Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Patent
1990-10-16
1993-12-21
Alexander, Lyle A.
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
435817, 435181, 435177, 435176, 435288, 204402, 204403, 20415312, C25B 1112
Patent
active
052720870
DESCRIPTION:
BRIEF SUMMARY
The present invention was made at the Laboratoire R.M.N. et Reactivite Chimique (NMR and Chemical Reactivity Laboratory) at the University of Nantes, Centre National de la Recherche Scientifique (National Scientific Research Center) associate unit No. 472.
the present invention relates to an enzymatic electrode, in particular to an electrode having a surface which is renewable by friction or cutting, such that its useful surface area always remains the same, whatever the shape and the application of the electrode. The invention also applies to an enzymatic electrode of the disposable type. The invention also relates to the preparation of this electrode.
An enzyme electrode results from the combination of an electrochemical sensor and an enzyme, in particular in immobilized form. The functioning of an electrode of this type is based on the principle than an enzymatic degradation of the substrate to be determined is effected and that, in parallel, the appearance or the disappearance of one of the reaction products is measured.
Thus, in the case of the glucose electrode containing glucose oxidase as enzyme, the enzymatic reaction is as follows: ##STR1##
Several types of electrochemical detection can be envisaged, namely measurement of the disappearance of O.sub.2, measurement of the appearance of H.sup.30 ions and the anodic oxidation of H.sub.2 O.sub.2 (platinum electrode) at a set potential and measurement of the resulting current.
Various techniques are known for immobilization of the enzymes, namely inclusion, according to which the enzyme is mechanically trapped in a polymer in the form of gel; crosslinking, according to which bifunctional chemical molecules, such as glutaraldehyde, permit crosslinking of enzyme molecules between one another or their co-crosslinking with inactive proteins, such as gelatin and albumin; confinement, according to which the enzyme is in solution inside a compartment separated from the solution to be determined by a porous membrane which allows the passage only of the small molecules; and covalent fixation which is effected by reaction between the functional groups carried by an activated surface and functional groups of the enzyme which do not participate in the catalytic activity of the latter.
Moreover, the dependence of the enzymatic reaction on the amount of oxygen dissolved in the solution can in some cases, in particular for determination in biological liquids, be a limiting factor for this determination technique. In fact, variations in the oxygen pressure can lead to significant fluctuations in the responses of the electrode; similarly, these responses are distorted if the amount of oxygen present becomes low. These disadvantages have led to the recommendation of the use of oxygen substitutes, in particular ferrocene and its derivatives, which are termed mediators. The reactions involved in this case are as follows in the case of the determination of glucose by means of a glucose oxidase GOD electrode using ferrocene as mediator: +2[C.sub.5 H.sub.5 ].sub.2 Fe+2H.sup.+ [ 2] +2e [3]
The electrochemical detection is the oxidation at the "naked" electrode of ferrocene to the ferricinium cation (reaction 3). Ferrocene can thus be introduced in a catalytic amount relative to the glucose and the current due to its electrochemical regeneration will serve as a glucose detection and determination signal.
This method has enabled satisfactory results to be obtained, but sometimes has the major drawback of introducing a substance which is foreign to the medium and which can prove toxic.
Conventionally, in enzymatic electrodes, the enzymes are kept near the surface of the electrode. This is the case in particular for the GOD electrode described by T. IKEDA et al. in Agric. Biol. Chem. 49(29), 541-543, 1985 and in Agric. Biol. Chem. 50(4), 883-890, 1986, the surface of the GOD-loaded electrode being covered by a film of nitrocellulose (cf. European patent application EP-A-177,743). In the publication "Analytical Sciences Dec. 1985, vol. 1", pages 455-457, IKEDA et al. describe a GO
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"Magnetic Enzyme Membranes as Active Elements of Electrochemical Sensors, Specific Amino Acid Enzyme Electrodes" Calvot et al., FEBS Letters 59(2); 258-262; 1975.
El Murr Nabil
Slilam Mohamed
Alexander Lyle A.
Centre National de la Recherche Scientifique (C.N.R.S.)
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