JPH0654304B2 - Biosensor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biosensor capable of quantifying specific components in various trace amounts of biological samples quickly and easily without diluting the sample solution. .

2. Description of the Related Art Conventionally, a biosensor as shown in FIG. 3 has been proposed as a method for highly accurately quantifying a specific component in a biological sample such as blood without performing operations such as dilution and stirring of a sample solution. ing. This biosensor has an insulating substrate 15
A measuring electrode 11 and a counter electrode 12 made of platinum or the like, and respective leads 13 and 14 are embedded in the porous body 1 and the exposed portions of these electrode systems contain a redox enzyme and an electron acceptor.
6 is covered with a permeation film 10 for separating the substance that interferes with the measurement. When the sample solution is dropped onto the porous body 16, the electron acceptor in the porous body is dissolved in the sample solution, an enzymatic reaction proceeds with the substrate in the sample solution, and the electron acceptor is reduced. In the sample solution after the reaction, giant membranes such as red blood cells and white blood cells in the blood that interfere with the measurement are overmembrane 1
0, and the sample reaction solution containing only low molecular weight substances such as electron acceptors and salts is dropped onto the electrodes 11 and 12.
The reduced electron acceptor was electrochemically oxidized on the electrode, and the substrate concentration in the sample solution was obtained from the oxidation current value obtained at this time.

Problems to be Solved by the Invention However, in such a conventional configuration, although it can be used as a sensor, the drop of the sample reaction solution onto the electrode becomes non-uniform, and the electrode surface is not sufficiently wet, so bubbles are not formed. Left over,
The phenomenon that the electrode area was reduced occurred, the measured values were unstable, and the reproducibility was poor.

The present invention is to solve such a problem, by providing a water-absorbing polymer layer on the measurement electrode and the counter electrode, to form a stable liquid film layer, the purpose of enabling stable measurement To do.

Means for Solving the Problems In order to solve this problem, the present invention provides a water-absorbing polymer that forms a stable gel liquid layer that sufficiently covers an electrode surface on an electrode system including at least a measuring electrode and a counter electrode. It is provided with layers. As a result, the water-absorbing polymer layer absorbs the reaction liquid in which the reaction between the enzyme, the electron acceptor and the sample liquid is completed, and a uniform gelled reaction liquid layer is formed on the electrode, which provides stable measurement. Is to do.

As polymers that absorb water and gel, natural polymers include starch-based, cellulose-based, alginic acid-based, gums and protein-based polymers, and synthetic polymers include vinyl-based, acrylic acid-based, and anhydrous polymers. There are various types such as maleic acid type, aqueous urethane type, polyelectrolyte type, but especially starch type,
Carboxymethylcellulose-based, gelatin-based, acrylate-based, vinyl alcohol-based, vinylpyrrolidone-based, and maleic anhydride-based ones are preferable. These may be homopolymers, mixtures, or copolymers. Since these polymers can be easily made into an aqueous solution, there is an advantage that a thin film having a required thickness can be directly formed on an electrode by applying and drying an aqueous solution having an appropriate concentration.

Operation With this configuration, the reaction liquid obtained by the reaction of oxygen, electron acceptor, and sample liquid falls onto the electrode, is absorbed by the water-absorbing polymer layer on the electrode, and is in close contact with the electrode, so that the electrode surface is sufficiently covered. Since the gel layer covered with the above is stably formed, nonuniformity of wetting of the electrode, residual bubbles, etc. can be eliminated, and stable electrochemical measurement can be performed. Furthermore, it is possible to perform highly reliable measurement, such as suppressing the fluctuation of the response current due to the vibration of the sensor.

Example One example of the present invention will be described below.

A glucose sensor will be described as an example of a biosensor. FIG. 1 shows an embodiment of the glucose sensor and is a sectional view of the structure of the sensor. A conductive carbon paste is printed on the insulating substrate 8 made of polyethylene terephthalate by screen printing, and dried by heating to form an electrode system including the measurement electrode 6 and the counter electrode 7, and a lead portion (not shown in the drawing). Form. Next, cover the electrodes partially to obtain a constant electrode area,
The insulating paste is printed and dried in the same manner as above to form the insulating layer 5. The porous body 1 and the porous membrane 2 made of polycarbonate and having a pore diameter of 1 μm are held by holding frames 3 and 4. The porous body 1 is glucose oxidase 1 which is an oxidoreductase.
00 mg and potassium ferricyanide as electron acceptor 15
A solution prepared by dissolving 0 mg in 1 ml of a phosphate buffer (pH 5.6) was impregnated into cellulose paper and dried. Reference numeral 9 denotes a water-absorbent polymer layer according to the present invention, which is obtained by directly coating a 1% aqueous solution of carboxymethyl cellulose on an electrode and drying it, and the film thickness after drying is 2 μ.

An aqueous glucose solution was dropped as a sample solution onto the porous body 1 of the glucose sensor having the above configuration, and after 2 minutes, the potential of the measurement electrode 6 was moved toward the anode. Swept at a rate of 2 V / sec. The dropped glucose reacts with potassium ferricyanide to produce potassium ferrocyanide by the action of glucose oxidase carried on the porous body 1. The sample reaction solution after the completion of this reaction passes through the permeation membrane 2 and is absorbed by the water-absorbing polymer layer 9 to be water-soluble by the water-absorbing polymer containing potassium ferrocyanide which is in close contact with the electrode and completely covers the electrode area. The gel layer 9 is formed. By the above sweeping in the anode direction, the produced potassium ferrocyanide is electrochemically oxidized to potassium ferricyanide, and the peak of the oxidation current is obtained. This oxidation peak current value corresponds to the glucose concentration in the sample.

FIG. 2 shows the relationship between the oxidation peak current value and the glucose concentration. In the figure, A is the case where the carboxymethyl cellulose thin film layer of the present invention is provided, and B is the case where the conventional thin film layer is not provided. The average value measured five times at each glucose concentration and the width of variation are shown. A shows a good linearity, and the variation in each glucose concentration is small, but in B of the conventional example, the variation is very large, and a partly abnormally small current value is shown. When the state on the electrode is examined when the current value is small like this, wetting on the electrode is poor,
It has been found that only part of the electrode is wet or there are bubbles remaining on and between the electrodes. On the other hand, when the gel layer 9 made of the water-absorbent polymer is formed, a stable and hard-to-flow liquid layer is formed on the electrode even when the amount of the liquid passed is small, and no bubbles remain. It was found that the electrode surface was completely wet. Further, when the sensor was vibrated during the measurement, a large variation in the response current corresponding to the vibration was observed in the conventional example B, but it is possible to perform a highly reliable measurement such as hardly observed in the A of the present invention. It was

It has been found that the water-absorbent polymer layer of the present invention works effectively in the range of a certain film thickness which is the basis of the dry state, and the range is slightly different depending on the polymer material. For example, in the case of the above-mentioned carboxymethyl cellulose, a film thickness of 0.5 to 50 μ is suitable, but acrylate-based polymer aquakeep 10
In the case of SH (manufactured by Iron and Steel Chemical Co., Ltd.), 0.1 to 2
A range of 0μ is suitable. As a result of various studies, it was found that the range of 0.1 to 100 μ is preferable for forming a stable gel layer. At a film thickness of 0.1 μ or less, a stable gel layer cannot be obtained because the liquid layer easily flows, and conversely, at a film thickness of more than 100 μ, when the sample liquid is a very small amount of several μ to several tens of μ, It was found to be unsuitable due to the insufficient diffusion of the liquid and the generation of a portion that does not gel.

Further, a stable value was obtained even when blood was used as a sample solution and measured by the glucose sensor. Although not shown in the drawing, it is preferable that a thin piece of hydrophilic porous material such as cellulose or rayon be interposed as a liquid-retaining layer between the overmembrane 2 and the water-absorbent polymer layer 9 to allow the sample solution to have an excessive velocity. Was faster, and the liquid could be absorbed into the water-absorbent polymer layer quickly and uniformly.

In the above embodiment, the bipolar electrode system having only the measurement electrode and the counter electrode was described, but more accurate measurement is possible by using the three-electrode system in which the reference electrode is added. Further, the shape of the filtration membrane, the holding frame, the porous body, or the like or the presence or absence thereof is not limited to the above embodiment. Furthermore, the same applies to the carrying state of the enzyme and the electron acceptor.

Further, as the electron acceptor, p-benzoquinone, phenazine methosulfate and the like can be used in addition to potassium ferricyanide used in the above examples. Furthermore, in the sensor of the above embodiment, if an alcohol oxidase other than glucose oxidase of the above embodiment, cholesterol oxidase, or the like is used as the enzyme, alcohol sensor,
It can also be used as a cholesterol sensor.

Effects of the Invention As described above, the biosensor of the present invention is a stable gel that sufficiently wets the electrode surface even with a small amount of liquid by providing a water-absorbing polymer layer that forms a stable gel liquid layer on the electrode system. The effect of forming a liquid layer and enabling stable and accurate measurement is obtained.

[Brief description of drawings]

FIG. 1 is a sectional view of a biosensor which is an embodiment of the present invention, FIG. 2 is a response characteristic diagram of the biosensor, and FIG. 3 is a sectional view of a conventional biosensor. 1 ... Porous body, 2 ... Permeable film, 5 ... Insulating layer, 6 ... Measuring electrode, 7 ... Counter electrode, 8 ... Insulating substrate, 9 ... Water absorbing polymer layer.

Claims (4)

[Claims] 1. An electrode system comprising at least a measuring electrode and a counter electrode, wherein a change in substance concentration during a reaction between an enzyme, an electron acceptor and a sample solution is electrochemically detected by the electrode system, A biosensor for measuring a substrate concentration, wherein a water-absorbing polymer layer forming a stable gel liquid layer is formed on the electrode system. 2. The water-absorbent polymer layer has a thickness of 0.1 to 100.
The biosensor according to claim 1, wherein the biosensor is μ. 3. The water-absorbing polymer is any one of the group consisting of starch type, carboxymethylcellulose type, gelatin type, acrylate type, vinyl alcohol type, vinylpyrrolidone type and maleic anhydride type or a mixture thereof. The biosensor according to claim 1. 4. The biosensor according to claim 1, wherein a liquid retaining layer made of a hydrophilic porous material is provided on the water absorbent polymer layer.