Choline oxidase and

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

The above-mentioned system has also been used for the indirect CL determination of some carbamate and organophosphorous pesticides that inhibit acetylcholinesterase. Acetylcholinesterase in solution or immobilized on methacrylate beads is coupled to immobilized choline oxidase and peroxidase [46],... 

In this system, choline formed by acetylcholinesterase is oxidized by choline oxidase and the hydrogen peroxide produced is determined using the luminol/peroxidase CL reaction. The sensor has been used for the analysis of Paraoxon and Aldicarb pesticides, with detection limits of 0.75 pg/L and 4 pg/ L, respectively. Recoveries in the range of 81-108% in contaminated samples of soils and vegetables were obtained. 

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

A colorimetric assay for lecithin and choline was described by Kotsira and Klonis (1998) using two enzymes (phospholipase and choline oxidase) and an indicator dye conjugate (bromothymol blue-glutathione) co-immobilised on a glutaraldehyde-activated polyacrylamide transparent gel. The change of the... 

Reagents. Choline oxidase and glucose oxidase and their respective substrates, choline chloride and glucose were purchased from Sigma Chemical Co. (St.Louis, USA). The cationic exchangers AQ 29D and 55D were kindly supplied by Eastman Chemical Inc. (Kingsport, USA) and were obtained as dispersed polymer solutions at concentration of 30 and 28% (w/v) in water, respectively. A blend of the AQ polymer solutions was prepared by mixing the AQ 29D with the AQ 55D in a ratio (1 1), and was further diluted with water to a final concentration % (w/v) indicated in the text. Nafion (equivalent mass 1100 g) 5% (w/v) in a mixture of lower aliphatic alcohols and 10% of water was obtained from Aldrich (St.Louis, USA) and diluted with methanol to yield a stock solution of 0.5% (w/v). 

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. 

Choline oxidase and acetylcholineesterase Enzymes immobilized on a nylon net attached to H202-selective amperometric sensor. ChO is used for choline and AChE and ChO for acetylcholine. Rectilinear response in the range of 1-10 pM. Response time 1-2 min. Interferences occur from ascorbic acid, primary amines, and most seriously from betaine aldehyde. [64]... 

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. 

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. 

A fluorimetric assay method for the determination of acetylcholine with picomole sensitivity was reported by MacDonald [44]. The method is based on the hydrolysis of acetylcholine to choline and acetate, catalyzed in the presence of acetylcholineesterase, oxidation of choline to betaine, and H202 in the presence of choline oxidase, and oxidation of 4-hydroxyphenylacetic acid by H202 to a fluorescent product, catalyzed by peroxidase. The interference in the analysis of brain homogenates was discussed. 

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 ... 

Quantitative measurement of phospholipids is rare in routine clinical practice but more common in research (e.g., in studies of dietary influences). The choline-containing phospholipids lecithin, lysolecithin, and sphingomyelin, which account for at least 95% of total phospholipids in serum, are readily measured by an enzymatic reaction sequence using phospholipase D, choline-oxidase, and horseradish peroxidase. Kit methods with this enzymatic sequence are available commercially. Before the availability of enzymatic reagents, the common quantitative method involved extraction and acid digestion with analysis of the total lipid-bound phosphorus. ... 

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. 

Immobilization of choline oxidase and glucose oxidase on platinum electrodes by entrapment into amorphous polyester cation exchange films Choline, glucose [25]... 

Flavodoxin from M. elsdenii was studied by the SERRS spectroscopy at liquid N2 temperature [98]. It has been shown, on the basis of comparison with the RR spectrum in the solution, that SERRS spectrum arises from the protein-bound FMN. The SERRS spectra of flavoproteins such as choline oxidase and sarcosine oxidase, whose FAD is covalently bound to the apoprotein, were reported by Taniguchi et al. [100]. The results of these two studies indicate the possibility of detecting the SERRS spectra of native flavoproteins. The close similarity between the solution resonance Raman and SERRS spectra [98] reveals that there is no strong chemical interaction between FMN and the silver electrode surface. It is thus reasonable to conclude that the electromagnetic enhancement contributes significantly to the overall enhancement of the SERRS spectra of flavoproteins under the conditions used in these studies. 

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. 

When enzyme sensors are combined with FIA they have applications in medicine and the food produce industry. For example, FIA has been combined with the glucose oxidase electrode to determine glucose in blood [270], with the choline oxidase electrode for the determination of cholinesterase in serum [271], and with a bienzyme electrode that uses choline oxidase and phospholipase for the determination of phospholipids in serum [272]. The technique has also been used with an alcohol oxidase electrode to determine alcohol in drinks (for example, beers) [273], or with a glucose oxidase electrode to determine glucose [274] in various foodstuffs [275] or in fermentation processes [276]. 

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