JPS6264940A - biosensor - Google Patents

Abstract

PURPOSE:To measure an objective specific substrate with high accuracy, by immobilizing the enzyme for recognizing the specific substrate and the enzyme for converting the substance obstructing the detection of the specific substrate to an innoxious substance in an enzyme immobilizing membrane. CONSTITUTION:The enzyme catalyzing the specific reaction with a specific substrate to be measured and the enzyme for converting the substance obstructing the measurement of the specific substrate to an innoxious substance are simultaneously immobilized in the enzyme immobilizing membrane of a biosensor. For example, ascorbic acid oxidase is immobilized in an enzyme fixing membrane for detecting glucose along with glucose oxidase. Even if ascorbic acid is present in a liquid specimen, ascorbic acid is oxidized by ascorbic acid oxidase to be prevented from obstructing the detection of glucose. Because the biosensor is constituted so as to remove the action of the substance obstructing the substance can be measured with high accuracy.

Description

[Detailed Description of the Invention] [Summary] Measurement accuracy is improved by immobilizing an enzyme that converts interfering substances into harmless substances in the enzyme-immobilized membrane of a biosensor.

[Industrial application field]

The present invention relates to a biosensor, and particularly to a biosensor that decomposes substances that interfere with biosensor measurements in an enzyme-immobilized membrane and provides highly reliable measurement values with small errors.

[Conventional technology]

A biosensor consists of a molecular recognition site that recognizes a specific substance and a transducer that converts the recognition result into an electrical signal.A biosensor catalyzes a specific reaction of a specific substance using an enzyme immobilized in the membrane. Substances, heat, light, etc. produced by this specific reaction are detected using electrodes, thermal change detectors, photodetectors, etc.

For example, an enzyme electrode for glucose detection is known, which consists of a membrane on which glucose oxidase is immobilized and a hydrogen peroxide electrode. When this enzyme electrode is immersed in a sample solution containing glucose, the next reaction is catalyzed by glucose oxidase as the glucose passes through the enzyme-immobilized membrane.

Glucose + 0□ + H, O → Gluconic acid + H20! Therefore, by knowing the amount of current generated by reducing the reaction products H and O□ with a hydrogen peroxide electrode, the degree of glucose) can be measured.

[Problems to be Solved by the Invention] However, in the above enzyme electrode for glucose detection, if a substance that is easily oxidized is mixed in the sample solution,
The drawback is that an extra current is generated in the hydrogen peroxide electrode depending on the agricultural level, resulting in a large error in the measured value of glucose concentration. In particular, when measuring glucose in blood, ascorbic acid in the blood is a substance that is easily oxidized, so its presence interferes with the measurement of glucose.

Such interference by ascorbic acid in the blood is not limited to the measurement of glucose; components such as urea and L-lysine in the blood are inhibited by specific enzymes (for example, uricase for urea and L-lysine carboxylase for L-lysine). ) and is detected using a hydrogen peroxide electrode. Furthermore, the problem of interfering substances is a common problem in biosensors.

[Means for solving problems]

In order to solve the above-mentioned problems, the present invention provides oxygen that catalyzes a specific reaction of a specific substrate to be measured, as well as harmless substances that interfere with the measurement of the specific substrate, in the enzyme-immobilized membrane of a biosensor. This method simultaneously immobilizes oxygen, which is converted into a chemical substance.

For example, in the example of the enzyme electrode for glucose detection described above, ascorbate oxidase and glucose oxidase are simultaneously immobilized in the enzyme immobilization membrane. This ascorbic acid oxidase oxidizes ascorbic acid present in the sample solution as shown in the following formula in the enzyme-immobilized membrane before the ascorbic acid present in the sample solution reaches the hydrogen peroxide electrode.

The following margin is HCOHHCOH I This reaction product (dehydroascorbic acid) is inert to the electrode reaction of the hydrogen peroxide electrode. Therefore, by immobilizing ascorbic acid oxidase in an enzyme-immobilized membrane, is it possible to easily prevent the enzyme from being interfered with by ascorbic acid? It pole is realized.

The biosensor to which the present invention is applicable identifies a specific substrate using an enzyme, catalyzes a reaction specific to that substrate,
There is no particular limitation as long as it is a device that converts a specific substance, heat, light, etc. that increases or decreases due to this reaction into an electrical signal. Furthermore, the enzyme that recognizes the specific substrate and the enzyme that detoxifies the interfering substance are not particularly limited, and an appropriate enzyme may be selected and used depending on the type and amount of the specific substrate and interfering substance.

〔Example〕

Glucose oxidase lQmI! and ascorbic acid oxidase 10mj! 5 ml of an aqueous solution of acrylamide monomer and N,N'-methylenebisacrylamide
The solution was cast onto a glass substrate to a thickness of 50 mm.
A 50 μm thick enzyme-immobilized membrane was prepared by pressing a glass plate using μm Teflon tape as a spacer. This membrane was attached tightly to the anode part of a hydrogen peroxide electrode to form a biosensor.

This biosensor was used to measure various concentrations of glucose solutions containing 10 −4 mol/l of ascorbic acid.

For comparison, glucose oxidase 10 was added in the same manner as above, but without the addition of ascorbic acid oxidase.
By adding only (1), an enzyme-immobilized membrane and a biosensor were created. Using this biosensor, the same glucose solution as above was measured.

The above measurement results are shown in FIG. From FIG. 1, the effect of ascorbic acid appears on the measured values with the conventional biosensor, but on the other hand, with the biosensor of the present invention, the interference caused by ascorbic acid is eliminated and a measured value proportional to the glucose concentration is obtained. It is recognized that

[Effects of the Invention] According to the present invention, the effects of substances that interfere with the measurement of a specific substrate in a biosensor can be removed by enzymes, so that precise measurement of the desired specific substrate can be performed. Furthermore, in the present invention, the effect of interfering substances can be removed by simply adding another enzyme to the enzyme-immobilized membrane, and the device is simple.

[Brief explanation of drawings]

FIG. 1 is a graph showing the measurement results of glucose solution samples by the biosensors of Examples and Comparative Examples. Biosensor output comparison Figure 1

Claims (1)

[Claims] 1. Consisting of a molecular recognition site that recognizes a specific substrate in the sample solution and a signal conversion site that converts changes occurring in the molecular recognition site into an electrical signal, and as a molecular recognition site that reacts specifically to the specific substrate. A biosensor using an enzyme-immobilized membrane on which a catalytic enzyme is immobilized, further comprising an enzyme that converts a substance that interferes with the detection of the specific substrate into a harmless substance in the enzyme-immobilized membrane. A biosensor featuring: