JPH0354447A - Biosensor and its manufacturing method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Industrial application field of the present invention Conventional technology related to manufacturing method Tonatsu: We proposed the following biosensor as a method that can easily quantify a specific amount in a biological sample such as blood without diluting or stirring the sample solution. (Patent application No. 63-38156).

This biosensor is one in which an electrode system is formed on an insulating substrate by a method such as screen printing, and an enzyme reaction layer consisting of a hydrophilic polymer layer, an oxidoreductase, and an electron acceptor is formed on the electrode system. When the Amo sample solution is dropped onto the enzyme reaction layer, the oxidoreductase and electron acceptor are dissolved in the sample solution.
An enzymatic reaction progresses with the substrate in the sample solution, and the electron acceptor is reduced. Enzyme reaction completed This reduced electron acceptor is electrochemically oxidized and the oxidation current value obtained at this time is used to determine the substrate concentration in the sample solution.

Problems to be Solved by the Invention With this conventional structure, the oxidoreductase and electron acceptor are already in contact when the biosensor is prepared. When exposed to water, enzyme activity was deactivated and a stable response could not be obtained1.
In addition, the presence of a substance that easily adsorbs to the electrode or a substance that is active on the electrode in the sample solution will affect the response of the sensor. above
An electrode system consisting of at least a measurement electrode and a counter electrode is provided (a first layer consisting of a hydrophilic polymer and an oxidoreductase, a second layer consisting of a hydrophilic polymer, and an electron acceptor are provided on the electrode system). In addition, an electrode system consisting of at least a measurement electrode and a counter electrode is provided on an insulating substrate, and a hydrophilic polymer and an oxidoreductase are provided on the electrode system. A first layer made of a hydrophilic polymer of the form 5!2,L is then spread on the first layer to form a second layer made of a hydrophilic polymer, and then an electron-accepting layer is formed. According to the present invention, a biosensor is produced by spreading an organic solvent dispersion of the body on a second layer to form a third layer containing an electron acceptor. A disposable type biosensor that can measure substrate concentration and has excellent storage stability can be used. As an example, a glucose sensor will be explained. Fig. 1 is a cross-sectional view of a glucose sensor manufactured as an example of the biosensor of the present invention, and Fig. 2 is a perspective view of the electrode portion used for manufacturing the sensor. Silver paste is printed on an insulating substrate 1 made of polyethylene terephthalate by screen printing, and leads 2,
3. Next, by printing a conductive carbon paste containing a resin binder and heating and drying it, an electrode system consisting of a measurement electrode 4 and a counter electrode 5 is formed.Furthermore, the electrode system is partially covered with L\ electrode. Print an insulating paste so that the area of the exposed part of the lead is constant and cover unnecessary parts of the lead, and heat-treat it to form the insulating layer 6.Next, 4.
The exposed part of 5 was polished and then heat treated in air at 100°C for 4 hours.The electrode part was constructed in this way.As a hydrophilic polymer, 0.5wt of carboxymethyl cellulose (hereinafter abbreviated as CMC) was used. % aqueous solution was spread on the electrode and dried to form a CMC layer.Next, glucose oxidase (hereinafter abbreviated as COD) was added as an enzyme to a phosphate buffer solution (pH=5.
6) Alternatively, spread the solution dissolved in water and dry it.C. In this case, CMC and COD are partially mixed to form a thin film having a thickness of several microns. Furthermore, this CMC
- Spread a 1% ethanol solution of polyvinylpyrrolidone (hereinafter abbreviated as PVP) on the GOD layer completely, dry it, and form the PVP layer 8. A mixture of microcrystals of potassium ferricyanide, which is an electron acceptor, was added dropwise to a 1% toluene solution of lecithin, spread, and dried to form potassium ferricyanide-lecithin layer 9. Here, lecithin is potassium ferricyanide dissolved in toluene. It plays the role of uniformly dispersing the liquid. PV is used as a solvent when forming this potassium ferricyanide-lecithin layer.
By using toluene in which P is poorly soluble, it is possible to uniformly spread the potassium ferricyanide-lecithin solution on the PVP layer, and a uniform potassium ferricyanide-lecithin layer can be obtained. If a solvent in which the hydrophilic polymer that forms the second layer is poorly soluble is used as a solvent for developing the third layer containing electron acceptors, it is possible to form a third layer with extremely high uniformity. In this case, the third layer is the second layer.
Although it is difficult to strictly control the amount of electron acceptors on the electrode system to the minimum amount required for the enzyme reaction, the glucose sensor constructed as described above After 1 minute, a pulse voltage of +0.6V was applied to the measurement electrode in the anode direction with reference to the counter electrode, and the current was measured after 5 seconds.When the sample solution was dropped, First, the potassium ferricyanide-lecithin layer dissolves in the glucose standard solution. When the sample solution reaches the 4 PVP layer, it instantly dissolves. This reaches the CMC-GOD layer, where glucose is oxidized and at the same time potassium ferricyanide is reduced to potassium ferrocyanide. However, by applying the pulse voltage described above, an oxidation current based on the concentration of the produced potassium cyanide was obtained, and this current value corresponded to the concentration of the substrate glucose. Among these glucose sensors, two types of glucose sensors without a second layer made of a hydrophilic polymer were subjected to a storage test for 35 tons and 30 days in a dry state, and a glucose standard solution (90 mg/ When looking at the sensor response after 30 days using di), there was variation in response for those without the second layer, and the Cv value was 5.3.
The Cv value of the second layer made of VP was 2.5, which was an extremely good value1, and the response current was measured by dropping a standard solution of glucose. A good linear relationship was obtained up to a high concentration of 0.05 mol/1) or more.
When we measured the response current 1 minute after adding 0μ1 drop, a response with very good reproducibility was obtained (Example 2) on an insulating substrate made of polyethylene terephthalate.
In the same manner as in Example 1, the same electrode portion as shown in FIG. 2 was formed by screen printing.Furthermore, in the same manner as in Example 1, a CMC-GOD layer and a PVP layer were formed on top of this PVP layer. A mixture of microcrystals of potassium ferricyanide, an electron acceptor, was added dropwise to a 0.5% ethanol solution of lecithin, a surfactant, spread, and dried to form a potassium ferricyanide-lecithin layer. - By using ethanol in which PVP is easily dissolved as a solvent when forming the lecithin layer, it was possible to develop the ferricyanide lecithin layer in a concentrated manner at one point on the PVP layer. This may be because the ethanol, which is a dispersion medium for the added potassium ferricyanide and lecithin, was quickly absorbed into the PVP layer, and the potassium ferricyanide and lecithin did not spread on the PVP, but formed a layer concentrated at the dropping point. to
It became possible to form a concentrated and uniform potassium ferricyanide-lecithin layer on the measurement electrode of the sensor, and it was possible to create a sensor that could obtain a stable response just by deploying the minimum necessary amount. If a solvent in which the hydrophilic polymer constituting the second layer is easily soluble is used as the solvent for developing the third layer containing the electron acceptor, the electron acceptor can be concentrated on the target point such as the electrode system. However, in this case, it is necessary for the second layer to absorb the developing solvent of the third layer. Therefore, if the amount of solvent that can be absorbed exceeds the amount of solvent that can be absorbed, a uniform third layer may not be obtained. Therefore, add 10μ1 drop of glucose standard solution as a sample solution to the glucose sensor prepared as described above. One minute after dropping, a pulse voltage of +0.6V was applied between the electrodes.The current was measured after 5 seconds.As in Example 1, a response current value corresponding to the concentration of glucose was obtained.
A good linear relationship was obtained up to a high concentration of 0 mg/di (o, o 5 mol/l) or higher.Even when blood was used as the sample solution, a very reproducible response was obtained. In the same manner as above, two types of samples, one with and without the second layer made of hydrophilic polymer, were subjected to a 30-day storage test in a dry state. Whole blood was used as the sample solution. Looking at the response after 30 days, we found that the sensor with the second layer made of PVP had an extremely good Cv value (Example 3). An exploded perspective view of this cover with the cover attached is shown in Figure 3. The cover 13 and spacer IO are made of polymer material, and the cover, spacer, and substrate on which the electrode system is printed have a positional relationship as shown by the broken line in the figure. It is possible to introduce the sample liquid from the end opening l2 of the spacer or the circular hole 14 of the cover in the sensor with the cover thus constructed. When the inlet at the tip of the is brought into contact with the sample liquid (human whole blood), the sample liquid is quickly introduced into the interior through the opening, and the space l1 surrounded by the cover and spacer is filled with the sample liquid without the formation of air bubbles. In addition, hydrophilic treatment enables smoother introduction of sample liquid by pre-treating the surfaces of parts such as covers and spacers with surfactants to make them hydrophilic. For example, the following method also achieved sufficient effects such that liquid absorption was completed in 0.5 to 1 second. In other words, Example 1 or Shape the first to third layers as in step 2, and drop a 0.5 wt% toluene solution of lecithin from the top of the third layer to the top of the insulating substrate corresponding to the end opening l2, and spread the surface. By drying, the area from the end opening to the surface of the reaction layer could be made hydrophilic. 1. By providing a cover in this way, it was possible to minimize the evaporation of the sample solution inside the cover. This enabled the measurement of glucose concentration quickly and with high accuracy without being affected by ambient humidity, etc. It becomes possible to measure the circle
Furthermore, by reducing the volume inside the cover, it is possible to reduce the amount of sample liquid and the amount of substances that make up the sensor carried to a very small amount.t4 (Example 4)
The same electrode portion as shown in FIG. 2 was formed by screen printing in the same manner as in Example 1. Furthermore, the CMC-COD layer and PVP layer were formed in the same manner as in Example 1. Q+ On top of this PVP layer, a mixture of microcrystals of potassium ferricyanide, which is an electron acceptor, and PVP is dropped into a 0.5% ethanol solution of lecithin, which is a surfactant, spread, and dried to form potassium ferricyanide. In an ethanol solvent formed with a lecithin-PVP layer, potassium ferricyanide is dispersed by lecithin. By adding a hydrophilic polymer such as PVP to this solution, a homogeneous solution can be obtained. Improved uniformity of the solution A potassium ferricyanide solution with a constant concentration could be developed with simple operations.Additionally, the addition of this hydrophilic polymer made it possible to form a thin film that was stronger and more difficult to peel off in the dry state, resulting in a stable solution. The sensor was now able to be constructed.A cover was attached to the glucose sensor constructed as described above in the same manner as in Example 3, and 10μ1 of the glucose standard solution was added as a sample solution.After 1 minute of the drop, a voltage of +0 was applied between the electrodes. A pulse voltage of .6 V was applied, and the current was measured after 5 seconds, and the response current value corresponding to the glucose concentration was obtained as in Example 1.
．． Even when blood with a good linear relationship was obtained up to a high concentration of 0.5 mol/1) or more was used as the sample liquid, a very reproducible response was obtained. Already C at the time of
However, since the OD-CMC layer and the potassium ferricyanide-lecithin layer were in contact with each other, it was difficult to improve the storage performance of the PV used in Examples 1, 2, or 3.
Hydrophilic polymer layer consisting of P (top) CO in dry state
It plays the role of completely separating the D-CMC layer and the potassium ferrinanide-lecithin layer, and although this has a great effect on improving the sensor storage life,
This hydrophilic polymer layer is extremely effective in ensuring a stable sensor response when there are substances that easily adsorb to the electrode or electrode active substances in the sample solution.The glucose sensor uses blood as a sample solution. When measuring the glucose concentration at L, a stable sensor response was obtained regardless of the viscosity of the sample liquid, etc. In Examples 1, 2, 3, or 4 above, the first layer
MC is used as the second layer and PVP is used as the hydrophilic polymer in the third layer, respectively.
Similar effects were obtained by using maleic anhydride nails, acrylamide nails, methacrylate resin, etc.A solution of these water-absorbing or water-soluble hydrophilic polymers at an appropriate concentration is applied.By drying, the required A hydrophilic polymer layer of a certain thickness can be formed on the electrode.

In the above example, lecithin was used to disperse the electron acceptors. In addition, polyethylene glycol alkyl phenyl ether/t < olein kai polyoxyethylene glycerin fatty acid ester/k cyclodextrin, etc. In addition, in the above example, a two-electrode system with only a measuring electrode and a counter electrode was described, but a three-electrode system with a reference electrode added would enable more accurate measurements. The material for constituting the mirror electrode is not limited to the carbon shown in the above examples, and noble metals such as platinum gold and metal oxides can also be used as electron acceptors (as shown in the above examples). E p other than potassium ferricyanide
-Penzoquinone, phenazine methosulfate, ferrocene, etc. can also be used.

Furthermore, if alcohol oxidase, cholesterol oxidase, etc. are used as the enzyme in addition to the glucose oxidase in the above embodiment, the biosensor of the present invention (in a dry state) can also be used for alcohol sensors, cholesterol sensors, etc. By completely separating the oxidoreductase layer and the electron acceptor layer, it is possible to improve the shelf life of the sensor. It is possible to perform stable and more accurate measurements without being affected by substances such as

[Brief explanation of drawings]

Fig. 1 shows a cross-sectional view of a biosensor that is an embodiment of the present invention. Fig. 2 shows a perspective view of an electrode portion. Fig. 3 shows an exploded perspective view of a biosensor with a cover. 2,
3...Lead, 4...Measurement screen 5...Counter vibration 6
・・・Insulation 凰 7...CMC-GOD 凰 8・PV
PJi 9-...Potassium ferricyanide-lecithin H to...Spacer- 11...Space enemy l2.
・・End opening 13・Cover 14・Cover Circular hole-cotton-bound pillar substrate 3-J-de”JIl Constant pole. Tatami 7-Ricyanized Karitsumureshite/Layer 3 2 3 j g +1 4 7 Fig. 3/4

Claims (7)

[Claims] (1) An electrode system comprising at least a measurement electrode and a counter electrode provided on an insulating substrate, and a reaction layer provided on the electrode system, the reaction layer comprising a hydrophilic polymer and an oxidoreductase. A biosensor comprising a first layer, a second layer made of a hydrophilic polymer, and a third layer containing an electron acceptor. (2) The biosensor according to claim 1, wherein the third layer containing an electron acceptor comprises at least an electron acceptor and a hydrophilic polymer. (3) The biosensor according to claim 1 or 2, further comprising a cover placed over the electrode system so as to contain the enzyme reaction layer inside. (4) The hydrophilic polymer is cellulose-based, vinylpyrrolidone-based, gelatin-based, acrylate-based, vinyl alcohol-based, starch-based, maleic anhydride-based, acrylamide-based,
Claim 1, wherein the resin is selected from methacrylate resins.
3. The biosensor according to 2 or 3. (5) After providing an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate, a first layer consisting of a hydrophilic polymer and an oxidoreductase is formed on the electrode system, and then An organic solvent solution of a hydrophilic polymer is spread on the first layer to form a second layer made of a hydrophilic polymer, and an organic solvent dispersion of an electron acceptor is further spread on the second layer. A method for producing a biosensor, comprising: forming a third layer containing an electron acceptor. (6) As a solvent for developing the third layer containing an electron acceptor, a solvent in which the hydrophilic polymer constituting the second layer is poorly soluble or easily soluble is used. Biosensor manufacturing method. (7) Dispersing an electron acceptor in an organic solvent in which a hydrophilic polymer is dissolved and spreading this on the second layer to form a third layer containing the electron acceptor and the hydrophilic polymer. The method for manufacturing a biosensor according to claim 5, characterized in that: