JPH0894574A - Biosensor and manufacturing method thereof - Google Patents

Abstract

(57) [Summary] [Objective] To provide a high-performance biosensor which is not affected by components other than the substrate coexisting in the sample solution and a method for producing the same. [Structure] An electrode system having a working electrode and a counter electrode formed on an electrically insulating substrate, a reaction layer containing a hydrophilic polymer, an enzyme, and an electron acceptor disposed on the electrode system, and a reaction layer A biosensor comprising a polymer gel particle layer formed of polystyrene, polyacrylamide, or the like formed on the substrate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor capable of performing a rapid and highly accurate quantification of a specific component in a sample, particularly a substrate in a biological sample by utilizing an enzymatic reaction, and a method for producing the same. .

[0002]

2. Description of the Related Art Conventionally, the following biosensor is known as a method for easily quantifying characteristic components in a sample without diluting or stirring the sample solution (Japanese Patent Laid-Open No. 3-202764). Gazette). That is, the conventional biosensor forms an electrode system consisting of a working electrode and a counter electrode on an insulating substrate by a method such as screen printing, and further,
After forming an insulating layer, an enzyme reaction layer comprising a hydrophilic polymer, a redox enzyme and an electron acceptor is formed on the electrode system. When the sample solution containing the substrate is dropped onto the enzyme reaction layer, the enzyme reaction layer is dissolved, the substrate and the enzyme react with each other, the substrate is oxidized, and the electric acceptor is reduced accordingly. After the completion of the enzymatic reaction, this reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate in the sample solution is determined from the oxidation current value obtained at this time.

[0003]

In the conventional biosensor having the above-mentioned structure, even if the substrate concentration of the sample solution is the same, the response characteristics of the sensor are different because the components other than the substrate contained in the sample solution are different. There was a problem that there was a difference in. In addition, a method of providing a filtration layer on the reaction layer to trap components other than the measurement target in the sample may be considered for a sample in which particulate components and the like exist, but it takes a long time to perform the measurement because of filtration time. It costs. As a method of relaxing the difference in the properties of the sample solution, a method of diluting with a constant diluting solution to reduce the difference between the properties is also effective, but it is not necessarily a good measure from the viewpoint of usability.

[0004]

The biosensor of the present invention comprises an electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a hydrophilic polymer arranged on the electrode system. And a reaction layer containing an enzyme and an electron acceptor, and a polymer gel particle layer formed on the reaction layer. Here, the polymer gel particles include polystyrene, polyacrylamide, polyhydroxyethyl methacrylate, glyceryl methacrylate copolymer, gelatin, dextran, agarose, polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, Particles containing as a main constituent at least one polymer selected from the group consisting of polyorthoester, starch, and human serum albumin are used. The polymer gel particles preferably have a diameter of 0.1 μm to 100 μm.

The method for manufacturing a biosensor of the present invention comprises a hydrophilic polymer, an enzyme and an electron acceptor in contact with the electrode system on an electrically insulating substrate provided with an electrode system having a working electrode and a counter electrode. A step of forming a reaction layer, and a step of applying a dispersion liquid in which polymer gel particles are dispersed at a ratio of 10 to 60 wt% on the reaction layer and drying to form a polymer gel particle layer. .

[0006]

In the sensor having the above structure, the spread of the sample solution on the sensor becomes the same with or without the particulate component due to the presence of the particles of the polymer gel. The difference in the response characteristics of the sensor due to the difference in the components other than the substrate, which is the specific component, can be reduced. In addition, since the method of laminating the polymer gel particle layer on the reaction layer on the electrode system is adopted, the polymer gel particle layer has a structure in which it is in close contact with the reaction layer, and the polymer gel particle layer can be easily formed. it can.

[0007]

The present invention will be described in more detail with reference to its examples. Example 1 A glucose sensor will be described as an example of a biosensor. FIG. 1 is a schematic cross-sectional view of a glucose sensor manufactured as an example of the biosensor of the present invention, excluding a cover and a spacer, and FIG. 2 is an exploded perspective view before forming a reaction layer. Reference numeral 1 denotes an insulating substrate made of polyethylene terephthalate. Leads 2 and 3 are formed on the substrate 1 by printing a silver paste by screen printing. An insulating layer 6 made of an insulating paste and an electrode system having a working electrode 4 made of a conductive carbon paste containing a resin binder and a counter electrode 5 is further formed on the substrate 1 by the same printing method. The insulating layer 6 keeps the exposed areas of the working electrode 4 and the counter electrode 5 constant and partially covers the leads.

After forming the electrode portion in this way,
A 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was dropped on the surface of the electrode system and dried in a warm air dryer at 50 ° C. for 10 minutes to CM.
Form the C layer. Then, on the CMC layer, glucose oxidase (EC 1.1.3.4;
(Hereinafter abbreviated as GOD) and a mixed aqueous solution of potassium ferricyanide as an electron acceptor dissolved in water are added dropwise at 50 ° C.
In a warm air dryer for 10 minutes to obtain a hydrophilic polymer,
A reaction layer 7 containing an enzyme and an electron acceptor is formed. As described above, when a mixed aqueous solution of an enzyme and an electron acceptor is dropped on the CMC layer, the CMC layer, which is a hydrophilic polymer, once dissolves and becomes hydrophilic when mixed with the enzyme in the subsequent drying process. A reaction layer composed of a polymer, an enzyme and an electron acceptor is formed. However, it does not become a complete mixed state because it does not involve stirring, and it is the CMC that directly coats the electrode system surface.
Will only be. This CMC coating layer can prevent the adsorption of proteins on the surface of the electrode system.

Next, a dispersion liquid in which particles composed of polystyrene gel having a diameter of 5 μm to 10 μm are dispersed in water at a concentration of 10 wt% is prepared, dropped on the reaction layer 7, and dried in a warm air dryer at 50 ° C. Dry for 10 minutes in. Even in this case, the reaction layer is partially dissolved and a portion mixed with the dispersion liquid of the particles is generated, but since the particles of the polymer gel are laminated on the surface of the reaction layer in the form of being in close contact with the reaction layer without stirring. . Reference numeral 8 represents the polymer gel particle layer thus formed. After forming the reaction layer 7 and the polymer gel particle layer 8 made of polymer gel particles as described above, the cover 12 and the spacer 11 are adhered to the substrate 1 in a positional relationship as shown by the one-dot chain line in FIG. Then, a glucose sensor was produced.

As a sample solution of this glucose sensor,
An aqueous glucose solution and blood were prepared. When 3 μl of the sample solution is supplied from the sample supply hole 13, the sample solution becomes an air hole 14
It reaches the part and penetrates into the polymer gel particle layer 8 and the reaction layer 7. As a result, the reaction layer 7 is dissolved. In the reaction layer 7, glucose in the sample solution is oxidized by glucose oxidase, and the electrons transferred there reduce the potassium ferricyanide to produce potassium ferrocyanide. The potassium ferrocyanide thus produced moves to the vicinity of the electrode surface. Therefore, one minute after supplying the sample, + between the counter electrode 5 and the working electrode 4 of the electrode system
When a voltage of 0.5 V was applied and the current value was measured after 5 seconds, a value dependent only on the glucose aqueous solution and the glucose concentration in the blood sample was obtained. The current response value to glucose concentration in the blood sample was about 98% of the glucose aqueous solution. [Comparative Example 1] A sensor having the same configuration as in Example 1 was prepared except that the polymer gel particle layer 8 was not provided. When using this sensor, the response to blood was 70-80% of the glucose aqueous solution.

[Example 2] As an example of a biosensor,
The glucose sensor will be described. In the same manner as in Example 1, an electrode system including a lead, a working electrode and a counter electrode and an insulating layer are formed on an insulating substrate made of polyethylene terephthalate. Further, in the same manner as in Example 1, CMC,
A reaction layer containing GOD and potassium ferricyanide is formed. Next, a dispersion liquid was prepared in which particles composed of polyacrylamide gel having an average diameter of about 20 μm were dispersed in water at a concentration of 40 wt%, and the dispersion liquid was dropped on the reaction layer and dried in a warm air dryer at 50 ° C. for 10 minutes. Let dry for minutes. When this layer is formed, a part mixed with the reaction layer also occurs, but since the polymer gel particles are not agitated, the particles of the polymer gel are laminated on the surface so as to be in close contact with the reaction layer. Further, as in Example 1, the cover and the spacer were integrated with the substrate to manufacture a glucose sensor. An aqueous glucose solution and blood were prepared as sample solutions for this glucose sensor. In the same manner as in Example 1, 1 minute after supplying 3 μl of the sample solution from the sample supply hole, a voltage of +0.5 V was applied between the counter electrode and the working electrode of the electrode system, and the current value after 5 seconds was measured. However, a value that depends only on the glucose aqueous solution and the glucose concentration in the blood sample was obtained. The current response value to glucose concentration in the blood sample was about 96% of the glucose aqueous solution.

[Example 3] As an example of a biosensor,
The glucose sensor will be described. In the same manner as in Example 1, a lead, an electrode system including a working electrode and a counter electrode, and an insulating layer are formed on an insulating substrate made of polyethylene terephthalate. Further, in the same manner as in Example 1, CM
A reaction layer containing C, GOD and potassium ferricyanide is formed. Next, a dispersion liquid in which particles composed of polyhydroxyethyl methacrylate gel having a diameter of 10 to 20 μm are dispersed in methanol at a concentration of 40 wt% is prepared and added dropwise onto the reaction layer, and the mixture is dried in a warm air dryer at 50 ° C. To dry for 10 minutes. When forming this layer, CMC, GOD, and potassium ferricyanide are not dissolved in methanol, so a polymer gel particle layer independent of the reaction layer is formed in close contact with the reaction layer. Further, as in Example 1, the cover and the spacer were integrated with the substrate to produce a glucose sensor. An aqueous glucose solution and blood were prepared as sample solutions for this glucose sensor. In the same manner as in Example 1, 1 minute after supplying 3 μl of the sample solution from the sample supply hole, a voltage of +0.5 V was applied between the counter electrode and the working electrode of the electrode system, and the current value after 5 seconds was measured. However, a value dependent only on the glucose concentration in the glucose aqueous solution and the blood sample was obtained. The current response value to the glucose concentration in the blood sample was about 97% of that of the glucose aqueous solution.

In the glucose sensor having the polymer gel particle layer formed as described above, almost the same response values were obtained with blood and the glucose aqueous solution, because the high value having the same size as the blood cell component in blood was obtained. It is presumed that the presence of the molecular gel particles on the sensor caused the glucose solution and the blood to have the same spread state of the sample on the sensor. In the above examples, polystyrene gel, polyacrylamide gel, and polyhydroxyethyl methacrylate gel were used as the polymer gel, but a copolymer of glyceryl methacrylate, gelatin, dextran,
Similar effects can be obtained by using other polymers capable of forming polymer gel particles such as agarose, polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, polyorthoester, starch and human serum albumin. can get.
Regarding the size of the particles, various investigations have revealed that it is 0.1
It is preferred to use those with a diameter between μm and 100 μm. Further, although water and methanol were used as the dispersion medium for dispersing the polymer gel particles, it is possible to use one or a mixture of two or more selected from water, methanol, acetone, toluene, ethyl ether and the like. When the concentration of the dispersion liquid in which the polymer gel particles used for forming the polymer gel particle layer are dispersed is 10 to 60 wt%, the polymer gel particle layer is easily formed and a uniform layer is formed. be able to.

Although the glucose sensor is shown in the above embodiments, the present invention can be widely used in reaction systems involving enzymes such as alcohol sensor, sucrose sensor, cholesterol sensor, lactate sensor and fructose sensor. The enzyme is not limited to glucose oxidase, depending on the specific component to be measured alcohol oxidase, invertase, mutarotase, cholesterol oxidase, cholesterol esterase, cholesterol dehydrogenase,
Lactate oxidase, lactate dehydrogenase, fructose dehydrogenase and the like can be used. Furthermore, although CMC was used as the hydrophilic polymer in the above examples, the present invention is not limited to this, and other cellulose derivatives such as hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose and carboxyethyl methyl cellulose may be used. Furthermore, polyvinyl pyrrolidone, polyvinyl alcohol, gelatin and its derivatives, polymers of acrylic acid and its salts and methacrylic acid and its salts, starch and its derivatives, and polymers of maleic anhydride and its salts can be used. However, the same effect can be obtained.

On the other hand, as the electron acceptor, in addition to potassium ferricyanide shown in the above examples, p-benzoquinone, phenazine methosulfate, indophenol and its derivatives, potassium β-naphthoquinone-4-sulfonate, methylene blue, ferrocene and The derivative etc. can also be used. Although water was used as a solvent for dissolving these hydrophilic polymers, enzymes, and electron acceptors in the above examples, various buffers such as phosphate buffer, citrate buffer, acetate buffer, and Tris-HCl buffer were used. A liquid can also be used. Further, in the above-mentioned Examples, the method of dissolving the enzyme and the electron acceptor in the sample solution was shown, but the method is not limited to this, and it can be applied to the case of being insolubilized in the sample solution by immobilization. . Further, by forming a layer containing lecithin on the polymer gel particle layer, it is possible to smoothly introduce the sample. Also,
In the above embodiment, the bipolar electrode system having only the working electrode and the counter electrode has been described, but more accurate measurement is possible by using the three-electrode system including the reference electrode.

[0016]

As described above, according to the present invention, it is possible to obtain a biosensor which can be quantified with high accuracy without being affected by a component coexisting with a specific component in a sample solution.

[Brief description of drawings]

FIG. 1 is a schematic vertical cross-sectional view of a biosensor according to an embodiment of the present invention without a cover and a spacer.

FIG. 2 is an exploded perspective view of the sensor except a reaction layer and a polymer gel particle layer.

[Explanation of symbols]

 1 Insulating Substrate 2 Lead 3 Lead 4 Working Electrode 5 Counter Electrode 6 Insulating Layer 7 Reaction Layer 8 Polymer Gel Particle Layer 11 Spacer 12 Cover 13 Sample Supply Hole 14 Air Hole

Claims (4)

[Claims] 1. An electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a reaction layer containing a hydrophilic polymer, an enzyme, and an electron acceptor disposed on the electrode system. And a polymer gel particle layer formed on the reaction layer. 2. The polymer gel particles are polystyrene, polyacrylamide, polyhydroxyethyl methacrylate, glyceryl methacrylate copolymer, gelatin, dextran, agarose, polyglycolic acid, polylactic acid,
The biosensor according to claim 1, wherein at least one polymer selected from the group consisting of glycolic acid / lactic acid copolymer, polyorthoester, starch, and human serum albumin is a main constituent element. 3. The polymer gel particles have a diameter of 0.1 μm to 1
The biosensor according to claim 1, which has a diameter of 00 μm. 4. A step of forming a reaction layer containing a hydrophilic polymer, an enzyme and an electron acceptor on an electrically insulating substrate provided with an electrode system having a working electrode and a counter electrode, in contact with the electrode system.
And 10-60 polymer gel particles on the reaction layer.
A method for producing a biosensor, comprising a step of applying a dispersion liquid dispersed in a weight ratio of wt% and drying it to form a polymer gel particle layer.