Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1990-07-12
1992-02-25
Lacey, David L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435288, 435817, 204403, G01N 2726, C12M 140, C12Q 100
Patent
active
050912992
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to bioelectrochemical reactions carried out in non-aqueous or microaqueous solvents. A microaqueous solvent is one formed by the addition of small quantities of water to a non-aqueous solvent (Yamane et al, 1988) and as used herein the term includes water-immiscible solvents which are saturated with water.
In particular, the present invention relates to a method of carrying out a bioelectrochemical reaction in a non-aqueous or microaqueous solution, the use of such a method for the determination of an analyte, and to an electrochemical cell and an enzyme electrode for carrying out such methods.
BACKGROUND ART
It is well kown to employ an enzyme electrode in order to perform and monitor a bioelectrochemical reacton in aqueous solution. For example, an enzyme electrode involving glucose oxidase may be used to effect the oxidaton of glucose, and consequently to monitor the concentration of glucose in aqueous solution (see e.g. Turner et al, 1985). In such electrodes the enzyme is, conventionally, immobilised on the electrode by means of covalent bonding, and electron transfer between the redox centre of the enzyme and the electrode surface may be effected by means of a mediator molecule such as ferrocene (Cass et al 1984)
The use of enzyme electrodes in aqueous solutions enables the concentration of chemical substances in samples to be determined without extensive preparation. The enzyme provides the specificity of a biochemical reaction and the electrode monitors the extent or progress of the reaction in a sensitive manner (Turner et al, 1987).
However, the methods used to date suffer from several disadvantages. For example, the method is limited to the determination of species which are relatively water soluble, the electrode material must be one which is stable and operable in an aqueous solvent, and the method is not appropriate for use at elevated temperatures because of poor thermal stability of many enzymes in aqueous environments.
The present inventors have found that it is possible to carry out bioelectrochemical reactions in organic or microaqueous solvents. Although enzyme reactions in organic and microaqueous solvents have been reported (Klibanov, 1986; Halling, 1987; Kazandijan et al, 1985) the possibility of employing enzyme electrodes in organic electrochemisty has not, previously, been explored.
DISCLOSURE OF THE INVENTION
According to one aspect of the present invention there is provided a method for carrying out a bioelectrochemical reaction in a non-aqueous or microaqueous solvent, the method comprising contacting a non-aqueous or microaqueous solution of a substrate for an enzyme with an electrode at which said enzyme is retained and allowing the substrate to undergo reaction at the electrode under the influence of said enzyme. One possibility is that the enzyme catalyses the conversion of the substrate into a porduct which then undergoes an electrochemical reaction directly at the electrode. An alternative is that the enzyme is one which can effect oxidation or reduction of the substrate, possibly with the intervention of a mediator, and is thus involved in the transfer of electrons between the substrate and the electrode. The enzyme may be present as a component of a whole cell, cell membrane, or organelle, or as a purified substance.
By carrying out the bioelectrochemical reaction in non-aqueous or microaqueous solvent the enzyme specificity may be made different from that in aqueous solution and the possibility exists of selecting particular specificities by making an appropriate choice of non-aqueous solvent. Furthermore, the solvent may be chosen so as to stabilise the enzyme substrate or product and hence enable the observation of otherwise difficult electrochemistry. Since thermal stability of enzymes is often enhanced in non-aqueous solvents reactions may also be carried out at elevated temperatures.
The method may be employed in the determination of an analyte in non-aqueous or microaqueous solution by including a non-aqueous o
REFERENCES:
patent: 4145255 (1979-03-01), Schiller et al.
patent: 4224125 (1980-09-01), Nakamura et al.
patent: 4556635 (1985-12-01), Hitzman et al.
Kazand Jian et al., J. Am. Chem. Soc. 107, 5448-5450, 1985.
Best David J.
Hall Geoffrey F.
Turner Anthony P. F.
Chan William K. Y.
Lacey David L.
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