JPH0652248B2 - Biosensor - Google Patents

Description

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

2. Description of the Related Art Conventionally, a biosensor as shown in FIG. 5 has been proposed as a method for easily quantifying a specific component in a biological sample such as blood without diluting or stirring the sample solution. In this biosensor, electrode systems 2 and 3 made of carbon or the like are formed on an insulating substrate 1 by a method such as screen printing, and an enzyme made of a hydrophilic polymer, a redox enzyme and an electron acceptor is formed on the electrodes. The reaction layer 5 is formed.
When the sample solution is dropped onto the enzyme reaction layer, the oxidoreductase and the electron acceptor are dissolved in the sample solution, and the enzyme reaction proceeds with the substrate in the sample solution to reduce the electron acceptor. After completion of the reaction, the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention However, in such a conventional configuration, the enzymatic reaction and the electrode reaction are influenced by the temperatures of the outside air and the sample solution at the time of the reaction, and the response varies.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system comprising at least a measuring electrode and a counter electrode on an insulating substrate, and a substance concentration in the reaction of an enzyme, an electron acceptor and a sample solution. In a biosensor that electrochemically detects a change in the electrode system and measures the substrate concentration in a sample solution, an enzyme reaction layer containing a redox enzyme, a hydrophilic polymer and an electron acceptor is formed on the surface of the electrode system. It forms a substance and further adds a substance that undergoes an exothermic reaction upon dissolution.

Effects According to the present invention, a disposable type biosensor including an electrode system can be constructed, and the substrate concentration can be measured very easily by adding a sample solution to the sensor. Moreover, the substance added at the time of adding the sample generates heat to mitigate the influence of the outside air and the temperature of the sample, so that a stable response can be obtained.

Examples Examples of the present invention will be described below.

Example 1 A glucose sensor will be described as an example of a biosensor. FIG. 1 and FIG. 2 show an embodiment of the glucose sensor, and are a perspective view and a longitudinal sectional view of the constituent parts. An electrically conductive carbon paste is printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate, and dried by heating to form an electrode system consisting of a counter electrode 2 and a measuring electrode 3. Next, an insulating paste is printed in the same manner as above so as to partially cover the electrode system and leave 2 ', 3' (each 1) which becomes an electrochemically acting portion of each electrode, and heating. The insulating layer 4 is formed by processing. An aqueous solution of CMC (carboxymethylcellulose), which is a kind of cellulosic hydrophilic polymer, is applied so as to cover the surface of the electrode system (2 ′, 3 ′), and the solution is applied at 45 ° C. for 30 minutes.
Min dried. A solution of glucose oxidase (GOD) dissolved in a phosphate buffer of pH 5.6 as an oxidoreductase was applied on the obtained CMC layer, and then dried at room temperature.
Then, a mixture of toluene as an organic solvent and fine crystals of potassium ferricyanide, which is an electron acceptor, was added dropwise.
The enzyme reaction layer was formed by leaving it at room temperature to evaporate toluene. Further, magnesium chloride 6 was supported near the electrode.

10 μl of glucose standard solution was dropped as a sample solution into the glucose sensor configured as described above, and after 2 minutes, a pulse voltage of +0.6 V was applied to the measurement electrode in the anode direction based on the counter electrode, and the current was measured 5 seconds later. To do. Potassium ferricyanide is dissolved in the glucose standard solution, glucose is oxidized by GOD, and potassium ferricyanide is reduced to potassium ferrocyanide at this time. Then, by applying the above-mentioned pulse voltage, an oxidation current based on the concentration of the produced potassium ferrocyanide is obtained, and this current value corresponds to the concentration of glucose as a substrate. When a standard solution of glucose was dropped and the response current was measured, good linearity was obtained up to a high concentration of 500 mg / dl. Furthermore, during the reaction, the added magnesium chloride melts at the same time to generate heat,
In order to accelerate the enzymatic reaction, the reaction completion time was accelerated by 30 seconds at a glucose concentration of 100 mg / dl. Add 10 μl of blood sample to the glucose sensor and add 2
When the response current was measured after a minute, a very reproducible response was obtained. Further, when the temperature of the outside air was changed to 15 to 30 degrees and measured using a molar solution of potassium ferricyanide and potassium ferrocyanide, the response was changed as shown in FIG. 3 with and without addition of magnesium chloride. There was a difference. This is because the enzymatic reaction is affected by the temperature and the electrode reaction is also dependent on the temperature. The influence of temperature could be reduced by supporting magnesium chloride. To keep the temperature constant,
A constant temperature heater or heating equipment can be considered, but the sensor becomes large and the price becomes high. Magnesium chloride can be prepared simply by supporting it near the electrodes, and is considered to be very advantageous when mass-producing sensors.

Example 2 After supporting CMC and GOD as described in Example 1, lecithin (phosphatidylcholine) was dissolved as a surfactant in toluene when supporting potassium ferricyanide to prepare a 1 wt% solution. A layer of potassium ferricyanide and lecithin was formed using a mixture of microcrystals of potassium cyanide. Lecithin concentration is 0.
When the content is 01 wt% or more, potassium ferricyanide is well dispersed in toluene, making it easy to add drops of 3 μl.
A thin film potassium ferricyanide-lecithin layer could be formed even with a small amount of the liquid. In the absence of lecithin, there was a defect that the potassium ferricyanide layer was formed nonuniformly and peeled off when the substrate was bent. However, by adding lecithin, a potassium ferricyanide layer which was difficult to be removed uniformly was easily formed. As the lecithin concentration increases, the potassium ferricyanide layer becomes difficult to peel off, but the dissolution rate of potassium ferricyanide also decreases, so 0.01-3 wt% is considered appropriate. When a glucose standard solution was dropped onto the above sensor and the response was measured in the same manner as in Example 1, linearity was obtained up to a glucose concentration of 500 mg / dl. Furthermore, when blood was dripped, the lecithin layer immediately spread and the reaction started.
A reproducible response was obtained even with a small amount of sample such as l. The smaller the amount of the sample, the smaller the amount of magnesium chloride supported, and the effect was observed. Polyethylene glycol alkyl phenyl ether instead of lecithin (trade name:
When Triton X) was used, 0.1% or more was required to disperse the fine particles of potassium ferricyanide in toluene, but a good potassium ferricyanide layer could be formed as with lecithin. As the surfactant, in addition to the above examples, those which disperse the electron acceptor in an organic solvent and do not affect the enzyme activity, such as oleic acid, polyoxyethylene glycerin fatty acid ester, and cyclodextrin. However, there is no particular limitation.

As the hydrophilic polymer, gelatin, methyl cellulose, etc. can be used in addition to CMC, and starch-based, carboxymethylcellulose-based, gelatin-based, acrylate-based, vinyl alcohol-based, vinylpyrrolidone-based, and maleic anhydride-based ones are preferable. Since these polymers can be easily made into an aqueous solution, a thin film having a required thickness can be formed on the electrode by applying and drying an aqueous solution having an appropriate concentration.

The organic solvent to be mixed with the electron acceptor may be toluene, petroleum ether, or the like as long as it has little effect on the GOD activity and the printed electrode.

Screen printing as a method of forming an electrode system can inexpensively produce a disposable type biosensor having uniform characteristics, and in particular, an electrode is formed using carbon, which is a cheap and stable electrode material. It is a convenient method to form.

In addition to magnesium chloride, sodium phosphate and sodium acetate could also be used as the substances that generate heat when dissolved in the sample solution. When the solution heats up to generate heat, the larger the heat of solution, the smaller amount it needs to be supported, but it is not desirable because the pH of the solution that affects the enzyme reaction must be isolated.

Example 3 The sensor having the structure shown in Example 2 was provided with a cover 7 as shown in FIG. Magnesium chloride 6 was added to the inside of this cover to assemble the sensor. When blood was supplied to the spotting part of the cover, it spread quickly to the electrode part and a response with good reproducibility was obtained. By reducing the volume in the cover, the sample amount could be made very small and a fixed amount could be supplied. In addition, since it can be shielded from the outside air by surrounding it with a cover, the effect of magnesium chloride in the cover can be further exerted.

The biosensor of the present invention is not limited to the glucose sensor shown in the above examples, but can be used in systems involving oxidoreductase such as alcohol sensor and cholesterol sensor. Glucose oxidase was used as the oxidoreductase in the examples, but other enzymes such as alcohol oxidase, cholesterol oxidase, xanthine oxidase and the like can be used. Further, as the electron acceptor, potassium ferricyanide used in the above-mentioned examples is suitable because it reacts stably, but when P-benzoquinone is used, the reaction rate is high, and therefore it is suitable for speeding up. Also, 2.6-dichlorophenol indophenol, methylene blue, phenazine methosulfate, β
-Naphthoquinone 4-sulfonate potassium, ferrocene and the like can be used.

Effects of the Invention As described above, the biosensor of the present invention prints an electrode system on an insulating substrate to form an enzyme reaction layer containing a redox enzyme, a hydrophilic polymer and an electron acceptor, and further dissolves it. By adding a heat-generating substance, the substrate concentration in the biological sample can be measured extremely easily, and the heat-generating substance alleviates the influence of temperature and improves the measurement accuracy. Further, when a surfactant is added when forming the electron acceptor layer, a small amount of the electron acceptor can be uniformly carried on the thin film layer which is difficult to peel off, which has a great effect on storability and mass production.

[Brief description of drawings]

FIG. 1 is a perspective view of a biosensor according to an embodiment of the present invention, FIG. 2 is a longitudinal sectional view of the biosensor, FIG. 3 is a graph showing the response characteristics of the biosensor, and FIG. FIG. 5 is a vertical sectional view of a biosensor of another embodiment, and FIG. 5 is a vertical sectional view of a biosensor of a conventional example. 1 ... Insulating substrate, 2 ... Counter electrode, 3 ... Measuring electrode, 4 ...
Insulation layer, 5 ... Enzyme reaction layer, 6 ... Magnesium chloride,
7 ... Cover

Claims (3)

[Claims] 1. An insulating substrate provided with an electrode system comprising at least a measuring electrode and a counter electrode, and an enzyme reaction layer containing a redox enzyme, a hydrophilic polymer and an electron acceptor is provided on the surface of the electrode system. Further, a substance that causes an exothermic reaction upon dissolution is added, and a change in the substance concentration during the reaction between the enzyme, the electron acceptor and the sample solution is electrochemically detected by the electrode system to measure the substrate concentration. And biosensor. 2. An enzyme comprising an insulating substrate provided with an electrode system comprising at least a measuring electrode and a counter electrode, and an electron acceptor containing a redox enzyme, a hydrophilic polymer and a surfactant on the surface of the electrode system. A reaction layer is provided, and a substance that causes an exothermic reaction upon dissolution is added, and a change in the substance concentration during the reaction between the enzyme, the electron acceptor and the sample solution is electrochemically detected by the electrode system to determine the substrate concentration. A biosensor characterized by measuring. 3. The biosensor according to claim 1, wherein the electrode system is made of a carbon-based material formed on the insulating substrate by screen printing.