesterase and choline oxidase Enzyme membrane in H20 was treated with 11% solution of PVA-SbQ (polyvinyl alcohol) with styryl pyridinium groups. Mixture was spread on a cellulose nitrate membrane and air dried. The membrane was exposed to UV radiation for 3 h and stored at 4°C. The enzyme membrane was fixed with a Pt electrode. Sample was dissolved in phosphate buffer and measured. The best results were obtained at pH 8 and at 30°. The calibration graph was rectilinear for 5mM ACh. The storage stability of the dry membrane was excellent. [77]... 

Acetylcholineesterase and choline oxidase Carbon-fiber micro-electrode for determination of ACh and choline, mounted in a glass capillary tube. Enzyme was immobilized on the surface of the carbon fiber with albumin. Electrode was dip-coated with Nafion. Evaluation of selectivity and dynamic range, at a fixed potential of 1.2 V versus Ag-AgCl. Calibration graph for ACh and Ch was rectilinear between 0.1 and 3mM. Interference from ascorbic acid was not observed in the range 0.1 to 0.3 mM. [79]... 

Acetylcholineesterase and choline oxidase Co-immobilized enzyme (AChE and ChO) on 7 pm diameter carbon-fiber electrode entrapped with polyvinyl alcohol quaternized stil-bazole. Sensor was used as an amperometric detector for ACh. Linear response in the range 0.1-1 mM of ACh. Response time was 5 s. [83]... 

Acetylcholineesterase and choline oxidase A Cross-linkable polymer, poly (vinyl pyridine) derivatized at the N atoms with a combination of iron-linking and redox functionalities was used to immobilize the enzymes and to shuttle electrons. Enzymes were deposited with the polymer and deposited onto C electrode. For peroxide selectivity over ascorbate is achieved by incorporation of Nafion. The microsensors if they can be successfully used in vivo will provide valuable information for brain diseases (Parkinsion s and Alzheimer s). [88]... 

Acetylcholineesterase and choline oxidase Au foil was treated with cystamine to produce a base layer of ami-nothiolate units, was derivatized by reaction of the amino group and disodium-4,4 -diisothiocyanato-trans-stilbene-2,2 -disulfonate. Enzymes were immobilized at the isothiocyanate group via thiourea link. The bifunctional sensor for ACh was prepared by stepwise immobilization of four layers of the enzyme ChO and three layers of AChE. Choline generated was detected amperometircally with the use of 2,6-dichloro-phenolindophenol as a mediator in solution. Electrical communication between the enzyme and the electrode is achieved either by the use of ferrocenecar-boxylic acid as mediator in the assay buffer or by immobilization of [(ferrocenyl methyl)amino] hexa-noic acid on the enzyme layer. [92]... 

Acetylcholineesterase and choline oxidase Enzyme immobilized over tetra-thiafulvalene tetracyanoquinodi-methane crystals packed into a cavity at the tip of a carbon-fiber electrode. The immobilization matrix consisted of dialdehyde starch/glutaraldehyde, and the sensor was covered with an outer Nafion membrane. The ampero-metric performance of the sensor was studied with the use of FIA system. An applied potential of +100 mV versus SCE (Pt-wire auxiliary electrode) and a carrier flow rate of 1 mL/min. The Ch and ACh biosensors exhibited linear response upto 100 pM and 50 pM, respectively. Response times were 8.2 s. [97]... 

Horseradish peroxidase (HPP), choline oxidase and acetylcholineesterase A three enzyme layered assembly on Au electrodes or Au quartz crystal, consisting of HRP, ChO and AChE is used to sense ACh by the HRP-mediated oxidation of 3,3, 5,5 -tetramethyl benzidine (1) by H202 and the formation of the insoluble product (2) on the respective transducers. Acetylcholine is hydrolyzed by AChE to choline that is oxidized by ChO and 02 to yield the respective betaine and H202. The amount of generated H202 and the resulting insoluble product on the transducer correlates with the concentration of acetylcholine in the samples. 

Mayer determined acetylcholine and choline by enzyme-mediated liquid chromatography with electrochemical detection [195]. The two compounds were separated by passing the eluted fractions through a post-column reactor containing immobilized Acetylcholineesterase and choline oxidase. In the presence of either compound, the dissolved oxygen was converted into hydrogen peroxide, which was detected amperometrically at a platinum electrode. This method was used to determine choline in rat brain homogenates. 

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