JPH03202764A - Biosensor and its manufacturing method - Google Patents

Abstract

PURPOSE:To measure the concentration of a substrate in the specimen of an organism readily and to improve measuring accuracy by forming an enzyme reaction layer comprising the mixture of oxidoreductase, hydrophilic macromolecules and an electron acceptor on the surface of an electrode system. CONSTITUTION:Conductive carbon paste is printed on an insulating substrate 1. The paste is heated and dried, and an electrode system comprising a counter electrode 2 and a measuring electrode 3 is formed. Then, an insulating layer 4 is formed so that parts 2' and 3' of the electrodes which are to become the electrochemically acting parts are made to remain. The aqueous solution of carboxymethylcellulose (CMC) which is one kind of hydrophilic macromolecules is applied so as to cover the surfaces of the electrode systems 2' and 3'. The mixture of oxidoreductase and an electron acceptor is dropped on the CMC, heated and dried. Thus an enzyme reaction layer 5 is formed. Glucose standard liquid as specimen liquid is dropped on the reaction layer 5 in this glucose sensor. A constant voltage is applied to the measuring electrode 3 with the counter electrode as a reference, and the current is measured. The current value corresponds to the concentration of the glucose which is a substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention 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.

BACKGROUND OF THE INVENTION A biosensor as shown in Figure 7 was proposed as a method for easily quantifying specific components in biological samples such as blood without diluting or stirring the sample solution.1
．． This biosensor (an electrode system 2 made of carbon or the like is formed by screen printing or other method on an insulating substrate 1).
3, an enzyme reaction layer consisting of a hydrophilic polymer layer 6, an oxidoreductase layer 9, and an electron acceptor layer 10 is formed on the electrode. When a sample solution is dropped onto the enzyme reaction layer, oxidation occurs. The reductase and electron acceptor are dissolved in the sample solution, and an enzymatic reaction progresses with the substrate in the sample solution, reducing the electron acceptor. The problem that the invention attempts to solve is to determine the substrate concentration in the sample solution from the oxidation current value obtained at this time. If the viscosity is high, the reaction may be delayed, or the electrode reaction may be affected by adhesion to the electrode surface, resulting in variations in response. Since the aqueous solution is applied and dried to form an electron acceptor layer, it takes time for each layer to dissolve and the start of the reaction is delayed.This solves the problem of not being able to shorten the measurement time. In order to solve the above-mentioned problems, the present invention provides an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate. In a biosensor that chemically detects with the electrode system and measures the substrate concentration in a sample solution, an enzyme reaction layer consisting of a mixture of a redox enzyme, a hydrophilic polymer, and an electron acceptor is formed on the surface of the electrode system. It is also characterized by
For samples containing solids, a filtration layer is added on top of the sample, and for the enzyme reaction layer, a woody polymer solution is applied and a hydrophilic polymer, enzyme, and electron acceptor solution is added. It is characterized by applying and drying a mixed solution.

According to the present invention, a disposable type biosensor including an electrode system can be constructed, and the substrate concentration can be measured extremely easily by adding a sample liquid to the sensor. At the time of addition, solid components such as blood cells are removed in the filtration layer so that they do not affect the response and a stable response can be obtained. When the biosensor is dissolved, the reaction begins.The measurement time can be shortened and the manufacturing process of the biosensor can be simplified.Example An example of the present invention will be described below.

<Example 1> A glucose sensor will be explained as an example of a biosensor. Fig. 1 and Fig. 2 EL An example of a glucose sensor is shown. A perspective view and a vertical cross-sectional view of a biosensor. Polyethylene terephthalate An electrode system consisting of a counter electrode 2 and a measuring electrode 3 is formed by printing a conductive carbon paste by screen printing on an insulating substrate 1 consisting of a substrate 1 and heating and drying it. , an insulating paste is printed in the same manner as described above so as to leave 2' and 3' (1 mm2), which are the electrochemically active parts of each electrode, and heated to form an insulating layer 4.

An aqueous solution of CMC (carboxymethyl cellulose), a type of cellulose-based hydrophilic polymer, is applied to cover the surface of this electrode system (2', 3'), and glucose oxidase (COD) as an oxidoreductase is applied to CMC.
A solution of potassium ferricyanide, which is an electron acceptor, was added dropwise and heated and dried at 40 degrees for 15 minutes to form an enzyme reaction layer 5. A glucose standard solution was added as a sample solution to the glucose sensor configured as described above. Drop L 5 μm onto the enzyme reaction layer 5 After 1 minute, add +0.5 to the measurement electrode toward the anode with reference to the counter electrode.
A constant voltage of V is applied and the current is measured after 5 seconds. As long as potassium ferricyanide is dissolved by the glucose standard solution and glucose is oxidized in the enzyme reaction layer, it is reduced to potassium ferrocyanide.Therefore, by applying the above constant voltage, an oxidation current based on the concentration of the generated potassium ferrocyanide is obtained.This current The value corresponds to the concentration of the substrate glucose. When I measured the response current, it was 90
Good linearity was obtained up to a high concentration of 0 mg/di.
To obtain linearity up to -900 mg/di, the reaction time required 2 minutes. This is probably due to the delay.

Therefore, when we mixed the enzyme, hydrophilic polymer, and electron acceptor and dried the enzyme reaction layer at 25 degrees, it took 25 minutes to dry it. When we investigated the response of a biosensor with an enzyme reaction layer that was dried at 25 degrees for 25 minutes, we found that when the glucose concentration was 100 mg/di, it was better to heat the biosensor by heating.
The reaction takes about a minute to complete without heating, compared to the reaction that completes in seconds.This is because when heating occurs, drying occurs quickly, so the particles of potassium ferricyanide are fine and uniformly distributed. It is thought that the potassium ferricyanide formed into large crystals, which slowed down the dissolution rate and the reaction rate.

When heated to -40 degrees, linearity was obtained up to 900 mg/di. Short-term heating did not affect the activity of the enzyme (the temperature for one heating was varied up to 100 degrees and the humidity was below 20%). We created a controlled biosensor and investigated the response after 1 minute to a glucose concentration of 600 mg/dl. As shown in Figure 3, when heated above 30 degrees, the response current increases, and up to L-70 degrees, the initial response deteriorates. 9 The response decreased when heated to 80 degrees or above. This may be because the enzyme is inactivated by heat.
The force exchange that took 25 minutes at 25 degrees could be shortened to 5 minutes at 70 degrees.On the other hand, if it was dried in an atmosphere with dry air flowing, it would dry in 15 minutes even at 25 degrees, improving the response speed and reducing the heating temperature to 40 degrees. A response similar to that of the sensor fabricated using the method was obtained. This is because the particles of potassium ferricyanide, etc., were formed in a fine-sized state when the evaporation of water was accelerated by the drying gas.

A similar effect was obtained by flowing nitrogen or argon instead of dry air.Furthermore, by using it in conjunction with heating, drying was completed in a short time of 5 minutes at 50 degrees without heating to 70 degrees, and the enzyme activity was increased. We were also able to reduce the impact on
A phenomenon was observed in which the enzyme reaction layer peeled off from the electrode surface as the drying time became longer.By introducing dry air and shortening the drying time, peeling could be prevented (Example 2) Example 1 After forming the electrode in the same manner as above, apply C to cover the electrode system.
A 0.5% aqueous solution of MC was applied and dried to form a hydrophilic polymer layer (CMC layer) 6 as shown in Figure 4. Furthermore, glucose oxidase (GOD) was added as an oxidoreductase to 1 g of a 0.5% aqueous CMC solution. A solution of 10 mg of potassium ferricyanide and 20 mg of potassium ferricyanide, an electron acceptor, was added dropwise and dried at 40 degrees for 10 minutes to form the enzyme reaction layer 5. In Example 1, COD and potassium ferricyanide were added dropwise without drying i&cMc. Itatabe The fact that the enzyme reaction layer has spread in the same way as the CMC layer has occurred. In order to keep the amount of enzymes and electron acceptors supported per unit area constant, it is necessary to control the spread of CMC. Once dried, add the same amount of enzyme reaction layer components dropwise.
It has become possible to form a uniform enzyme reaction layer that spreads over approximately the same area, which is an advantage when producing sensors in large quantities.

In addition, by drying the CMC, the amount of liquid used to dry the enzyme reaction layer decreases, and the drying is completed in 7 minutes at 40°C.The shorter the drying time, the finer the particle size of potassium ferricyanide becomes during the reaction. It is a food that can be dissolved quickly.Measurements can be carried out in a short time.It also reduces the effect on the enzyme by shortening the heating time.It suppresses the deterioration of the enzyme reaction rate.It is effective in maintaining storage characteristics. , Furthermore, by using dry air in combination, the drying time was shortened as in Example 1. Example 3 A sensor was produced in the same manner as in Example 1, but blood was used instead of the glucose standard solution. B The linearity slope decreased by 20% and the variation in response increased.The linearity slope did not change for plasma components. This is thought to be due to a decrease in the response speed due to the high viscosity of the sample.Furthermore, slight hemolysis was observed due to contact between blood cells and potassium ferricyanide.Therefore, polyvinylpyrrolidone (PVP) was placed on top of the enzyme reaction layer.
) is dried to form a filtration layer as shown in 7 in Figure 5. When blood is dropped onto the PVP layer, the PVP layer swells due to the moisture and the effect of blood cells on the electrode area is reduced. In addition to PVP, gelatin and methyl cellulose can also be used as hydrophilic polymers, such as starch, carboxymethyl cellulose, gelatin, acrylate, and vinyl alcohol. Vinyl pyrrolidone nails Maleic anhydride-based ones are preferable u℃ These polymers can be easily made into aqueous solutions, so apply an aqueous solution of an appropriate concentration and dry to form a thin film of the required thickness. be able to.

Furthermore, when dissolved in an organic solvent such as ethanol and applied, a filtration layer could be formed without disturbing the enzyme reaction layer, and the variation in response could also be improved. Since the spread of the enzyme reaction layer is controlled, the spread of the filtration layer can also be easily controlled.As an organic solvent for dissolving the material of the filtration layer, 1-yotoluene, ethanol, petroleum ether, etc. can be used to reduce the impact on COD activity and printed electrodes. As long as it's small, it's tIXC.

<Example 4> A 0.05% toluene solution of polystyrene was applied as a filtration layer to the sensor with the enzyme reaction layer formed in the same manner as in Example I. The dried polystyrene film was not water-soluble.
Since the concentration of polystyrene is low, a porous thin film can be formed on the surface of the enzyme reaction layer, making it possible to filter blood cells. If the temperature is raised to 100%, the film becomes thicker and the porosity decreases, so it takes time to filter the blood cells and the reaction is delayed, so it is necessary to make the film thinner.Polycarbonate can also be used instead of polystyrene to form a porous thin film, allowing the blood cells to be filtered. The resulting polystyrene has a greater solubility in toluene, making it easier to adjust its concentration. Celluloses such as cellulose acetate, cellulose nitrate, and polyvinyl chloride can also be used as materials that are insoluble in water but soluble in organic solvents to form porous thin films.

<Example 5> A mixture of 10 mg of 5ins in 1 g of 1% polystyrene toluene solution was added dropwise to the sensor with the enzyme reaction layer formed in the same manner as in Example 1 and dried to form a filtration layer. When blood was supplied, the polystyrene Insoluble power < SiO2
△ Plasma components are filtered and reach the enzyme reaction layer, and A instead of SiO2
A similar filtration layer can be formed using 120s,
If a porous thin layer is used as in Example 4, blood cells can be quickly filtered, but because the layer is thin, it is easily broken. A hard-to-break sensor could be formed (Example 6) A sensor formed up to the enzyme reaction layer in the same way as in Example 1 was coated with 0.1% lecithin (0.01% polystyrene in toluene solution).
phosphatidylcholine) was added dropwise and dried to form a filtration layer.1. Furthermore, the cover 8 is installed as shown in Figure 6.The gap between the cover 8 and the board l is 0.3.
When blood is applied to the tip of the cover, the lecithin in the filtration layer quickly draws it onto the sensor and spreads over the entire surface of the filtration layer.By adding lecithin as a surfactant to the filtration layer, the blood is It is now possible to expand quickly.1. When polyethylene glycol alkyl ether (trade name: Triton X) was used instead of lecithin, the result was 0. If the amount is 5% or more, the same effect as lecithin can be obtained.In addition to the above examples, oleic acid, polyoxyethylene glycerin fatty acid ester, cyclodextrin, etc. can be used as a surfactant. By installing a cover, the volume inside the cover can be reduced, and the amount of sample can be reduced to a very small amount.Furthermore, by surrounding it with a cover, it is possible to prevent evaporation of the sample inside the cover, which can be isolated from the outside air. The biosensor of the present invention is not limited to the glucose sensor shown in the above embodiments, but also includes alcohol sensors, cholesterol sensors, etc.
It can be used in systems involving oxidoreductases. In the Examples, glucose oxidase was used as the oxidoreductase, but 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 if P-benzoquinone is used,
Since the reaction speed is high, it is suitable for increasing speed.
2. Effects of the invention in which 6-cyclophenol indophenonok methylene blue, phenazine methosulfate, β-naphthoquinone potassium 4-sulfonate, ferrocene, etc. The system is printed, and a mixed solution consisting of oxidoreductase, hydrophilic polymer, and electron acceptor is applied and dried to form an enzyme reaction layer.Furthermore, a filtration layer is provided to remove solid components present in the biological sample in advance. The substrate concentration in biological samples can be measured extremely easily using this method, and the measurement accuracy has been improved.Also, by adding a surfactant when forming the filtration layer, it is possible to improve the spread of the sample. Enzyme reaction layer (top layer) Formed by mixing the enzyme and electron acceptor.The two are close to each other, which limits the reaction rate.The manufacturing process can be simplified.

[Brief explanation of drawings]

FIG. 1 shows a perspective view of a biosensor according to an embodiment of the present invention. FIGS. 2, 4, 5, and 6 show vertical cross-sections of the same biosensor. FIG. 7 is a longitudinal sectional view of a conventional biosensor. 1. Substrate 2. Counter-oscillation 3. Measuring device 4. Insulating screen 5. Enzyme reaction layer 6. Hydrophilic polymer layer 7. Filtration layer 8. Cover, 9. Enzyme [10.・Electron acceptor layer

Claims (1)

[Claims] (1) An insulating substrate provided with an electrode system consisting of at least a measurement electrode and a counter electrode, the surface of the electrode system comprising a mixture of an oxidoreductase, a hydrophilic polymer, and an electron acceptor. A biosensor that includes an enzyme reaction layer and electrochemically detects a change in substance concentration during a reaction between the oxidoreductase, an electron acceptor, and a sample liquid using the electrode system to measure the substrate concentration. (2) comprising an insulating substrate provided with an electrode system consisting of at least a measurement electrode and a counter electrode, and providing an enzyme reaction layer comprising a mixture of an oxidoreductase, a hydrophilic polymer, and an electron acceptor on the surface of the electrode system; A biosensor further includes a filtration layer, and the substrate concentration is measured by electrochemically detecting a change in substance concentration during the reaction between the oxidoreductase, the electron acceptor, and the sample liquid using the electrode system. (3) The biosensor according to claim 2, wherein the filtration layer is made of a hydrophilic polymer. (4) The biosensor according to claim 2, wherein the filtration layer is a porous polymer layer. (5) The biosensor according to claim 2, wherein the filtration layer contains a surfactant. (6) An insulating substrate provided with an electrode system consisting of at least a measurement electrode and a counter electrode, an enzyme reaction layer consisting of an oxidoreductase, a hydrophilic polymer, and an electron acceptor provided on the surface of the electrode system, and the enzyme In a biosensor that electrochemically detects a change in substance concentration during a reaction between an electron acceptor and a sample liquid using the electrode system, a hydrophilic polymer solution is applied onto the electrode system, and a hydrophilic polymer solution is applied on top of the hydrophilic polymer solution. A method for producing a biosensor, which comprises applying a mixed solution of an oxidoreductase and an electron acceptor and drying it to form an enzyme reaction layer. (7) An insulating substrate is provided with an electrode system consisting of at least a measurement electrode and a counter electrode, an enzyme reaction layer consisting of an oxidoreductase, a hydrophilic polymer, and an electron acceptor is provided on the surface of the electrode system, and the enzyme In a biosensor that electrochemically detects a change in substance concentration during a reaction between an electron acceptor and a sample solution using the electrode system, a hydrophilic polymer solution is coated on the electrode system, dried, and then a hydrophilic polymer solution is applied on top of the hydrophilic polymer solution. A biosensor production method characterized by applying a mixed solution of molecules, oxidoreductase, and electron acceptor and drying to form an enzyme reaction layer. (8) The method for producing a biosensor according to claim 6 or 7, characterized in that after forming the enzyme reaction layer, a polymer solution is further applied and dried to form a filtration layer. (9) The method for producing a biosensor according to claim 6 or 7, characterized in that the enzyme reaction layer is formed in an atmosphere of 30 to 70 degrees. (10) The method for producing a biosensor according to claim 6 or 7, wherein the enzyme reaction layer is formed in a dry gas.