Enzyme electrodes cholesterol

Although the primary focus of oxidase based enzyme electrodes has been the determination of glucose, the list of extensions to other analytes is considerable. Systems have been described for cholesterol 123,132-136) galactose ii -i35-i37) dd i38.i39) lactatepyruvatecreatinine serum lipaseethanoland amino acids... 

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

Chemically binding enzymes to nylon net is very simple and gives strong mechanically resistant membranes (135). The nylon net is first activated by methylation and then quickly treated with lysine. Finally, the enzyme is chemically bound with GA. The immobilized disks are fixed direcdy to the sensor surface or stored in a phosphate buffer. GOD, ascorbate oxidase, cholesterol oxidase, galactose oxidase, urease, alcohol oxidase (135), and lactate oxidase (142) have been immobilized by this procedure and the respective enzyme electrode performance has been established. 

Cholesterol can be determined by immobilizing the enzyme cholesterol oxidase (COD) in a layer over an oxygen electrode or a hydrogen peroxide electrode. Cholesterol oxidase is chemically immobilized onto nylon net (135, 265) or collagen membranes (267) and fixed onto an O2 electrode ... 

For application in enzyme electrodes COD has been mainly immobilized by surface fixation (Table 7). As early as 1977, Clark had patented the polarographic analysis of free and esterified cholesterol by means of free as well as immobilized COD and CEH with anodic H2O2 indication. Cholesterol in food and serum samples has been determined by using COD bound to a collagen membrane via glutaraldehyde and coupled to a Pt electrode (Clark, 1978). A similar probe has been devised by Bertrand et al. (1979). In this sensor the enzyme membrane was not protected by a semipermeable membrane. Interferences were compensated for by difference measurements between an enzyme sensor and an enzyme-free membrane electrode. The lower detection limit was 0.05... 

Enzyme electrodes comprising coimmobilized COD and CEH can be used for the determination of total cholesterol. On the other hand, coupling of COD with HRP enables cholesterol measurement at low electrode overvoltage, which avoids electrochemical interferences. Fig. 89 shows the diversity of the potential sequences of enzymatic and electrochemical reactions in cholesterol electrodes. 

Fig. 89. Coupling of enzymatic reactions with electrochemical detection in amperometric enzyme electrodes for cholesterol. (Redrawn from Wollenberger et al., 1983).

ADP AFP ab as ALAT AP ASAT ATP BQ BSA CEH CK CME COD con A CV d D E E EC ECME EDTA EIA /e FAD FET FIA G GOD G6P-DH HBg HCG adenosine diphosphate a-fetoprotein antibody antigen alanine aminotranferase alkaline phosphatase aspartate aminotransferase adenosine triphosphate benzoquinone bovine serum albumin cholesterol ester hydrolase creatine kinase chemically modified electrode cholesterol oxidase concanavalin A coefficient of variation (relative standard deviation) layer thickness diffusion coefficient enzyme potential Enzyme Classification enzyme-chemically modified electrode ethylene diamine tetraacetic acid enzyme immunoassay enzyme loading factor flavin adenine dinucleotide field effect transistor flow injection analysis amplification factor glucose oxidase glucose-6-phosphate dehydrogenase hepatitis B surface antigen human chorionic gonadotropin... 

Other examples of amperometric enzyme electrodes based on the measurement of oxygen or hydrogen peroxide include electrodes for the measurement of galactose in blood (galactose oxidase,enzyme), oxalate in urine (oxalate oxidase), and cholesterol in blood serum (cholesterol oxidase). Ethanol is determined by reacting with a cofactor, nicotinamide adenine dinucleotide (NAD" ) in the presence of the enzyme alcohol dehydrogenase to produce the reduced form of NAD", NADH, which is electrochemically oxidized. Lactate in blood is similarly determined (lactate dehydrogenase enzyme). 

Singh, S. Chaubey, A. Malhotra, B. D. Preparation and characterization of an enzyme electrode based on cholesterol esterase and cholesterol oxidase immobilized onto conducting polypyrrole films. J. Appl. Polym. Sci. 2004,6, 3769-3773. 

An enzyme electrode using collagen immobilized cholesterol oxidase for the microdetermination of free cholesterol has been proposed by Coulet and his co-workers [234, 235]. A combined system with collagen bound oxidase and soluble esterase was used by Clark et al. [236]. 

Cholesterol Enzyme electrode Fixed cholesterol oxidase Diagnosis (commercially available) Oxygen electrode... 

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

A.L. Crumbliss, J.Z. Stonehuerner, R.W. Henkens, J. Zhao, and J.P. O Daly, A carrageenan hydrogel stabilized colloidal gold multi-enzyme biosensor electrode utilizing immobilized horseradish peroxidase and cholesterol oxidase/cholesterol esterase to detect cholesterol in serum and whole blood. Biosens. Bioelectron. 8, 331-337 (1993). 

Although freeze drying has often been used to prepare stable dry enzyme preparations we found that when preparing enzyme powders and electrodes it was preferable to vacuum dry the enzyme in the presence of stabilizers (Fig. 8). However this was not true in all cases. Cholesterol oxidase was found to be stabilized best by freeze drying in stabilizers and buffer at pH 5.5 well below the pH for optimal activity (Gibson and Woodward unpublished results). 

Fig. 14.25. (A). The scheme of enzyme adsorption of the electrode with different lipid interlayers. (B). The relative oxygen reduction rate vs. the distance between the electrode and the enzyme for lactase adsorbed (1) on soot, (2) on cholesterol, (4) on lecithin. The curves a and b are calculated for barrier heights of 4 and 5 eV, respectively. (Reprinted from J. O M Bockris, M. Szklonzyk, and Szucs, in Electropharmacology, G. M. Eckert, F. Gutmann, and H. Keyzar, eds., Figs. 25,29, 30,1990. Reproduced with permission of CRC Press.)...

Sensors have also been constructed from some oxidases directly contacted to electrodes to give bioelectrocatalytic systems. These enzymes utilize molecular oxygen as the electron acceptor for the oxidation of their substrates. Enzymes such as catechol oxidase, amino acid oxidase, glucose oxidase, lactate oxidase, pyruvate oxidase, alcohol oxidase, xanthine oxidase and cholesterol oxidase catalyze the oxidation of their respective substrates with the concomitant reduction of O2 to H2O2 ... 

These ferrocene modified polysiloxane polymers were also used to construct glycolate [6,7], lactate [7], acetylcholine [12,81], glutamate [12] and cholesterol [81] sensors. All these electrodes showed that ferrocene containing siloxane polymers efficiently shuttled electrons between redox center(s) of enzyme and the electrode surface. 

The inactivation of certain enzymes such as cholesterol oxidase (CO) on interaction with Au nanoparticles is overcome by immobilizing CO and cholesterol esterase (CE) in a carragenean hydrogel and coupling with Au particles loaded with HRP. This multienzyme device has been shown to be very useful in the determination of cholesterol in serum and whole blood [172]. The sensor operates at low potentials avoiding major interferences. The presence of the hydrogel enables the analysis of whole blood with no fouling of the electrode surface. 

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