Glucose oxidase fabrication

In contrast to the molecular wire of molecular interface, electron mediators are covalently bound to a redox enzyme in such a manner as an electron tunneling pathway is formed within the enzyme molecule. Therefore, enzyme-bound mediators work as molecular interface between an enzyme and an electrode. Degani et al. proposed the intramolecular electron pathway of ferrocene molecules which were covalently bound to glucose oxidase [ 4 ]. However, few fabrication methods have been developed to form a monolayer of mediator-modified enzymes on the electrode surface. We have succeeded in development of a novel preparation of the electron transfer system of mediator-modified enzyme by self-assembly in a porous gold-black electrode as schematically shown in Fig.12 [14]. 

Enzymes can lose their activity because of protein denaturation and loss of active site.26 Glucose oxidase can also lose its activity when exposed to excessive amounts of hydrogen peroxide. Most enzymes utilized in the fabrication of biosensors for implantation are derived from nonmammalian proteins, which can lead to an allergic response if the enzymes leach (or leak out of the sensor) into the body. Enzyme leaching can result when the attachment or entrapment of the enzyme is not robust. For example, if a method of entrapment via cross-linking polymers is utilized and the reaction does not go to completion during entrapment, then the enzyme can leach out and cause a loss of signal.27... 

Liu H, Liu Y, Qian J, Yu T, Deng J. Feature of entrapment of glucose oxidase in regenerated silk fibroin membranes and fabrication of a L -dimethylferrocene-mediating glucose sensor. Microchemical Journal 1996, 53, 241-252. 

Film Fabrication. The platinum electrode (0.28 cm area) was fabricated and cleaned as previously described (19). Thin films of AQ-enzyme were prepared by dissolving an amount of the enzyme, as indicated below, in 10 il of 1.5% AQ polymers solution at room temperature. Two aliquots of 5 il were deposited atop the platinum electrode and the first aliquot was allowed to dry before the second addition. This procedure corresponds to the first protocol. In addition, for the second protocol, 10 il of the 0.5% Nafion solution was casted atop the dried AQ-enzyme film and the methanol was allowed to evaporate at room temperature. The third protocol consisted in the deposition of 10 il of a 1% of AQ solution containing the enzyme, atop the platinum electrode followed by heating in an oven at 50°C during 30 min. In each case, 2 U of glucose oxidase were used. 

Electrode and Nafion film Platinum disk electrode (area = 0.28 cm2) was fabricated as previously described (27). A solution of Nafion (EW = 1100) 5% w/v in a mixture of lower aliphatic alcohols and 10% water was obtained from Aldrich and diluted with methanol to give a stock solution of 0.35% w/v. Films of Nafion-glucose oxidase were formed by syringing aliquot (20 pi) of the mixed solution (86.7 pi of Nafion stock solution and 13.3 pi of glucose oxidase solution) at the surface of the platinum electrode. The solvents were left to evaporate at room temperature for at least 30 min. The extra amount of GOD was... 

The influence of different spacer and coupling molecules on the relative performances of seventeen glucose oxidase-nylon electrodes (NGO) fabricated from the same batch of fresh enzyme have been conveniently established in the FIA mode with standard glucose( 1 mM) ... 

Multienzyme tylon Electrodes. Di- and polysaccharides require more than one enzyme to realise the amperanetrically detectable hydrogen peroxide and even glucose really needs the back up of mutarotase with glucose oxidase. It is fortunate that all the necessary enzymes can be immobilized simultaneously on just one nylon net. Thus a viable starch electrode has been fabricated (6) from a nylon net immersed in a cocktail of glucose oxidase, mutarotase and amylogluoosidase (Figure 1). Its response to a continuous flow of 0.1% m/v starch remained steady for over a period of 60 h. 

M.A. McRipley and R.A. Linsenmeier, Fabrication of a mediated glucose oxidase recessed microelectrode for the amperometric determination of glucose, J. Electroanal. Chem., 414 (1996) 235-246. 

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