JPH02157645A - Biosensor - Google Patents

Abstract

PURPOSE:To relieve the influence of temp. and to improve the measurement accuracy of the substrate concn. in a living body forming electrode systems and an enzyme reaction layer consisting of an oxidation reduction enzyme, hydrophilic high polymer and electron acceptor on an insulating substrate and further, adding a heater and temp. control function thereto. CONSTITUTION:Conductive carbon paste is printed on an insulating substrate 1 and is dried by heating to form the electrode system consisting of a counter electrode 2 and a measuring electrode 3. An aq. soln. of carboxymethylcellulose CMC which is one kind of the hydrophilic high polymers of a cellulose system is applied on the substrate to cover the surfaces of the electrode systems 2b and 3b and is dried to form the CMC layer. A soln. prepd. by dissolving glucose oxidase as the oxidation reduction enzyme into a phosphoric acid buffer soln. of, for example, pH 5.6 is applied on the resulted CMC layer and is dried. Further, a mixture formed by mixing the fine crystals of potassium ferricyanide which is the electron acceptor and lecithin of a surfactant with toluene as an org. solvent is dropped thereon to form the potassium ferricyanide layer. The enzyme reaction layer contg. the oxidation reduction enzyme, etc., on the surface of the electrode system is formed in such a manner.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a biosensor that can quickly and easily quantify specific components in various minute amounts of biological samples without diluting the sample liquid.

2. Description of the Related Art Conventionally, a biosensor as shown in FIG. 5 has been used as a method for easily quantifying a specific component in a biological sample such as blood without diluting or stirring the sample liquid.

This biosensor is manufactured by using an electrode system 12.13 made of carbon or the like on an insulating substrate by a method such as screen printing.
An enzyme reaction layer 15 consisting of a hydrophilic polymer, an oxidoreductase, and an electron acceptor is formed on the electrode. When the sample solution is dropped onto the enzyme reaction layer, the oxidoreductase and electron acceptor are dissolved in the sample solution, and an enzymatic reaction proceeds with the substrate in the sample solution to reduce the electron acceptor. After the reaction is completed, the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

Problems to be Solved by the Invention However, with this conventional biosensor, the enzymatic reaction and electrode reaction are affected by the temperature of the outside air and sample solution during the reaction, resulting in variations in response and difficulty in obtaining a stable response. There was a point.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate, and
An enzyme reaction layer consisting of an oxidoreductase, a hydrophilic polymer, and an electron acceptor is formed on the surface of the electrode system, and changes in substance concentration during the reaction of the oxidoreductase, electron acceptor, and sample liquid are electrochemically measured. In the biosensor that detects with the electrode system and measures the substrate concentration in the sample liquid, a first feature is that the biosensor is provided with a heating device, and the insulating substrate itself has a temperature adjustment function. As the second feature,
More preferably, the third feature is that the electrode system in the first or second feature is made of a carbon-based material with screen printing formed on an insulating substrate.

Function According to the present invention, since the sensor can be controlled by a heating device or a temperature adjustment function, the influence of the temperature of the outside air and the sample is alleviated, a stable response is obtained, and the substrate concentration in the sample solution can be measured more accurately. It can be done. Furthermore, since the electrode system can be formed by screen printing from a material mainly composed of carbon, disposable biosensors can be mass-produced at low cost.

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

As an example of a biosensor, an embodiment of a glucose sensor will be described based on FIGS. 1 and 2.

On an insulating substrate l made of polyethylene terephthalate,
By printing a conductive carbon paste by screen printing and heating and drying it, an electrode system consisting of a counter electrode 2 and a measurement electrode 3 is formed. Next, an insulating paste is printed in the same manner as above to partially cover the electrode system and leave 2b and 3b, which are the electrochemically active parts of each electrode, and is heated to form an insulating layer. form 4. This electrode system 2b and 3
By applying an aqueous solution of CMC (carboxymethylcellulose), a type of cellulose-based hydrophilic polymer, to cover the surface of b, and drying it at 45°C for 30 minutes, CMC
formed a layer. On the obtained CMC layer, a solution of glucose oxidase (COD) as an oxidoreductase dissolved in a phosphate buffer solution of pH 5 or 6 was applied, and then dried at room temperature. Furthermore, a mixture of microcrystals of potassium ferricyanide, which is an electron acceptor, and lecithin (phosphatidylcholine), which is a surfactant, is added dropwise to toluene as an organic solvent, and the mixture is left at room temperature to vaporize the toluene. formed a layer. In this way, an enzyme reaction layer 5 consisting of an oxidoreductase, an electron acceptor containing a hydrophilic polymer, and a surfactant is formed on the surface of the electrode system.

Place the glucose sensor configured as above on a heating device, drop 10 μQ of glucose standard solution as a sample solution, and after 2 minutes, set the counter electrode to 1 & 2 and apply a pulse voltage of +0.6 V to the measurement electrode toward the anode. is applied and the current is measured after 5 seconds. The heating device consisted of a PTC thermistor (positive temperature coefficient thermistor), the Curie temperature was set at 40° C., and when a constant voltage was applied, the temperature rose in about 30 seconds. Potassium ferricyanide is dissolved in the glucose standard solution, which reaches the CMC-COD layer where glucose is oxidized, and at this time potassium ferricyanide is reduced to potassium ferrocyanide. Therefore, by applying the above pulse voltage, an oxidation current is obtained based on the concentration of the generated potassium ferrocyanide, and this current value corresponds to the concentration of glucose, which is the substrate. When measured, good linearity was obtained up to a high degree of 500 mg/dll. When 10 μg of blood sample was dropped into the above glucose sensor and the response current was measured 2 minutes later, a response with very good reproducibility was obtained. Got it. Another 1
Glucose 4 by changing the temperature of the outside air to 15°C to 30°C
When measured using a 00mg/dll solution, PTC
As shown in FIG. 3, there was a difference in response between when the thermistor was activated and when it was not activated, indicating that the PTC thermistor was able to alleviate the influence of temperature. In order to warm up,
In addition to PTC thermistors, irradiation with light and the use of heating elements have also been effective, but the enzyme activity decreases at high temperatures, so it is necessary to control the temperature around 40°C. Therefore, a temperature sensor was required.

In addition to lecithin, surfactants include polyethylene glycol alkyl phenyl ether (trade name: Triton There is no particular restriction as long as it does not affect the activity.

In addition to CMC, gelatin, methylcellulose, and the like can be used as hydrophilic polymers, and starch-based, carboxymethylcellulose-based, gelatin-based, acrylate-based, vinyl alcohol-based, vinylpyrrolidone-based, and maleic anhydride-based polymers are preferred.

Since these polymers can be easily made into an aqueous solution, a thin film of a required thickness can be formed on the electrode by applying an aqueous solution of an appropriate concentration and drying.

The organic solvent in which the electron acceptor is mixed may be any organic solvent other than toluene, such as ethanol or petroleum ether, as long as it has little effect on GOD activity and the printed electrode.

Screen printing as a method for forming electrode systems can produce disposable biosensors with uniform properties at low cost, especially using carbon, which is a low-cost and stable electrode material. It is a convenient method for forming electrodes.

Example 2 In Example 1, a heating device was provided to adjust the temperature of the sensor from the outside, but as shown in Figure 4, a PTC thermistor with a temperature adjustment function was attached directly to the sensor board. By doing so, we were able to control the temperature even more efficiently. Therefore, the temperature becomes constant in about 10 seconds, and if the outside temperature is 15°C, the glucose concentration will be 100 m
g/dR, the reaction took 2 minutes to complete,
The reaction was completed in 1 minute using the PTC thermistor, and a stable response was obtained. PTC thermistors can be formed directly on the substrate as shown in Figure 4 if the substrate is heat resistant.
Suitable for mass production.

Example 3 A cover 7 was attached to the sensor having the configuration shown in FIG. 1.2 as shown in FIG. 4. When blood was supplied to the spotting part of the cover, it quickly spread to the electrode part and a response with good reproducibility was obtained. By reducing the volume of the inside of the cover 7, the amount of sample could be kept small and a constant amount could be supplied.Furthermore, by surrounding it with the cover 7, it can be isolated from the outside air, so the effect of the PTC thermistor 6 can be improved. I was able to perform even better.

Note that the biosensor of the present invention is not limited to the glucose sensor shown in the above embodiments, but can be used in systems involving alcohol sensors. Although glucose oxidase was used as the oxidoreductase in the examples, other enzymes such as alcohol oxidase, cholesterol oxidase, xanthine oxidase, etc. can be used.

In addition, as an electron acceptor, potassium ferricyanide used in the above example is suitable because it reacts stably, but P-
Using benzoquinone has a high reaction rate, so it is suitable for speeding up the reaction. In addition, the biosensor of the present invention can use 2,6-cyclophenol indophenophenol, methosulfate, potassium 4-sulfonate, ferrocene, etc. As described above, the biosensor of the present invention has an electrode system and an oxidized material on an insulating substrate. By forming an enzyme reaction layer consisting of a reductase, a hydrophilic polymer, and an electron acceptor, and adding a heating device and temperature control function (temperature control device, etc.), the influence of temperature is alleviated and The measurement accuracy of substrate concentration can be improved. Furthermore, since the electrode system can be formed from a material mainly composed of carbon by screen printing, disposable biosensors can be mass-produced at low cost.

[Brief explanation of the drawing]

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 same biosensor, FIG. 3 is a response characteristic of the same biosensor, and FIG. 4 is another embodiment of the present invention. FIG. 5 is a longitudinal sectional view of an example biosensor, and FIG. 5 is a sectional view of a conventional biosensor. l...Fist insulating board, 2...Fist counter electrode, 3...Measuring electrode,
4ΦΦ・Insulating layer, 5... Enzyme reaction layer, 6... PTC
Thermistor, 7-φ・Name of cover agent Patent attorney Shigetaka Kurino Haka1 person Figure 1 112 Figure 6-... Suggestion 4 Law number Passing sign + I9 Mitsu name line Akebono 1 Ship gA S Forget place P2O”7- Mister

Claims (3)

[Claims] (1) An electrode system consisting of at least a measurement electrode and a counter electrode is provided on an insulating substrate, and an enzyme reaction layer consisting of an oxidoreductase, a hydrophilic polymer, and an electron acceptor is formed on the surface of the electrode system, and the oxidation A biosensor that electrochemically detects a change in substance concentration during a reaction between a reductase, an electron acceptor, and a sample solution using the electrode system and measures the substrate concentration in the sample solution, and a heating device is attached to the biosensor. A biosensor characterized by: (2) An electrode system consisting of at least a measurement electrode and a counter electrode is provided on an insulating substrate, and an enzyme reaction layer consisting of an oxidoreductase, a hydrophilic polymer, and an electron acceptor is formed on the surface of the electrode system, and the oxidation In a biosensor that electrochemically detects a change in substance concentration during a reaction between a reductase, an electron acceptor, and a sample liquid using the electrode system and measures the substrate concentration in the sample liquid, the insulating substrate itself has a temperature control function. A biosensor characterized by having. (3) The biosensor according to claim 1 or 2, wherein the electrode system is made of a carbon-based material formed by screen printing on an insulating substrate.