FR2656423A1 - Electrochemical biosensor - Google Patents

Abstract

Electrochemical biosensor consisting of a single-piece electrode comprising an anode and a cathode; a cap provided with a central perforation and a neutral membrane permitting the diffusion of substrate is attached to the end of the sensor; the biosensor contains, in the space available between the cap and the body of the electrode and adjacent to the working electrode, enzymes immobilised on particles of a polymer which is non-soluble in the reaction medium. Use for the assay of organic substrates liberating hydrogen peroxide by enzymatic reaction.

Description

ELECTROCHEMICAL BIOSENSOR
The present invention relates to a biosensor consisting of an enzymatic electrode and its application for the determination of organic substrates.

I1 is known to measure glucose for example using an amperometric sensor to H2O2 immersed in a tank in which is carried out the enzymatic oxidation of glucose to gluconic acid with release of
H2O2; the amount of H2O2 formed is directly proportional to the amount of glucose used. H202 is detected amperometrically v: a fixed potential difference (ddp), of approximately 600 mV, between a working electrode (platinum) and a reference electrode (Ag / AgCl) allows the electrochemical oxidation of H202 on l 'platinum anode; a variation in anode current proportional to the concentration of H 2 O 2 formed by the enzymatic reaction can thus be demonstrated on a recorder. Such a method involving a relatively not very fragile enzyme such as glucose oxidase is particularly effective.

By cons it is poorly suited to dosages involving fragile enzymes causing inactivation phenomena or interaction between substrate and product of the enzymatic reaction. This is the case with the determination of pyruvate using pyruvate oxidase. Indeed it has been found that the substrate (pyruvate) and the hydrogen peroxide formed interact with the formation of a peracid.

If pyruvate oxidase is introduced into the measuring tank, the hydrogen peroxide formed reacts quickly with the pyruvate; this results in a decrease in the concentration of the two species in the tank; the dosage is then inaccurate.

The Applicant has solved this problem by introducing the enzymes inside the electrochemical sensor.

According to the invention, it is an electrochemical biosensor consisting of a single-body electrode comprising an anode and a cathode, and attaching to the end of the sensor, a nozzle provided with a central perforation and a neutral membrane allowing the diffusion of the substrate, biosensor characterized in that it contains in the space available between the tip and the body of the electrode and bordering the "working electrode" of the enzymes immobilized on particles of polymer not soluble in the reaction medium.

The biosensor of the invention based on amperometric detection is very particularly advantageous for the determination of organic substrates releasing by enzymatic reaction of oxygenated water. The "working electrode", the anode, consists of a platinum disc where the electrochemical reaction takes place.

The reference electrode, the cathode, consists of a silver spiral wire covered with silver chloride.

An electrolyte present in the space between the tip and the body of the electrode ensures the conductivity between the anode and the cathode; the presence of potassium chloride in the electolyte ensures permanent chlorination of the reference electrode.

The body and the tip of the electrode are preferably made of synthetic resin of the polyvinyl chloride type.

The membrane is neutral; it can be made of synthetic fibers (polyester, polyamide ...); its thickness is generally of the order of 50 to 150 micrometers with a pore diameter smaller than that of the polymer particles and generally of the order of 0.1 to 0.8 micrometers.

The polymers which can constitute the support particles are derived from water-immiscible monomers (that is to say solubility in water of less than 5% by weight).

Among these monomers, mention may be made
- vinyl aromatic monomers (styrene, vinyltoluene ...)
- alkyl esters of unsaturated a-ss acids (methyl, ethyl acrylates and methacrylates ...)
- esters of unsaturated carboxylic acids (vinyl acetate, etc.)
- vinyl chloride; vinylidene chloride
- dienes (butadiene ...)
- those with nitrile functions (acrylonitrile ...)
- siloxanes.

- COOR, - PO(OR)2,

- # CH2C1,
R représentant un radical alkyle en Cl - C4, de préférence en Cl - C2
A titre d'exemple on peut citer
- le vinylbenzène sulfonate, les sulfoalkylesters d'acides insaturés (2-sulfoéthylméthacrylates...)
- les acides carboxyliques insaturés (acide acrylique, méthacrylique, maléfique, itaconique...)
- les hydroxyalkylacrylates ou méthacrylates (acrylate d'hydroxyéthyle, hydroxypropyle...)
- les aminoalkylesters d'acides insaturés (2-aminoéthylméthacrylate...)
- l'acrylamide
- le chlorure de vi nyl benzène
- le méthacrylate de glycidyle.The monomer composition from which said polymer is derived also contains up to 10% of its weight (preferably up to 4% of its weight) of at least one monomer carrying ionogenic or reactive groups such as - SO 3 H, - OSO 3 H, - NR3, - COOH, - OH, - NH2, - NR2,
CONH2, - SH,

- COOR, - PO (OR) 2,

- # CH2C1,
R representing a C1 - C4, preferably C1 - C2 alkyl radical
As an example we can cite
- vinylbenzene sulfonate, sulfoalkyl esters of unsaturated acids (2-sulfoethylmethacrylates ...)
- unsaturated carboxylic acids (acrylic, methacrylic, malefic, itaconic ...)
- hydroxyalkylacrylates or methacrylates (hydroxyethyl acrylate, hydroxypropyl ...)
- aminoalkylesters of unsaturated acids (2-aminoethylmethacrylate ...)
- acrylamide
- vi nyl benzene chloride
- glycidyl methacrylate.

Said polymer particles can have a particle size of the order of 0.2 to 20 microns and preferably of the order of 0.2 to 3 microns.

A variant consists in using, as support particles, non-water-soluble magnetizable polymer particles.

The particles of magnetizable polymer contain from 0.5 to 70% by weight (preferably from 5 to 50%) of a magnetic charge whose size is less than 1 ian and preferably between 0.002-0.02 ian; the magnetic charge is obviously fine enough to be able to be included in the polymer particles. This magnetic charge can be constituted for example by
- metals or their alloys such as: iron, iron-silicon, nickel, cobalt, samarium or their alloys with molybdenum, chromium, copper, vanadium, manganese, aluminum, titanium, neodymium, etc. .

- iron oxides: Fe304 or y-Fe03 pure or in combination or in mixture with other oxides such as cobalt, manganese, zinc, barium, rare earth oxides
- chromium dioxide.

The particles of magnetizable polymer can be obtained in the form of latex according to known methods for example according to the methods described in European patent no 38,730 or American patent n 4,157,323.

The immobilization of the enzymes on the particles of magnetizable or non-magnetizable polymer can be carried out by adsorption or else by coupling, the coupling reaction involving the reactive or functional surface groups of the polymer and the functional groups of the enzyme to be fixed.

The coupling reaction can be carried out according to well-known methods, for example by most often calling on the activation of the polymer functions using coupling agents (diazotization, cyanogen bromide, tosyl chloride, glutaraldehyde, water-soluble carbodiimide,
N-hydroxybenzotriazole ...) with or without the use of spacers of type 1-6 diaminohexane, polysaccharide ..., then reaction with the enzyme to be fixed.

Examples of coupling reactions are given in the US patents no 3,857,931, 4,045,384, 4,140,662, 4,046,723, 4,421,896, English no. 2,004,892, French no. 2,333,567, 2,345,459, 2,378,094, European no 15,841 ...

The immobilization protocol must be adapted according to the specific characteristics of the enzyme used (optimum pH, specific activity, etc.)
The amount of enzyme immobilized on the polymer particles is generally of the order of 10 to 500 units of enzyme per gram of polymer (preferably from 100 to 200).

The biosensor forming the subject of the invention being particularly advantageous for the assays of organic substrates releasing by enzymatic reaction of oxygenated water, the enzymes used are in particular the oxidases such as for example
- lactate oxidase to measure L-lactate
- glucose oxidase to measure glucose
- pyruvate oxidase to dose pyruvate
- alcohol oxidase to measure short alcohols (methanol, ethanol, propanol ...)
During its use, the biosensor connected to a polarostat imposing a potential difference between the anode and the cathode, is immersed in a thermostated tank (generally around 30 ') containing the substrate to be assayed.

Since the enzymes immobilized inside the biosensor cannot diffuse outside the electrode, hydrogen peroxide is formed in the vicinity of the anode.

In the case of pyruvate the reaction scheme is as follows pyruvate + O2 + phosphate

<tb> pyruvate <SEP> oxidase,
<tb> acetylphosphate + C02 + H202
The amount of H2O2 formed is directly proportional to that of the pyruvate present.

02 + 2H+ + 2e
Ag/Ag Cl/C1
Un enregistreur relié au polarostat permet de lire la variation du courant en fonction du temps.Oxygenated water, as it is formed, is oxidized at the anode according to the reaction
ddp (+ 500 mV) H2O2

02 + 2H + + 2nd
Ag / Ag Cl / C1
A recorder connected to the polarostat makes it possible to read the variation in current as a function of time.

The same operation carried out with a conventional biosensor (not containing the immobilized enzyme) immersed in a tank containing pyruvate and pyruvate oxidase immobilized on polymer particles cannot lead to a satisfactory assay of the substrate because the latter interacts on the hydrogen peroxide formed.

The implementation of this biosensor is easy and rapid; it can be reused.

Claims (8)

1) Electrochemical biosensor consisting of a single-body electrode comprising an anode, a cathode, and attaching to the end of the sensor, a nozzle provided with a central perforation and a neutral membrane allowing the diffusion of the substrate, biosensor characterized in that it contains in the space available between the tip and the body of the electrode and surrounding the working electrode of the enzymes immobilized on polymer particles not soluble in the reaction medium. 2) Biosensor according to claim 1 characterized in that the working electrode is the anode and in that it consists of a platinum disc. 3) Biosensor according to claim 1 or 2 characterized in that the reference electrode is the cathode and in that it consists of a silver wire covered with silver chloride. 4) Biosensor according to any one of the preceding claims, characterized in that the polymer particles on which the enzymes are immobilized derive from a water-immiscible monomer composition containing up to 10% by weight of at least a monomer carrying ionogenic or reactive groups. 5) Biosensor according to claim 4 characterized in that the polymer particles have a particle size of the order of 0.2 to 20 microns. 6) Biosensor according to claim 5 characterized in that the polymer particles contain from 0.5 to 70% of their weight of a magnetizable charge. 7) Biosensor according to any one of the preceding claims, characterized in that the quantity of enzyme immobilized on the polymer particles is of the order of 10 to 500 units of enzyme per gram of polymer. 8) Use of the biosensor which is the subject of any one of the preceding claims for the determination of organic substrates releasing by enzymatic reaction of hydrogen peroxide.