Choline oxidase

A flow injection optical fibre biosensor for choline was also developed55. Choline oxidase (ChOX) was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) after adsorption on weak anion-exchanger beads (DEAE-Sepharose). In this way, the sensing layer was directly created at the surface of the working glassy carbon electrode. The optimization of the reaction conditions and of the physicochemical parameters influencing the FIA biosensor response allows the measurement of choline concentration with a detection limit of 10 pmol. The DEAE-based system also exhibited a good operational stability since 160 repeated measurements of 3 nmol of choline could be performed with a variation coefficient of 4.5%. 

Tsafack V.C., Marquette C.A., Pizzolato F., Blum L.J., Chemiluminescent choline biosensor using histidine-modified peroxidase immobilized on metal-chelate substituted beads and choline oxidase immobilized on anion-exchanger beads co-entrapped in a photocrosslinkable polymer, Biosens. Bioelectron, 2000 15 125-133. 

EC1 Oxidoreductases dehydrogenase, acceptor ECU Acting on the CH-CH (cholestenone 5a- Choline oxidase choline H202 Luminol 17... 

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs), AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide (OP) biosensor [40, 41], Another OP biosensor has also been constructed by adsorption of AChE on MWNTs modified thick film [8], More recently AChE has been covalently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite film [11], in which biopolymer chitosan provides biocompatible nature to the enzyme and MWNTs improve the conductive nature of chitosan. Even though these enzyme immobilization techniques have been reported in the last three decades, no method can be commonly used for all the enzymes by retaining their complete activity. 

C. Cremisini, A.D. Sario, J. Mela, R. Pilloton, and G. Paleshci, Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an amperometric choline oxidase based biosensor. Anal. Chim. Acta 311, 273—280 (1995). 

F.N. Kok and V. Hasirci, Determination of binary pesticide mixtures by an acetylcholinesterase-choline oxidase biosensor. Biosens. Bioelectron. 19, 661-665 (2004). 

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. 

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],... 

Choline, acetylcholine Choline oxidase Acetylcholine esterase 48-50... 

Choline-containing phospholipids Phospholipase D Choline oxidase 51... 

Choline-containing phospholipids have been determined in human serum using an IMER consisting of coimmobilized phospholipase D and choline oxidase [51]. Recently, immobilized glutamate oxidase was used to determine L-glutamic acid in culture media [52],... 

Pesticides Immobilized choline oxidase/peroxidase, luminol-H202 CL 4 pg/L aldicarb 0.75 pg/L paraoxon 55... 

T piece Choline Choline oxidase column, luminol-H202 CL 0.1 pM 62... 

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... 

Phospholipids Phospholipase/choline oxidase Catalase (increases stability of main en2yme)... 

In 1989, to the best of our knowledge, the first report appeared on the use of an avidin-biotin system for the immobilization of enzymes in the preparation of biosensors. Walt et al. have immobilized biotin-modified enzymes (urease, esterase, and penicillinase) on the surface of biotin-modified optical fiber using avidin as a binder (Figure 5a). They have demonstrated the general use of this procedure in immobilizing several types of enzymes. At nearly the same time, Gunaratna and Wilson used an enzyme column in which choline esterase and choline oxidase were immobilized through avidin-biotin complexation for the determination of acetyl-... 

Numerous postcolumn enzymatic reactors have been designed for LCEC. Enzymes can be used to produce an electroactive compound from the analyte of interest or, alternatively, to generate an electroactive species that is proportional to analyte concentration. An example of the latter is the detection of acetylcholine [46]. In this case, acetylcholinesterase is used to convert acetylcholine to choline. The resulting choline is reacted with choline oxidase to produce hydrogen peroxide. The amount of hydrogen peroxide produced is directly proportional to the initial concentration of acetylcholine. Detection limits are in the 100 femto-mole range. 

---