Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1989-02-24
1992-06-16
Nucker, Christine
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 4, 435 14, 435 25, 435 174, 435176, 435177, 435181, 435288, 435291, 435180, 435817, C12Q 132, C12Q 100, C12Q 154, C12Q 126
Patent
active
051224569
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method for the quantitative determination of 1,4-dihydronicotinamide adenine dinucleotide (NADH) in solution.
NADH and its oxidised counterpart NAD are cofactors in numerous enzyme catalysed redox reactions. In some, an enzyme substrate is oxidised in the presence of cofactor NAD and a suitable oxidase or dehydrogenase to yield NADH in solution; in others an enzyme substrate is reduced in the presence of cofactor NADH to yield NAD in solution. In many cases, determination of the NADH concentration can be used as an indicator of substrate concentration, or as a means of following the course of an enzyme reaction involving NADH (or NAD).
It is known that NADH concentration in solution can be measured colorimetrically, but colorimetric methods on the whole are disadvantageous. Much more advantageous are electrochemical methods, but attempts to determine NADH electrochemically have so far not met with any very great degree of success. It is known, for example, that NADH concentration can be determined by an amperometric assay in which NADH is oxidised at an electrode at a fixed, controlled, potential, the current passing under suitable conditions being proportional to NADH concentration. Unfortunately the electrochemical oxidation of NADH requires a high overpotential, and the NADH is generally not oxidised cleanly at the electrode surface; for example, in many cases the surface of the electrode is quickly fouled by formation of a surface film which affects the size and speed of the electrochemical response: I. Moiroux and P. J. Elving, J. Amer. Chem. Soc. (1980) 102, 6533-6538, and D. G. Johnson, M. D. Ryan and G. S. Wilson, Analyt. Chem. (1986) 58, 42R.
There have been many attempts to avoid these problems. For example, it has been proposed to use modified electrodes coated with a layer of conducting organic salts: J. J. Kulys, Biosensors (1986) 2, 3-13. Alternatively it has been proposed to use an adsorbed redox mediator such as Meldola's blue to couple the oxidation reaction more effectively to the electrode and/or to lower the oxidation potential: L. Gorton et al., J. Electroanalyt. Chem. (1984), 161, 103-20. In another proposal redox mediators have been used in free solution. For example, methoxy phenazine methosulphate has been used with a modified pyrolitic graphite electrode: Y. Kimura and K. Nihi, Analytical Sciences (1985), 1, 271-4. Other experiments have been carried out with platinum, graphite and glassy carbon electrodes, but as yet no electrochemical method for the determination of NADH has been developed which is both rapid and reproducible.
In accordance with the present invention it has been discovered that NADH can be oxidised cleanly, with good amperometric response, both in buffer solutions containing NADH alone, and in solutions containing enzyme, enzyme substrate and NADH, using an activated carbon electrode of a type used in fuel cell technology and comprising a heterogeneous resin-bonded layer of noble metal containing, preferably platinised or palladised (which terms as used herein include materials containing or treated with platinum and/or palladium oxide, as well as materials containing or treated with platinum or palladium metal) carbon or graphite particles bonded with a natural or synthetic resin binder, preferably a synthetic, hydrophobic binder, such as a fluorocarbon resin, most preferably polytetrafluoroethylene. Preferably a platinised or palladised activated carbon electrode is used in which the carbon or graphite particles are platinised or palladised by adsorbing or depositing colloidal platinum or palladium metal, or platinum or palladium oxide, onto the surface of the powder particles before bonding, the resultant electrode comprising a heterogeneous porous activated carbon powder layer with colloidal platinum or palladium, or the corresponding oxides, distributed substantially uniformly throughout the layer. The resin-bonded layer of platinised or palladised activated carbon or graphite particles may be self-supporting, but will usually be
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Bennetto Hugh P.
DeKeyzer David R.
Delaney Gerard M.
Mason Jeremy R.
Mullen William H.
Cambridge Life Science plc
Nucker Christine
Preston David R.
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