JPH0814562B2 - 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, there is a biosensor as shown in FIG. 5 as a method for easily quantifying a specific component in a biological sample such as blood without diluting or stirring the sample solution. This biosensor has an electrode system 12 and 13 made of carbon or the like formed on an insulating substrate 11 by a method such as screen printing, and an enzyme made of a hydrophilic polymer, a redox enzyme and an electron acceptor on the electrode. The reaction layer 15 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, which is detected electrochemically by the electrode system and corresponds to the reduction amount of the electron acceptor.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention However, in this conventional biosensor, there is a problem in that the enzymatic reaction and the electrode reaction are affected by the temperature of the outside air and the sample solution at the time of the reaction and the response varies, and a stable response is difficult to obtain. there were.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system comprising at least a measurement electrode and a counter electrode on an insulating substrate, and the surface of the electrode system contains a redox enzyme and a hydrophilic polymer. And an electron acceptor are formed, and a change in the substance concentration during the reaction between the oxidoreductase, the electron acceptor and the sample solution is electrochemically detected by the electrode system to determine the substrate concentration in the sample solution. In the biosensor to be measured, a heating device is integrally attached to the back surface of the insulating substrate. In the present invention, a PTC thermistor is attached to the surface of the insulating substrate, and a cover surrounding the electrode system and the PTC thermistor is attached.

Effect According to the present invention, since the sensor can be controlled by the heating device or the temperature adjusting function, the influence of the temperature of the outside air or the sample is mitigated,
A stable response is obtained, and the substrate concentration in the sample solution can be measured more accurately. Further, since the electrode system can be formed by screen printing with a material mainly containing carbon, disposable type biosensors can be mass-produced at low cost.

Example 1 An example of the present invention will be described below.

As an example of a biosensor, an example of a glucose sensor will be described with reference to FIGS. 1 and 2.

Insulating substrate 1 made of polyethylene terephthalate
Then, a conductive carbon paste is printed by screen printing and heated and dried to form an electrode system composed of the counter electrode 2 and the measurement electrode 3. Then partially cover the electrode system,
An insulating paste is printed in the same manner as above so as to leave 2b and 3b which become the electrochemically acting portions of each electrode, and heat treatment is performed to form the insulating layer 4. An aqueous solution of CMC (carboxymethylcellulose), which is a kind of cellulosic hydrophilic polymer, was applied so as to cover the surfaces of the electrode systems 2b and 3b, and dried at 45 ° C. for 30 minutes to form a CMC layer. A solution of glucose oxidase (GOD) dissolved in a phosphate buffer of pH 5.6 as an oxidoreductase was applied onto the obtained CMC layer and then dried at room temperature. Furthermore, a mixture of fine crystals of potassium ferricyanide, which is an electron acceptor, in organic solvent together with lecithin (phosphatidylcholine), which is a surfactant, is added dropwise to the mixture as an organic solvent. Layers were formed. In this way, the enzyme reaction layer 5 composed of the redox enzyme and the electron acceptor containing the hydrophilic polymer and the surfactant is formed on the surface of the electrode system.

Insulating substrate 1 of glucose sensor configured as described above
The heating device 6 is integrally attached to a portion corresponding to the electrode system on the back surface of the. Glucose standard solution (10μ) was dropped on the enzyme reaction layer 5 of this biosensor, and after 2 minutes, a pulse voltage of + 0.6V was applied to the measurement electrode based on the counter electrode toward the anode, and the current was measured 5 seconds later. To do. The heating device consists of a PTC thermistor (positive temperature coefficient thermistor), and the Curie temperature is set to 40 ° C.
The temperature was raised in seconds. Potassium ferricyanide is dissolved in the glucose standard solution, which reaches the CMC-GOD layer to oxidize glucose, at which time potassium ferricyanide is reduced to potassium ferrocyanide. 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. When 10 μm of a blood sample was dropped on the glucose sensor and the response current was measured 2 minutes later, a very reproducible response was obtained. Also, when the temperature of outside air was changed to 15 ℃ to 30 ℃ and a glucose 400mg / dl solution was used, the response was different as shown in Fig. 3 when the PTC thermistor was activated and when it was not activated. , PTC thermistor could reduce the effect of temperature. In addition to the PTC thermistor for heating, the effect was obtained by irradiating light or using a heating element, but at high temperature, the activity of the enzyme decreases, so the temperature is around 40 ° C. It was necessary to control the temperature sensor, so a temperature sensor was required.

As the surfactant, in addition to lecithin, polyethylene glycol alkyl phenyl ether (trade name: Triton X), oleic acid, polyoxyethylene glycerin fatty acid ester, cyclodextrin, and other electron acceptors dispersed in an organic solvent, and an enzyme There is no particular limitation as long as it does not affect the activity.

In addition to CMC, gelatin or methylcellulose can be used as the hydrophilic polymer, 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.

As the organic solvent to be mixed with the electron acceptor, other than toluene, ethanol, petroleum ether, or the like may be used as long as it has little influence on the GOD activity and the printed electrode.

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

Example 2 In this example, as shown in FIG. 4, a PTC thermistor having a temperature adjusting function was directly attached to the substrate of the sensor, whereby the temperature could be controlled more efficiently. Therefore, it took about 10 seconds to reach a constant temperature, and when the outside air temperature was 15 ° C and the glucose concentration was 100 mg / dl, it took 2 minutes to complete the reaction. Completed and a stable response was obtained. If the substrate is heat resistant, the PTC thermistor will directly
Since it can be formed on the substrate as shown, it is suitable for mass production.

Example 3 A cover 7 was attached to the sensor having the structure shown in FIGS. 1 and 2 as shown in FIG. 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 inside the cover 7, the sample amount could be made minute and a fixed amount could be supplied. Furthermore, since it can be shielded from the outside air by surrounding it with the cover 7, the effect of the PTC thermistor 6 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. Also,
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
-Potassium naphthoquinone 4-sulfonate, ferrocene, etc. can be used Effect of the invention As described above, the biosensor of the present invention is an enzyme reaction consisting of an electrode system, an oxidoreductase, a hydrophilic polymer and an electron acceptor on an insulating substrate. By forming a layer and further attaching a heating device integrally to the back surface of the insulating substrate, the enzyme reaction layer portion can be efficiently heated. Therefore, it is possible to significantly reduce the influence of the temperature change of the sample and the surroundings on the sensor response, promote the enzyme reaction, and improve the measurement accuracy of the substrate concentration in the sample. Further, according to the structure in which the PTC thermistor is attached to the surface of the insulating substrate, and further the cover surrounding the electrode system and the PTC thermistor is attached, the enzyme reaction layer portion and the PTC thermistor can be shielded from the outside air, so that it is more efficient. The temperature can be controlled. In addition, since the electrode system can be formed by screen printing using a material mainly containing carbon, disposable type biosensors can be mass-produced at low cost.

[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 response characteristic of the biosensor, and FIG. 4 is another embodiment of the present invention. FIG. 5 is a vertical sectional view of an example biosensor, and FIG. 5 is a sectional view of a conventional biosensor. 1 ... Insulating substrate, 2 ... Counter electrode, 3 ... Measuring electrode, 4 ...
Insulating layer, 5 ... Enzyme reaction layer, 6 ... PTC thermistor, 7
……cover.

 ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Shiro Nankai 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Inventor Takashi Iijima 1006 Kadoma, Kadoma City, Osaka Matsushita Electric Industrial Co., Ltd. 56) References JP 62-142264 (JP, A) JP 54-139286 (JP, A) JP 55-166141 (JP, A) JP 62-232554 (JP, A) JP Sho 63-139242 (JP, A) Actually opened Sho 52-170092 (JP, U)

Claims (2)

[Claims] 1. An electrode system comprising at least a measuring electrode and a counter electrode is provided on an insulating substrate, and an enzyme reaction layer comprising an oxidoreductase, a hydrophilic polymer and an electron acceptor is formed on the surface of the electrode system, A biosensor for electrochemically detecting a substance concentration change during the reaction of the oxidoreductase, electron acceptor and sample solution with the electrode system, and measuring the substrate concentration in the sample solution, on the back surface of the insulating substrate. A biosensor characterized in that a heating device is integrally attached. 2. An electrode system comprising at least a measuring electrode and a counter electrode is provided on an insulating substrate, and an enzyme reaction layer comprising a redox enzyme, a hydrophilic polymer and an electron acceptor is formed on the surface of the electrode system, In a biosensor for electrochemically detecting a change in substance concentration during the reaction of the oxidoreductase, electron acceptor and sample solution with the electrode system, and measuring the substrate concentration in the sample solution, the surface of the insulating substrate is , A PTC thermistor, and a cover surrounding the electrode system and the PTC thermistor.