Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Patent
1993-04-28
1996-06-11
Beisner, William H.
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
435817, 4352877, 204403, C12M 140
Patent
active
055255116
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to an electrochemical biosensor of the kind wherein an enzyme-containing reagent system is applied to an electrically conductive support member and which responds amperometrically to the catalytic activity of the enzyme in the presence of its substrate.
BACKGROUND OF THE INVENTION
Immobilisation of an enzyme upon an electrode impedes conduction of electrons between the active site of the enzyme and the electrode. Electron carriers or mediators have been employed to overcome this disadvantage and to enhance the reliability and sensitivity of the electron transfer.
Mediator assisted biosensors do not give consistent results upon repeated use, especially those made by screen printing methods. Commercial products are designed for use once or twice only, after which they must be discarded.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention an electrochemical biosensor comprises:
a conductive support member;
an enzyme system including a mediator immobilised on the support member; and
a covering membrane,
characterised in that the membrane incorporates a quantity of a mediator.
The mediator contained in the membrane may be the same or different to that of the enzyme system. Preferably the mediators are the same.
According to a second aspect of the present invention an electrical biosensor comprises:
a conductive support member;
an enzyme system incorporating a mediator immobilised on the support member; and
a conductive membrane;
characterised in that the biosensor provides a substantially quantitative and amperometric response when used for 50 repeat tests with an analyte.
We have found surprisingly that provision of a quantity of mediator within the membrane provides a biosensor which may be used repeatedly, for example preferred biosensors may be used 1000 times or more, without significant reduction in the quantitative and amperometric response. Biosensors in accordance with this invention may be incorporated into automatic analytical equipment for example for monitoring analytes in a sample stream, or implantation to allow in vivo metabolic analysis. Further applications include blood analysis for example for glucose, lactate, creatinine, urea, pyruvate etc. The invention may also be used for process control for industrial fermentation and brewing, amino acid production and for food processing, for example for monitoring glucose levels in meat. Monitoring of mammilarian cell cultures is also facilitated.
Membranes which may be applied to the enzyme containing layer of the biosensor act as a diffusion barrier, preventing loss of the components of the enzyme containing layer.
The membrane may be composed of various materials. Preferred materials include ethyl hydroxyethyl cellulose, ethyl cellulose, cellulose acetate, polyvinylchloride, polyurethane, polycarbonate, cellulose nitrate, polyurethane, cellulose acetate, aryl polyethers for example polyether sulphone or polyether ketone including functionalised polyaryl ethers, such as sulphonated polyether sulphonate.
Anionic or cationic ion exchange membrane materials may be used. For example Nafion membranes may be used to retain the mediator in the charged state.
In most applications non-ionic membranes are preferred.
Membrane thickness of 0.1 to 100 microns may be used.
In preferred embodiments of the invention wherein the enzyme system is partially soluble in aqueous solvents, the membrane and mediator are preferably soluble in a non aqueous solvent. This prevents leaching of the enzyme reagents during casting of the membrane. It also prevents solubilisation of the printed electrode matrix. The solvents for the membrane and mediator may be a mixture of two or more solvents, one of which may be a good solvent for the mediator and another being a good solvent for the membrane material, the volatilities of the solvents being selected so that the concentration of the mediator across the membrane may be selectively controlled.
The concentration of the mediator in the membrane may be up
REFERENCES:
patent: 3838033 (1974-09-01), Mindt et al.
patent: 4820399 (1989-04-01), Senda et al.
Patent Abstracts of Japan. vol. 14, No. 309(p.-1071) (4252) Jul. 4, 1990.
Mizutani et al. "Ferrocene-Mediated Enzyme Electrode . . . " Bull. Chem. Soc. Japan vol. 61, No. 12 (1988) pp. 4458-4460.
Pickup et al. "Potentially-Implantable, Amperometric Glucose Sensors . . . " Biosensors, vol. 4 (1989) pp. 109-119.
Beisner William H.
Environmental & Medical Products Ltd.
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