JP4036384B2 - Biosensor manufacturing method - Google Patents

Description

  The present invention relates to a biosensor. More specifically, the concentration of various liquid components is electrochemically measured using enzymes, etc., for home self-diagnosis blood glucose meter, urine sugar meter, glycated hemoglobin meter, lactic acid meter, cholesterol meter, uric acid meter, The present invention relates to a protein sensor, a single nucleotide polymorphism sensor, a DNA chip used for genetic diagnosis, and a biosensor used for an alcohol meter, a glutamic acid meter, a pyruvic acid meter, a pH meter, and the like. The present invention also relates to a method for manufacturing a biosensor having a bending step. More specifically, the present invention preferably relates to a manufacturing method including a bending step and a cutting step, or a bending step and an immobilization step.

Conventionally, a disposable sensor (Patent Document 1; Japanese Patent Laid-Open No. 47-500, Patent Document 3; Japanese Patent Laid-Open No. 52-142588) has a three-dimensional structure to ensure quantitativeness, and further a capillary phenomenon (Patent Document 5). Japanese Patent Application Laid-Open No. 56-79242, Patent Document 6; Japanese Patent Publication No. 61-502419) and the like are known in which a sample solution is automatically introduced into the sensor (FIG. 1, Patent Document 7; JP-A-1-291153). The sensor having such a configuration is assembled by stacking the spacer 2 and the cover 3 on the electrically insulating substrate 1. An electrode pattern 4 is formed on the substrate, and air holes 5 necessary for air necessary for capillary action are formed on the cover. A sample introduction port 7 having an air hole 5 on one side and a sample transport path 8 are formed by a substrate, a spacer, and a cover to introduce a certain amount of sample solution 6 into the detection portion by capillary action. In addition, each of these components must be punched into a predetermined shape in advance, and positioning for accurate overlaying of each component in 3D processing is also required. The process becomes complicated. Further, coating of reagents such as molecular identification elements and mediators on these sensors (Patent Document 2; JP-A-48-37187, Patent Document 4; JP-A-54-50396) and films for avoiding the influence of interfering substances When it is necessary to form (Patent Document 8; Japanese Patent Laid-Open No. 3-202864), the process becomes more complicated.
JP 47-500 Miles disposable sensor, dry reagent JP-A-48-37187, Roche, Enzyme, Mediator JP-A-52-142854 Kodak disposable sensor JP 54-50396, Matsushita, Enzyme, Mediator JP 56-79242 UNITED CAPILLARY Special Table Sho 61-502419, Unilever, Capillary JP-A-1-291115 / Matsushita / Basic structure, Sample inlet, Multi-item measuring sensor Japanese Patent Application Laid-Open No. 3-202864 / Matsushita / Basic Structure, Blood Cell Filtration Membrane Japanese Patent Laid-Open No. 5-199898 / Toshiba / DNA chip JP-A-9-222414, NOK, pH sensor JP 2001-204494 Glycated hemoglobin sensor WO 01/33216 A1, Cerasense, facing electrode, sample inlet US 4225410 Technicon array electrode US 5563864 NOK protein sensor US 6071391, NOK, sample inlet, facing electrode A. Ahmadian et al., Biotechniques, 32,748 (2002), SNPs

  The conventional sensor described above requires many processes and materials for manufacturing, and has to take a complicated structure. As a result, a large capital investment was required for the production line, the product yield was not sufficient, and the cost was high. Naturally, the environmental load at the time of material procurement and manufacturing was also large. Furthermore, due to the complicated process (especially alignment during substrate lamination) in terms of characteristics, the coefficient of variation (CV), which is an indicator of variations in manufactured sensor characteristics, was not sufficient. In addition, since the change in shape of the biosensor causes a decrease in measurement accuracy and reproducibility, it has been required to ensure long-term shape stability in the biosensor without causing warping of a cover or the like after manufacturing. .

  In order to solve the above-described problems, the present invention provides a biosensor manufactured by folding, bending, or bending a single electrically insulating flat substrate.

Biosensor The biosensor of the present invention includes an electrode installed in a space sandwiched between a substrate and a cover, a sample introduction port for injecting a sample into the space, and the sample introduction port extending through the electrode. A biosensor comprising a sample transport path, wherein the substrate and the cover are formed by bending a single electrically insulating plate member, and the electrode is fixed to the surface of the plate member, The substrate is bent so that the surface is inward, and is disposed in a space sandwiched between the substrate and the cover. The sample transport path is provided on the surface of the plate member, and the substrate and the cover are arranged to face each other. Is defined by an adhesive layer.

  According to the said invention, it becomes possible to manufacture a biosensor simply by forming an electrode and an adhesive bond layer on the surface of one sheet of an electrically insulating plate, and bending it.

  In the above, in order to manufacture a biosensor more simply, a perforation can be formed in a bent portion of the plate member.

  The biosensor of the present invention includes a sensor main body formed by bending a single electrically insulating plate member into a cylindrical structure, an electrode disposed on the inner wall of the sensor main body, one end of the cylinder or A sample introduction port formed on a side surface and a sample transport path extending from the sample introduction port through the electrode are provided. The cylindrical structure is a cylinder, an elliptical cylinder, a semi-cylindrical column, a fan column, a crescent column, a triangular column, a quadrangular column, or a polygonal column.

  According to the above invention, a cylindrical biosensor having a sample transport path can be manufactured by fixing an electrode to the surface of a single electrically insulating plate member and processing it into a cylindrical shape. .

  The present invention further provides a biosensor in which a reagent layer is provided on an electrode or a cover through which a sample transport path passes. According to the present invention, the sample and the sample react with each other when the sample fed from the sample transport path comes into contact with the reagent layer on the electrode or the cover. This reaction is monitored as an electrical change at the electrode.

  The sample introduction port may be one end of the sample conveyance path or an intermediate point as long as the sample can be injected into the sample conveyance path.

  In the above invention, a surfactant and / or lipid may be applied to the periphery of the sample introduction port, the surface of the sample conveyance path, and the reagent layer or the periphery thereof. By applying a surfactant or lipid, the sample can be moved smoothly. As the lipid, lecithin is preferable. The lipid application is preferably performed by dissolving the lipid in a solvent. When lecithin is used, 2-butanol is preferred as the solvent. Moreover, the tip part of the sample introduction port can have a structure having a curved part.

  The plate member can be selected from plastic, biodegradable material, and paper as long as it is electrically insulating. A suitable example of plastic is polyethylene terephthalate.

  The electrode can be composed of carbon, silver, silver / silver chloride, platinum, gold, nickel, copper, palladium, titanium, iridium, lead, tin oxide, or platinum black. Carbon may be carbon nanotubes, carbon microcoils, carbon nanohorns, fullerenes, dendrimers, or derivatives thereof. Such an electrode can be formed on the plate member by any one of a screen printing method, a vapor deposition method, a sputtering method, a foil attaching method, and a plating method.

  The electrode may be defined by a resist layer. The resist layer can be formed by a screen printing method or the like.

  The adhesive layer can also be formed by a screen printing method. Moreover, you may contain a reagent in an adhesive bond layer. As the adhesive, for example, an acrylic resin is preferable, and among these, a thermosetting resin or a photocurable resin is more preferable, and a visible light curable acrylic resin is more preferable.

The reagent layer is formed by a screen printing method or a dispenser method, and the reagent layer can be immobilized on the electrode surface, the plate member surface, or the cover by an adsorption method involving drying or a covalent bonding method.
The reagent layer is preferably formed by purification before formation. Examples of the purification method include a method such as filtration through a membrane. Impurities are removed by purification.

  The reagent layer is not limited to one place, and two or more places can be provided. In that case, two or more kinds of different reagent layers may be provided. In addition, when two or more reagent layers are provided, a convex partition portion can be provided between them. The convex partition portion can be formed by a screen printing method. The partition part of this convex part can be comprised from either carbon, a resist, or a water absorbing material.

  The reagent layer includes enzymes, antibodies, nucleic acids, primers, peptide nucleic acids, nucleic acid probes, microorganisms, organelles, receptors, cell tissues, crown ethers and other molecular identification elements, mediators, intercalators, coenzymes, antibody labeling substances, substrates, Any of inorganic salts, surfactants, lipids, or combinations thereof can be included. Further, the enzyme includes an enzyme such as oxidase or dehydrogenase, such as glucose oxidase, fructosylamine oxidase, lactate oxidase, urate oxidase, cholesterol oxidase, alcohol oxidase, glutamate oxidase, pyruvate oxidase, glucose dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase. In addition, any of cholesterol esterase, protease, DNA polymerase, or a combination thereof can be used.

  Further, the reagent layer may be contained not as an enzyme alone but as a combination of mediators. This mediator is selected from potassium ferricyanide, ferrocene, and benzoquinone. The reagent layer may contain a combination of inorganic salts such as sodium chloride and potassium chloride and quinhydrone.

  The reagent layer may contain a combination of primer, DNA polymerase, and deoxyribonucleotide triphosphate. Further, the reagent layer may contain a combination of inorganic salts such as sodium chloride and potassium chloride and quinhydrone in primer, DNA polymerase, deoxyribonucleotide triphosphate.

  When a biosensor is used as a DNA chip, a nucleic acid probe can be immobilized as a sample layer. In this case, it is preferable to arrange the electrodes in an array.

  The present invention relates to a biosensor device comprising any one of the biosensors of the present invention, a measurement unit that measures an electrical value in an electrode of the biosensor, and a display unit that displays a measurement value in the measurement unit. . Any of potential step chronoamperometry, coulometry, and cyclic voltammetry is used as a measurement method in the measurement unit. Furthermore, Bluetooth can be mounted on the apparatus as a wireless means.

Biosensor manufacturing method The biosensor manufacturing method according to the present invention includes an electrode, a sample introduction port for injecting a sample into the space, and a sample introduction port through the sample introduction port. A sample transport path extending through the electrode, and the sample transport path is a biosensor manufacturing method defined by an adhesive layer for disposing the substrate and the cover opposite to each other. A method including a step of bending a member;
The plate member is folded so that the electrode formed on the surface of the electrically insulating plate member is on the inside, and the electrode is disposed in a space sandwiched between the substrate and the cover. Forming a substrate and a cover;

  According to such a manufacturing method, a biosensor can be easily manufactured. In addition, the formation method of an electrode and an adhesive bond layer (sample conveyance path) is as above-mentioned.

  Moreover, the manufacturing method of the biosensor according to the present invention can include the bending step and a step (cutting step) of cutting a folded portion that is a portion where the plate member is bent.

In the specification, the “folded portion” means a bent portion of the plate member.
In this way, if the folded portion is cut, the stress applied to the folded portion can be removed, and the adhesion between the substrate and the cover can be performed firmly and for a long time.

  Moreover, it is preferable to cut | disconnect a folding part along a perforation. Examples of the cutting method include a method of cutting with a scalpel or the like along the perforation after bending.

  In addition, the biosensor manufacturing method according to the present invention includes a step of bending the plate member, and compression or modification of the substrate or cover, and mounting of a curing agent or heat shrink agent or a fixture to the folded portion. It is preferable to include a step of fixing the cover (an immobilization step).

  Moreover, you may apply compression, modification | denaturation processing, a hardening | curing agent or a heat shrink agent, or mounting | wearing of a fixing tool individually by 1 type or in combination of multiple.

  Since the biosensor manufacturing method according to the present invention includes a step of bending a single plate member to form a substrate and a cover to form a biosensor, immediately after the bending, the bent portion of the plate member ( (Folded part) may be restored and the substrate or cover may be warped. For this reason, it is possible to prevent the substrate or the cover from warping by including the fixing step.

  Examples of the immobilization method include compression of a substrate or cover, modification processing, curing agent or heat shrink agent, or mounting of a fixture.

<Compression>
Compression is a method of pressure-bonding at least a part of the biosensor. The pressure applied to the substrate or the cover may be uniform and large enough not to destroy the biosensor.

<Modification processing>
The denaturing process means a processing method in which the physical properties of the constituent members of the biosensor or the physical properties of the material added to the constituent members of the biosensor are modified by heat, light, chemicals, or the like. By the modification process, the stress of the warping applied to the folded portion can be removed or reduced, and the warping of the substrate or the cover can be prevented.
Hereinafter, the modification | denaturation processing method which can be used by this invention is shown to (1)-(4).

(1) Modification by heat or thermocompression bonding
[1] A method of heating or thermocompression-bonding the fold part or the fold part of the biosensor and its periphery, or another part of the biosensor.
According to such modification by heating, for example, the folded portion (folded portion) of the plate member after folding, or the folded portion and its surroundings are overheated using a mold that has been molded in the shape of the folded portion. Thus, the member of the folded portion itself can be denatured to remove the warping force. Further, instead of using a mold, the folded portion can be modified using heat rays.

Further, “the other part of the biosensor” is preferably a surface on the surface of the substrate and the cover where no reagent layer is present immediately below. In the heating method, it is preferable to denature the member by applying heat to the position where the reagent layer does not exist using the mold and heat rays.
The thermocompression bonding method is also preferably carried out by pressing the heated mold from above or below the biosensor substrate surface or both to the same site as in the case of heating.
Although the temperature of heating or thermocompression bonding depends on the material of the plate member, it is usually preferably 50 to 300 ° C, more preferably 50 to 150 ° C.

[2] The biosensor adhesive layer includes a thermosetting resin, and the modification process is performed by heating or thermocompression-bonding the biosensor folding part or the folding part and the periphery thereof, or another part of the biosensor. A method of curing all or part of the adhesive layer.
In the case where the biosensor according to the present invention has an adhesive layer, a thermosetting resin is mixed in the adhesive layer, or the plate member is bent by using the thermosetting resin itself as an adhesive. After the biosensor is formed, the above heating or thermocompression bonding is performed.

In this case, examples of the thermosetting resin include an epoxy resin, a urea resin, a melamine resin, a phenol resin, and an acrylic resin, and among these, an acrylic resin can be preferably used. In the case of an epoxy resin, it can also be used as the adhesive itself.
A crosslinking agent, a polymerization initiator, etc. can be suitably included with a thermosetting resin.

(2) Modification by light
[1] The plate member of the biosensor is made of a light-transmitting material, the adhesive layer contains a photocurable resin, and the modification process cures the adhesive layer by irradiating the biosensor with light. How to make.
When the biosensor according to the present invention has an adhesive layer, a photocurable resin is mixed with the adhesive layer, or the photocurable resin itself is used as an adhesive layer, and the plate member is bent to form the biosensor. After the formation, the adhesive layer is irradiated with light to cure the photocurable resin. In this case, the plate member constituting the substrate and the cover needs to be a light transmissive material. Examples of the light transmissive material include vinyl chloride, polycarbonate, polyester, and polyethylene terephthalate. The light irradiation is preferably performed around the folded portion after bending.

  Examples of the photocurable resin include an ultraviolet curable epoxy resin, an ultraviolet curable acrylic resin, an ultraviolet curable silicone resin, an ultraviolet curable silicone gel, a light delayed curable resin, a visible light curable dental resin, and a visible light curable resin. An acrylic resin etc. are mentioned. In the case of an ultraviolet curable acrylic resin, a light delayed curable resin, or a visible light curable acrylic resin, it can also be used as the adhesive itself.

Moreover, a crosslinking agent, a polymerization initiator, etc. can be suitably included with photocurable resin.
The light to be irradiated varies depending on the type of the photocurable resin. For example, ultraviolet rays can be used when an ultraviolet curable resin is used, and visible light can be used when a visible light curable resin is used. In the case of ultraviolet rays, deuterium lamps, high-pressure mercury lamps, low-pressure mercury lamps, metal halide lamps, electrodeless ultraviolet lamps can be used for ultraviolet rays, and in the case of visible rays, halogen lamps, xenon lamps, metal halide lamps, An incandescent bulb (tungsten lamp), a fluorescent lamp, a light emitting diode, an organic light emitting element, or the like can be used.
The type of plate member that transmits light varies depending on the type of light used, and examples thereof include vinyl chloride, polycarbonate, polyester, and polyethylene terephthalate.

(3) When the plate member itself is a heat or light modifying material
[1] A method in which the plate member of the biosensor includes a thermosetting resin, and the modification process heats and hardens all or part of the plate member.
[2] A method in which the plate member of the biosensor includes a photo-curable resin, and the modification process cures the plate member by light irradiation.

  In addition, the plate member itself may be a thermosetting resin or a photocurable resin. When the plate member is made of a thermosetting resin, the biosensor is bent and formed, and then, as shown in (1) [1] above, the folded portion or the folded portion and its surroundings, or another biosensor. The part is heated or thermocompression bonded. The same thermosetting resin as described above can be used.

  When the plate member is made of a photocurable resin, the biosensor is bent and formed, and then light irradiation is performed in the same manner as described in (2) [1] above. The same thing as the above can be used for a photocurable resin.

(4) Permeation of solvent into the fold
[1] A method in which a solvent is applied to a fold portion or a fold portion of the biosensor and a surface around the fold portion, and the solvent is infiltrated into the fold portion.

In this case, a solvent can be applied to the folded portion or the folded portion and the periphery thereof, and the warping force remaining on the plate member can be removed or reduced by infiltrating the solvent into the plate member.
Any solvent can be used as long as it can penetrate into the plate member, and an organic solvent is preferable although it depends on the material of the plate member. For example, the following is mentioned as a preferable combination of the plate member and the organic solvent.
Polytetrafluoroethylene ether
Polyethylene amylbenzene
Polyisobutylene xylene
Polystyrene methyl chloride
Chlorinated rubber Methylene chloride
Vinyl acetate resin Ethylene chloride
Methyl methacrylate dioxane
Vinyl chloride resin cyclohexane
Epoxy resin Acetone
Acetylcellulose Isopropyl alcohol
Nitrocellulose Dimethylformamide
Phenol resin Nitromethane

  As described above, the biosensor according to the present invention is preferably modified by the above method.

<Application of curing agent or heat shrink agent>
The curing agent or heat shrink agent is applied to the folded part by applying a curing agent or heat shrink agent to the folded part of the biosensor component and curing the curing agent or semi-curing the heat shrink agent. Yes, it is possible to suppress the stress of warping applied to the folded portion and prevent the substrate or the cover from warping.
(1) A method of covering the folded portion with a solidifying agent (thermosetting resin or photocurable resin) or a heat shrinking agent
[1] A method in which a thermosetting resin is applied to a folded portion of the biosensor or a folded portion and the periphery thereof, and the thermosetting resin is further heated to cure the thermosetting resin.
[2] A method in which a photocurable resin is applied to the folded portion of the biosensor or the folded portion and its periphery, and the photocurable resin is further irradiated with light to cure the photocurable resin.
[3] A method of semi-curing the heat-shrink agent by applying a heat-shrink agent to the folded portion of the biosensor or the fold-portion and its periphery, and further heating the heat-shrink agent.
The application is preferably performed uniformly on the folded portion or the folded portion and the surrounding external surface.

In this case, the thermosetting resin or the photocurable resin is applied to the folded portion or the folded portion and the periphery thereof, and the resin attached to the folded portion is heated or irradiated with light in the same manner as described above. The resin is cured.
Alternatively, the heat shrink agent is applied to the folded portion or the folded portion and the periphery thereof, and the heat shrink agent adhered to the folded portion is heated in the same manner as described above to semi-cur the heat shrink agent. Examples of the heat shrink agent include polyolefin, fluororesin, and polyethylene.
In this way, it is possible to prevent the substrate or the cover from being bent back from the folded portion.

<Fixing tool>
Examples of the fixing method by mounting the fixing tool include pinching processing, surrounding processing, mounting of a cap (lid), mounting of a fastening tool by an elastic body, processing by a heat shrink agent, and mounting of an adhesive tape.

  The pinching process means a method using a stopper that holds and holds at least a part of a biosensor with a clip or the like.

  Encircling means a processing method that encloses at least a part of a biosensor using a fixed fixture.

  When attaching a cap, it is preferable to attach it to the end of the folded portion of the biosensor. Examples of the material of the cap include an elastic body, a thermal photocuring agent, a photocuring agent, and a heat shrinking agent described below.

  Among the fasteners made of an elastic body, examples of the elastic body include natural rubber, synthetic rubber (such as butyl rubber), and silicone.

Examples of the heat shrink agent include polyolefin, fluororesin, and polyethylene.
In carrying out the fixing method, for example, an adhesive such as an acrylic adhesive is applied to the inside of the fixture (for example, a portion that comes into contact with an inner sensor such as an elastic material or a heat shrink agent). Is more preferable. Thereby, fixation becomes more reliable and it can prevent more reliably the curvature of a board | substrate or a cover.

  Examples of the adhesive tape include cellophane tape, polypropylene tape, acetate tape, Kapton (polyimide) tape, metal tape (aluminum, copper, etc.), paper tape, and non-woven tape. In general, an acrylic pressure-sensitive adhesive is preferably used as the pressure-sensitive adhesive for these tapes.

  By such an immobilization method, the biosensor of the present invention may have a fixture that prevents the substrate and the cover from warping.

  As is clear from the above description, a biosensor that is superior in productivity and economy and has a low environmental impact is obtained by folding or bending or bending a single electrically insulating flat substrate to produce a biosensor. The sensor can be manufactured. In the measurement with this biosensor, it is possible to measure with high accuracy by introducing the sample solution quantitatively into the structure of the biosensor using the capillary phenomenon, and reproducibility by a simple manufacturing process. An excellent biosensor can be realized.

  In addition, since the biosensor manufacturing method according to the present invention is manufactured by bending a single plate member, a biosensor can be obtained very simply. Furthermore, when it has the process of fixing the bent plate member, the biosensor can be prevented from warping.

  Or the manufacturing method of the biosensor which concerns on this invention can also prevent the curvature of a biosensor by having the process of cut | disconnecting the bent board member.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 2 shows a typical structure of the biosensor of the present invention, in which a is a folded biosensor and b is an example of a cylindrical biosensor.

  The development view of the biosensor is shown on the left of FIG. 2a. This biosensor is composed of a plate member 1 having a flat surface. Here, “the surface is flat” means that there are no irregularities formed artificially by mold, cutting, bonding, etching, or the like. The plate member 1 is provided with a perforation 9 for facilitating the bending process. The plate member 1 includes a substrate portion 1a which becomes a substrate after bending processing, which will be described later, with a perforation 9 interposed therebetween, and a cover portion 1b whose lower portion functions as a cover after bending processing.

  A pattern 4 including electrodes is formed on the surface of the substrate portion 1 a of the plate member 1. The electrode pattern 4 is bent in an L shape at the lower end of the drawing, and this L-shaped portion is orthogonal to a reagent transport path 8 described later. Moreover, the reagent layer 11 can be provided in the L-shaped part of this electrode pattern 4 as needed.

  On the other hand, an adhesive layer 10 is provided on the surface of the cover portion 1b. The adhesive layer 10 has a role of defining the sample transport path 8 in addition to the role of bonding and fixing the surfaces of the substrate portion 1a after bending and the cover portion 1b. Therefore, the adhesive layer 10 is provided on both sides of the cover portion 1b except for the central portion that becomes the sample transport path 8. Further, a sample introduction port 7 for injecting the sample 6 into the sample transport path 8 is formed where the sample transport path 8 and the perforation 9 intersect. After the electrode 4, the adhesive layer 10 that defines the sample transport path 8, and the sample introduction port 7 are formed on the plate member 1 in this way, the perforation 9 is bent, whereby a biosensor as shown in the right of FIG. Is manufactured. The opposite of the sample inlet (air outlet) functions as an air outlet during production. The reagent layer 11 can be provided on the electrode as described above, but can also be provided on the cover portion 1b.

  When this biosensor is used for inspection or the like, the sample introduction port 8 formed at the lower end is brought into contact with the sample 6 to suck up the sample 6. The sucked sample 6 comes into contact with the reagent layer 11 when passing through the sample transport path 8, and the reagent reacts with a target component in the sample, and electrochemical changes such as potential and current generated by the reaction are detected by the electrodes. To do. When there is no reagent layer, the target component is detected only by the electrode.

  According to the configuration of the above biosensor, since it can be manufactured by one turn, the complicated positioning at the time of stacking, which is found in the conventional sensor, is not necessary, and not only the manufacturing process can be simplified but also the product yield. Can be improved.

  Other embodiments of the folding type biosensor are shown in FIGS. In FIG. 2, the sample transport path is provided in the longitudinal direction of the plate member 1, but in FIG. 3, the cover 1 b is formed by dividing the adhesive layer 10 in the vertical direction. It is formed so as to cross the plate member 1. Since the sample transport path 7 extends in the lateral direction as described above, the electrode pattern 4 does not need to be bent in an L shape as shown in FIG. 2A, and is provided as a pattern of two electrodes extending in parallel. The plate member 1 on which the electrode and the adhesive layer are thus formed is bent at the perforation 9 to form the sample transport path 8 and the sample introduction port 7 is formed once. In the case of this configuration, it is not necessary to process and form the sample introduction port into the plate member 1 as shown in FIG. 2, so that the manufacturing process can be further simplified.

  As shown in FIGS. 2 and 3, the sample introduction port does not necessarily have to be provided at the ends of the upper and lower sides and the side surfaces. For example, as shown in FIG. 4, the sample introduction port 7 can be formed in the middle of the sample transport path 8. Specifically, the sample introduction port 7 can be formed not on the perforation 9 as shown in FIG. 2A but on the sample conveyance path 8 of the cover portion 1b. In the case of this configuration, the sample introduction port 8 is formed on the cover surface as shown in the right side of FIG.

  Moreover, the board | substrate part and cover part arrangement | positioning in a board member do not need to be up-and-down as shown to FIG. 2 a, 3 and 4, and may be arrange | positioned right and left like FIG. FIG. 10 is an application example of FIG. 3, but when the substrate part and the cover part are arranged on the left and right as shown in FIG. 10, the air discharge port 13 for discharging the sample 6 injected from the sample introduction port 7. It is necessary to provide.

The electrode may be installed not only on the substrate portion 1a side of the plate member 1 but also on the cover portion 1b side. Furthermore, as shown in FIG. 5, it can also be set as the facing arrangement | positioning structure which fixed the electrode to the board | substrate part 1a and the cover part 1b, respectively. In the case of the biosensor shown in FIG. 5, the lower end portion is formed in a triangular shape as shown on the right side of the drawing, and the tapered portion at the lower end functions as a sample introduction port. The sample sucked up from the sample introduction port spreads over the entire sample introduction path on the triangle where the adhesive layer 10 is not provided, and is stored between the electrodes forming the facing structure for measurement.
Other examples of the facing electrode type are shown in FIGS. 6, 7, 8, 9, 14b and f, and FIGS. 15a, b, c, d, e, f, and g. Here, in FIG. 6, the electrodes are respectively fixed to the substrate portion 1 a and the cover portion 1 b of the plate member 1 to form a facing structure. And the sample conveyance path 8 prescribed | regulated to the adhesive bond layer 10 is formed so as to be orthogonal to this electrode. 7 has the same electrode arrangement as that of FIG. 6, but the sample introduction port 7 is provided on the perforation 9, and the adhesive layer 10 is defined from the sample introduction port 7 along the electrode pattern 4. A sample transport path 8 is formed. FIG. 8 is also an example of a biosensor in which the electrodes have a face-to-face structure. In this example, the adhesive layer 10 is provided leaving a slight electrode portion in the vicinity of the sample introduction port 7. In the case of the biosensor of FIG. 8, the measurement is performed by bringing the sample 6 injected from the sample introduction port into contact with the end of the electrode having the facing structure. FIG. 9 shows that a U-shaped adhesive layer 10 is provided on either the cover part or the substrate part, and the sample transport path extends from the U-shaped depression toward the sensor end along the electrode. 7 is formed. In addition, a sample introduction port 7 is formed on the other side of the cover portion or the substrate portion at a position facing the U-shaped depression. In the configuration of FIG. 9, as shown on the right side of the drawing, the sample 6 injected from the sample introduction port 7 is sent to the sample transport path 8 formed between the facing electrodes, and measurement is performed at this time.

  In addition, the example in which the sensor unit is configured by the two electrodes composed of the working electrode and the counter electrode is shown above, but the electrode does not need to be two electrodes, and the three electrodes composed of the working electrode, the counter electrode, and the reference electrode. A sensor unit can also be configured. Examples provided with these three electrodes are shown in FIGS. 14 (e) to 14 (g) and FIGS. 15 (e) to 15 (g). In order to reduce the size of the sensor, a sensor unit comprising two working electrodes and a counter electrode is preferable. On the other hand, in order to increase the reliability of measurement, it is preferable to employ a sensor unit composed of three electrodes including a reference electrode. In either case of two poles or three poles, the width and arrangement of the electrodes are arbitrary. Further, in the case of three poles, the electrode arrangement may be any of a parallel arrangement (FIGS. 14E and 14G) and a facing arrangement (FIGS. 14F and 15E to 15G).

  In addition, the plate member 1 has been described by taking a suitable example provided with a perforation. However, the configuration for facilitating the bending process is not limited to the perforation, and a triangular groove as shown in FIG. Alternatively, a fan-shaped groove (c) shown in (b) can be provided on the back surface of the plate member 1.

  As shown in FIGS. 12, 13, 15 (b) and (f), when there is a sample inlet at the tip of the sensor (all drawings are shown on the lower end side in the assembled structure), contact with the human body is required. In consideration, it can be formed into a shape having a curved surface.

  The biosensor is not limited to the above-described folded laminated type, and may be a cylinder formed by bending a single plate member. A typical configuration example is shown in FIG. That is, an electrode pattern 4 is formed on the surface of one sheet of electrically insulating plate member 1, and an adhesive layer 10 is provided on one side end. In this state, the cylindrical sensor is simply configured by bending once and attaching the adhesive layer to the back surface of the other end. In the case of this cylindrical sensor, since the ends of the cylinder function as a sample inlet and outlet, a step of forming a sample inlet or the like in the plate member in advance can be omitted. Other examples of the cylindrical sensor are shown in FIGS. FIG. 16 shows an example of a cylindrical sensor, FIG. 17 shows an example of a cylindrical sensor having a triangular cross section, and FIG. 18 shows an example of a cylindrical sensor having a quadrangular cross section. A sensor having a fan, a crescent moon, or a polygonal cylinder structure may be used. Also in the case of a cylindrical sensor, the electrodes may be two or three, and any one can be selected. Further, the arrangement of the electrodes may be either parallel or facing. Examples of the cylindrical biosensor provided with three electrodes include the configurations shown in FIGS. 16e, f, g, h, FIGS. 17e, f, g, h, and 18e, f, g, h. In the case of a cylindrical structure, as a method of maintaining the shape of a three-dimensionally processed sensor, a side portion parallel to the electrode of the sensor pattern or a paste margin portion is extended from the side surface, and an adhesive is applied thereto to apply three-dimensional processing. Sometimes, the three-dimensional shape of the sensor can be maintained by pasting at a predetermined position. A double-sided adhesive tape can be used instead of the adhesive.

  Although the reagent layer is not necessarily illustrated in the above-described drawings, the reagent layer can be provided as necessary in the drawings that are not illustrated. For example, when a nickel electrode is used as the electrode, it can be used as a protein sensor (US Pat. No. 5,653,864) for detecting protein even without a reagent layer. When a platinum electrode is used, this sensor can be used as an oxygen sensor by combining a conductivity sensor, a hydrogen peroxide sensor, an oxygen permeable membrane, and an electrolyte. On the other hand, if a reagent layer is used, a blood glucose sensor using an enzyme and a mediator, a urine sugar sensor, a glycated hemoglobin sensor (Japanese Patent Laid-Open No. 2001-204494), a lactic acid sensor, a uric acid sensor, a cholesterol sensor, an alcohol sensor, a glutamic acid sensor, a pyruvate sensor, PH sensor using silver / silver chloride electrode and quinhydrone, inorganic salt (Japanese Patent Laid-Open No. 9-222414), single nucleotide polymorphism sensor using pH sensor and primer, DNA polymerase, DNA chip using immobilized nucleic acid probe, etc. And can be applied as a sensor for detecting various chemical and physical states. The reagent layer is formed on or around the electrode through which the sample transport path passes.

  A surfactant and lipid can be applied around the sample inlet and the surface of the sample transport path so that the sample can be easily introduced.

  Hereinafter, materials, manufacturing methods, applications, and the like of the biosensor according to the above-described embodiment will be described in detail.

  As the plate member, a plastic such as polyethylene terephthalate, a biodegradable material such as polylactic acid, paper, or the like can be used.

  Examples of the electrode material used in this sensor include metals such as platinum, gold, silver / silver chloride, silver, copper, palladium, iridium, lead, nickel, titanium, tin oxide, and platinum black. These are excellent in conductivity and can be formed by vapor deposition, sputtering, plating, CVD, coating and drying. In addition, carbon powder is slightly inferior in conductivity compared to platinum and gold, but it can be formed into a paste and applied to a substrate to form electrodes easily by screen printing, etc., as with silver powder. it can. Furthermore, a particulate material such as platinum or gold can be pasted and processed by printing.

  Carbon nanotubes, carbon microcoils, carbon nanohorns, fullerenes, dendrimers, and derivatives thereof can be used as the carbon material. These are suitable for immobilization of molecular identification elements and electrode materials because of their unique properties (structure, conductivity, etc.).

  In the case of a protein sensor, nickel is preferably used as the electrode material. Nickel oxidizes the amino group of a protein under a predetermined condition to become a protein sensor. FIA (flow injection analysis) is also possible.

  The adhesive layer is preferably formed by a screen printing method. Double-sided adhesive tape is also possible. Here, examples of the adhesive include a bond and a pressure-sensitive adhesive.

  It is also possible to include a reagent in the adhesive layer and form the adhesive layer and the reagent layer simultaneously by screen printing.

  The reagent layer is preferably formed by dropping the reagent in the form of an aqueous solution with a dispenser or the like and drying it. It is also possible to adjust the viscosity and form by screen printing.

  When there is only one reagent layer, there is only one substance to be detected. However, when two kinds of substances are detected at the same time, for example, as shown in FIGS. It can also be formed on an insulating flat substrate (Japanese Patent Laid-Open No. 1-291153). In this case, as shown in the figure, in order to prevent mixing of the dissolved reagent layers in the middle of the reagent layer, the convex portion (12) is formed by a screen printing method or the like with carbon, a resist, a water-absorbing material, or the like. Is also possible. In this case, the initial thickness of the convex portion should be thinner than the adhesive layer, and when bent, the upper and left and right sides of the convex portion must be open. This is to facilitate the passage of the sample. In the case of a water-absorbing material, after passing through the sample, it has a function of preventing the dissolving reagents from mixing with each other due to swelling. Further, when the number of electrodes is not four (a and b in FIGS. 19, 20, and 21) but three, for example, the middle one can be used as a common counter electrode (FIGS. 19, 20, and 21). C, d).

  The binding between the reagent and the electrode surface or the substrate can be carried out by an adsorption method after drying or a covalent bonding method.

  The reagents include enzymes, antibodies, nucleic acids, primers, peptide nucleic acids, nucleic acid probes, microorganisms, organelles, receptors, cell tissues, crown ethers and other molecular identification elements, mediators, intercalators, coenzymes, antibody labeling substances, substrates, inorganic Salts, surfactants, lipids and the like are used.

  For example, in an enzyme sensor, the type of enzyme serving as a molecular identification element is changed depending on the subject to be measured. For example, glucose oxytase or glucose dehydrogenase when the measurement target is blood glucose (glucose), urine sugar, a mixture of fructosylamine oxidase and protease when the measurement target is glycated hemoglobin, lactate oxidase when the measurement target is lactic acid, measurement Mixture of cholesterol esterase and cholesterol oxidase when the target is total cholesterol, urate oxidase when the measurement target is uric acid, alcohol oxidase when the measurement target is ethanol, glutamate oxidase when the measurement target is glutamic acid, and pyruvine In the case of acid, pyruvate oxidase or the like is used.

  In the enzyme sensor, an electron carrier (mediator) is used together with the enzyme. As the mediator, potassium ferricyanide, ferrocene, ferrocene derivatives, nicotinamide derivatives, flavin derivatives, benzoquinone, quinone derivatives and the like are used.

  In the case of a pH sensor, an inorganic salt such as sodium chloride or potassium chloride and a quinhydrone reagent layer are used on a substrate provided with a silver / silver chloride electrode and another electrode. In this case, the change in the interelectrode potential is measured.

  In the case of single nucleotide polymorphism (SNPs) sensors (A. Ahmadian et al., Biotechniques, 32, 748, 2002), a mixture of primer, DNA polymerase, deoxyribonucleotide triphosphate, etc. is used as a reagent on the above pH sensor. The change in pH when the analyte DNA in the sample and the primer are complementary is measured.

  An immunosensor uses an antigen-antibody reaction, and for example, when measuring human serum albumin, anti-albumin is used as a molecular identification element. In an immunosensor, an interelectrode potential that varies with the formation of an antigen-antibody complex is measured.

  In the microorganism sensor, microorganisms such as Pseudomonas fluorescence (measuring object: glucose) and Trichosporon brassicae (measuring object: ethanol) are used as the molecular identification element. Since these microorganisms breathe oxygen (aerobic) or produce metabolites in an oxygen-free environment, they will capture oxygen respiration or metabolites electrically.

  In the organelle sensor, an organelle is used as a molecular identification element. For example, NADH can be measured by using mitochondrial electron transfer particles. As this principle, NADH is oxidized by mitochondrial electron transfer particles, and oxygen is consumed at this time. Therefore, NADH and NADPH can be measured using this oxygen as an index.

  In the receptor sensor, a receptor such as a cell membrane is used as a molecular identification element. Specimens include hormones and neurotransmission. As a measurement principle, a change in acceptance is converted into a potential and measured through an electrode.

  Tissue sensors use animal and plant tissues as molecular identification elements. As animal and plant tissues, for example, frog skin, animal liver slices, cucumbers, banana peels and the like can be used. As a measurement principle, for example, in a sodium sensor using frog skin tissue, the frog skin tissue selectively permeates sodium ions, and the potential of the skin tissue changes at that time. Find the amount.

  Another example of the application of the biosensor described here is a DNA chip (Japanese Patent Laid-Open No. 5-199898). Many kinds of single-stranded nucleic acid probes having complementarity to many kinds of target genes to be detected are immobilized on an electrode array (US 4225410) as shown in FIG. One type of nucleic acid probe is immobilized on one electrode. In order to confirm the presence or absence of a large number of target genes, a single-stranded denatured gene sample and a nucleic acid probe are hybridized, and then specifically bound to a double-stranded nucleic acid and electrochemically An active double-stranded recognizer is added to After washing, the substrate is folded in the presence of buffer solution, the array electrode is the working electrode, the upper electrode is the counter electrode, and voltage is applied to each electrode sequentially. The insertion agent is oxidized and an oxidation current flows. No current due to the intercalating agent flows at the electrode where the double strand is not formed. Since the type of the nucleic acid probe is known at the position of the electrode where the current is generated, the presence or absence of the target gene and the qualitativeness are possible. In addition, as said double strand recognition body, metallointercalators, such as intercalators (inserting agent), such as acridine orange, a tris (phenanthroline) cobalt complex, etc. can be used.

  The biosensor according to the present invention is preferably stored in a dry state, and preferably stored with a desiccant. Examples of the desiccant include activated alumina, zeolite, silica gel, calcium chloride and the like.

  When measurement is performed using the biosensor as described above, the biosensor is attached to the apparatus, and the electrical value generated in the biosensor is measured. This measurement includes a measurement unit that measures the electrical value of the biosensor and a display unit that displays the measured value. As a measuring method of the measuring unit, potential step chronoamperometry, coulometry, voltammetry, or the like can be used. In addition, this apparatus can be provided with a memory for storing measurement values. In addition, when managing the measurement values remotely, wireless means such as Bluetooth may be installed.

FIG. 24 is an example of a diagram illustrating a method for preventing the warpage of the plate member by fixing the biosensor manufactured by folding from one elastic plate member by heat modification.
FIG. 24A is an example of the biosensor 101 manufactured by bending a plate member. The biosensor includes a plate member 102 having insulating elasticity, an electrode 103 on the plate member, a sample inlet 104, and a folded portion 105. Here, although not shown, a spacer layer for forming a sample introduction port and a reaction detection unit exists between the upper and lower plate members 102 of the biosensor. The spacer layer may be formed of only an adhesive layer, or may be a plate member and an adhesive layer provided on both upper and lower surfaces thereof.

Here, the folding part 105 is formed, for example, when the plate member is formed by bending along the perforation made on the substrate.
FIGS. 24b and 24c show parts where the folding part 105 or the folded part and its periphery are heated and modified by inserting the biosensor folding part 105 into the groove of the denaturing processing device 106 (mold) for heat denaturing. An example of (107) is shown.

The mold 106 does not necessarily require a groove, and may be a flat mold (heat source). Biosensor folding part end ("folding part end" is a part excluding the periphery of the sample inlet 104 and the reaction detection part, and includes the folding part 105 or the folding part 105 and its surroundings. The end portion of the folded portion may be modified by heat from the mold without contacting the mold.
Here, since the enzyme may be inactivated by heating, the range of heating is the folding part 105 or the folding part of the biosensor 101 and the periphery thereof, or the end part of the cover opposite to the folding part (cover end part; The “cover end” is a portion excluding the periphery of the sample introduction port 104 and the reaction detection unit, and is preferably a region around the end of the cover opposite to the folded portion. As a result, the sample introduction port and the reaction detection unit can be controlled to be less susceptible to the influence of heating.

FIG. 25 is a part in which the folding part 105 or the folding part 105 and its periphery are subjected to thermal denaturation by the end part of the biosensor 101 and three denaturing processing devices (heat rays) 106 above and below the biosensor 101. An example of obtaining a biosensor having 107 is shown. In this case, the relevant part of the biosensor may be thermally denatured by contact with the heat rays, or the relevant part may be thermally denatured by the radiant heat without contacting.
Similarly to the above, it is preferable that the heat rays are arranged so that the enzyme in the biosensor 101 is not affected by heat.

FIG. 26 shows that a pair of upper and lower denaturing processing devices (types) 106 for denaturing by thermocompression bonding are thermocompression-bonding the end portions of the folded portions of the biosensor 101 to cure and denature the corresponding portions of the plate member 102 and the spacer. An example of obtaining a biosensor having a modified part 107 is shown.
In FIG. 26, a pair of upper and lower molds 106 are used, but only the upper or lower molds may be used for heat denaturation to reduce the influence of warping.
As described above, it is preferable to arrange the mold so that the enzyme in the biosensor 101 is not affected by heat.

In FIG. 27, similarly to FIG. 26, two sets of upper and lower molds 106 for denaturation by thermocompression are heated at two places around the fold end portion 106 including the fold portion of the biosensor 101 and around the cover end 109. The plate member 102 and the corresponding portions of the spacer are cured and denatured by pressure bonding.
In FIG. 27, two sets of upper and lower molds 106 are used. However, only the upper or lower molds may be used to reduce the influence of warping due to thermal denaturation.
As described above, it is preferable to arrange the mold so that the enzyme in the biosensor 101 is not affected by heat.

  FIG. 28 shows a photocuring resin in which the folding portion 105 or the folding portion 105 formed of the substrate 102 having the photocurable resin in the folding portion 105 or the plate member 102 made of the photocurable resin and its periphery receive light. Shows an example in which photocuring occurs.

FIG. 29 shows an example in which the fold 105 or the fold 105 and its periphery are subjected to denaturation by being immersed in a chemical reagent contained in the groove of the denaturing apparatus 106 from the fold of the biosensor 101. As the chemical reagent used here, an organic solvent that dissolves the substrate is suitable. The solvent can permeate into the folded portion, and the warping force (stress) can be removed or reduced.
In addition, when the chemical reagent in FIG. 29 is a thermosetting resin or a photocurable resin, warping can be suppressed by curing the resin through a predetermined process such as heating or light irradiation.

FIG. 30 shows an example in which the fold 105 or the fold 105 and the side surface around the fold 105 are denatured by coming into contact with the surface of a rotating roller moistened with a chemical reagent.
When the chemical reagent in FIG. 30 is a thermosetting resin or a photocurable resin, warping can be suppressed by curing the resin through a predetermined process such as heating or light irradiation. In this case, thermal curing or photocuring can be caused by replacing with a rotating roller heater or a light source.

  FIG. 31 to FIG. 36 show a method of fixing a substrate or a cover by using a clip as a fixture and sandwiching a terminal having a biosensor folding part in whole or in part to prevent warping.

  FIG. 31 shows an example of a fixing method using a fixing tool (clip) 108 of a size that can be fixed with the entire end of the folded portion of the biosensor 101 as a fixing tool. The clip material used in this case has a pinch strength that can fix the folds and suppress warping, and the biosensor body can be excessively damaged by dents, especially the sample inlet and the reaction detector. It suffices if the change in shape can be prevented. For example, metal or plastic can be used, and plastic is preferable.

  FIG. 32 is the same as FIG. 31 except for the size of the clip. In this case, it is preferable that the fixture (clip) 108 is sandwiched in the center of the end of the folded portion of the biosensor 101 in consideration of the left / right balance. Furthermore, it is also possible to fix the end of the folded portion of the biosensor 101 by using a small clip or a hairpin or the like, and to prevent warping.

  FIG. 33 shows an example of a fixing method using a fixing tool (clip) 108 of a size that can be fixed by sandwiching the entire width of the biosensor from one side of the end of the folded portion of the biosensor 101 as a fixing tool.

  FIG. 34 shows an example of a fixing method in which the side surface of the end portion of the folded portion of the biosensor 101 can be fixed by being sandwiched from both sides using two fixing tools (clips) 108.

  FIG. 35 shows an example of a fixing method in which the entire end portion side of the biosensor 101 is covered with a fixing tool (cap) 108. The material of the cap used in this case is not particularly limited, such as metal or plastic, but plastic is preferred. Further, by replacing the cap material with a heat-shrinkable material, the end of the folded portion of the biosensor can be uniformly fixed with an appropriate force.

  FIG. 36 shows an example of a method for fixing the end of the folded portion of the biosensor 101 as a fixture with a fixture (frame) 108. The material of the frame used in this case is not particularly limited, such as metal or plastic, but plastic is preferable.

  FIG. 37 shows an example of a method for fixing the end of the folded portion of the biosensor 101 with a fixture 108 (ring) 108 as a fixture. The material of the ring used in this case is not particularly limited, but plastic is preferable. In particular, use is made of a ring having elasticity such as rubber, or a ring made of a heat shrink agent and fixed by heat shrink treatment.

  FIG. 38 shows an example of a method of fixing the end of the folded portion of the biosensor 101 as a fixture with a fixture (tube-shaped ring) 108. The material of the ring used in this case is not particularly limited, but plastic is preferable. In particular, use is made of a ring having elasticity such as rubber, or a ring made of a heat shrink agent and fixed by heat shrink treatment.

  FIG. 39 shows an example of a method of winding and fixing the end of the folded portion of the biosensor 101 as a fixture with a fixture (adhesive tape) 108. The material of the adhesive tape used in this case is not particularly limited, but plastic is preferable. Furthermore, the use of a ring having elasticity such as rubber or a ring made of a heat-shrinking agent and fixed by heat-shrinking is also suitable.

  Hereinafter, although it demonstrates in detail using the Example of this invention, this invention is not limited to a present Example. In addition, in the Example, the example of the glucose biosensor was shown as an enzyme sensor.

[Example 1]
FIG. 2a shows a glucose sensor according to an embodiment of the present invention. As the reagent layer, glucose oxidase and potassium ferricyanide are used. The measurement principle of the glucose sensor shown in FIG. 2a is as follows.

  In this sensor, a sample is introduced into the inside through a sample inlet by capillary action. In the introduced glucose solution, ferricyan ions are converted to ferrocyan ions as the glucose is oxidized as shown in the following formula 1 by the catalytic action of GOD in the reagent layer.

[Formula 1]
GOD
Glucose + ferricyan ion → Gluconolactone + ferrocyan ion

  The produced ferrocyanian ions are oxidized at the carbon electrode according to the electrode reaction of the following formula 2 and detected electrochemically.

[Formula 2]
Electrode ferrocyanide ions → ferricyanide ion + e ^-

  In the detection method using the glucose sensor of the present invention, the produced ferrocyan ion is oxidized by the anode electrode, an anode current is generated, and the ferrocyan ion becomes ferricyan ion again. As described above, glucose can be quantified by observing the change in the current value of the ferrocyan ion concentration generated from the enzyme reaction.

Next, a manufacturing method and a measuring method will be described.
PET having a length of 65 mm, a width of 6 mm, and a thickness of 188 μm was used as a sensor substrate. Two carbon electrodes having a width of 1.3 mm were formed on the sensor substrate by a screen printing apparatus with an interval of 2.6 mm. The adhesive was also formed as an adhesive layer by screen printing. The perforated part was perforated. The sample volume was 0.5 μl.

  The method for forming the reagent layer of the enzyme and mediator is as follows. Glucose oxidase (GOD) and potassium ferricyanide (mediator) were dissolved in distilled water and applied to the electrode surface. 4 units GOD 0.1 mg potassium ferricyanide is dissolved in 10 μL of distilled water. 3 μl of this GOD solution was applied to the electrode surface and vacuum-dried to form a reagent layer comprising an enzyme / mediator on both electrodes.

  An example in which blood glucose (blood glucose) is measured using this glucose sensor will be described. Blood glucose measurement using this glucose sensor used whole blood with a hematocrit value of 40%, prepared so that the glucose concentration was 50, 100, 200, 300, 400, 500 mg / dl as the sample liquid. The potential step chromoamperometry was used as the measurement method. Five seconds after 0.5 μl of blood was introduced into the sample inlet by capillary action, a potential of 900 mV was applied between the two electrodes in the sensor, and the current value 5 seconds after the application was taken as the measured value.

  FIG. 23 shows a change in current value according to blood glucose concentration of the sensor of the present invention. Referring to FIG. 23, a current value change of 1 to 2.5 μA was observed in the range of blood glucose 50, 100, 200, 300, 400, and 500 mg / dl. When the measurement was performed 10 times with 100 mg / dl whole blood, the reproducibility of the measured value was 4.1% in terms of coefficient of variation.

[Example 2]
In Example 1, a reagent layer composed of glucose oxidase and potassium ferricyanide was formed on the electrode and evaluated. In Example 2, the reagent layer was formed not on the electrode but on the cover part. That is, referring to FIG. 2a, the reagent layer is formed on a part of the cover portion of the sample transport path 8 between the adhesive layers 10 formed on the cover portion. Except that the reagent layer was formed on the cover, the method for forming the sensor substrate, the enzyme and mediator reagent layers, and the measurement conditions were the same as in Example 1. As a result, a change in current value of 1 to 3.0 μA was observed in the range of blood glucose 50, 100, 200, 300, 400, and 500 mg / dl, almost as in FIG. When the measurement was performed 10 times with 100 mg / dl whole blood, the reproducibility of the measured value was 5.9% as a coefficient of variation.

The structure of a conventional sensor (Japanese Patent Laid-Open No. 1-291153) is shown. The biosensor representative example of this Embodiment is shown. a is a development view (left) and a configuration diagram (right) of a folding biosensor, and b is a development view (left) and a configuration diagram (left) of a cylindrical biosensor. The development view (left) of the biosensor of another folding type (bonding structure) and the configuration diagram after assembly (right) are shown. The development view (left) of the biosensor of another folding type (bonding structure) and the configuration diagram after assembly (right) are shown. The expansion | deployment figure (left) of the biosensor of the facing electrode of another bending type | mold (bonding structure) and the block diagram (right) after an assembly are shown. The development view (left) and configuration diagram (right) after assembly of the biosensor of the facing electrode in another folding mold (bonding structure) are shown. The expansion | deployment figure (left) of the biosensor of the facing electrode of another bending type | mold (bonding structure) and the block diagram (right) after an assembly are shown. The expansion | deployment figure (left) of the biosensor of the facing electrode of another bending type | mold (bonding structure) and the block diagram (right) after an assembly are shown. The expansion | deployment figure (left) of the biosensor of the facing electrode of another bending type | mold (bonding structure) and the block diagram (right) after an assembly are shown. The development view (left) of the biosensor of another folding type (bonding structure) and the configuration diagram after assembly (right) are shown. The configuration of the bent portion is shown in (a) to (c) using a cross-sectional view taken along the line A-A ′ of the developed view shown at the top. A development view (left) and a configuration diagram (right) of a folding biosensor having a curved tip at the tip are shown. The expansion | deployment figure (left) and the block diagram (right) of the bending type biosensor with which the other front end was provided with the curved surface are shown. Fig. 2 shows another folding type (bonded structure) biosensor, (a) and (c) showing examples of parallel electrodes, (b) showing examples of facing electrodes, and (e) to (g) showing 3 examples. A development view (left) and a configuration diagram after assembly (right) in an example provided with polar electrodes are shown. The form of the biosensor of another bending type (bonding structure) is shown. Various forms of cylindrical biosensors are shown, and (a) to (h) all show a developed view (left) and a configuration diagram after assembly (right). The form of the cylindrical biosensor with various cross-sectional triangles is shown, and (a) to (h) all show a developed view (left) and a configuration diagram after assembly (right). The form of the cylindrical biosensor with various cross-sectional squares is shown, and (a) to (h) all show a development view (left) and a configuration diagram after assembly (right). (A) to (d) are development views of a folding type biosensor provided with a plurality of reagent layers so that multiple items can be measured. (A) to (d) are development views of a folding type biosensor provided with a plurality of reagent layers so that multiple items can be measured. (A) to (d) are development views of a folding type biosensor provided with a plurality of reagent layers so that multiple items can be measured. An example in which a bonded biosensor is applied to a DNA chip is shown. It is a graph which shows the blood glucose calibration curve of an Example. A method for thermally denaturing a folded portion of a biosensor using a mold will be described. A method of thermally denaturing a folded part of a biosensor using heat rays will be described. The method of denature | folding the folding part edge part of a biosensor by thermocompression bonding is shown. A method of modifying both the folded end portion and the cover end portion of the biosensor by thermocompression bonding will be described. A method of photocuring a biosensor fold and its surroundings using light rays is shown. A method of immersing in a chemical reagent and curing the folded portion of the biosensor and its periphery by chemical treatment will be described. A method is shown in which a biosensor fold and its periphery are cured by chemical treatment by contacting a roller impregnated with a chemical reagent. The method of pinching and fixing the folding part edge part of a biosensor using a clip (large) is shown. The method of pinching | pinching and fixing the folding part edge part of a biosensor using a clip (small) is shown. The method of pinching and fixing the folding part edge part of a biosensor from one side using a clip (large) is shown. A method of using two clips (small) to fix a biosensor folding end from both sides is shown. A method of using a cap to cover and fix the end of the folded portion of the biosensor will be described. The method of fixing the folding part edge part of a biosensor using a frame is shown. The method of fixing the fold part edge part of a biosensor using a ring-shaped elastic material is shown. The method of fixing the folding part edge part of a biosensor using a tube-shaped elastic material is shown. A method for fixing the end of the folded portion of the biosensor by winding an adhesive tape is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Board | substrate 2 Spacer 3 Cover 4 Electrode pattern 5 Air hole 6 Sample liquid 7 Sample introduction port 8 Sample conveyance path 9 Perforation 10 Adhesive layer 11 Reagent layer 12 Convex part 13 Air discharge port
DESCRIPTION OF SYMBOLS 101 Biosensor 102 Plate member 103 Electrode 104 Sample introduction port 105 Folding part 106 Denaturation processing apparatus 107 Denatured part 108 Fixing tool 109 Cover edge part

Claims (16)

A space between the substrate and the cover includes an electrode, a sample introduction port for injecting a sample into the space, and a sample conveyance path extending through the electrode from the sample introduction port, the sample conveyance path Is a method of manufacturing a biosensor defined by an adhesive layer for opposingly arranging the substrate and the cover, and includes a bending step of the following plate member;
The plate member is folded so that the electrode formed on the surface of the electrically insulating plate member is on the inside, and the electrode is disposed in a space sandwiched between the substrate and the cover. Forming a substrate and a cover; The bending step; and
The method for manufacturing a biosensor according to claim 1, comprising a step of cutting a folded portion that is a portion where the plate member is bent.   The method according to claim 2, wherein the fold is cut along a perforation. Bending the plate member; and
The biosensor according to claim 1, comprising a step of immobilizing the substrate and the cover by compressing the substrate or the cover, applying a denaturing process, applying a curing agent or a heat shrink agent to the folded portion, or attaching a fixture. Production method.   The method according to claim 4, wherein the compression is a method of crimping at least a part of the biosensor.   The method according to claim 4, wherein the denaturing process is a method of heating or thermocompression-bonding the folded portion of the biosensor or the folded portion and the periphery thereof, or another part of the biosensor.   The adhesive layer of the biosensor includes a thermosetting resin, and the denaturing process is performed by heating or thermocompression bonding the biosensor folding part or the folding part and the periphery thereof, or another part of the biosensor, The method of Claim 4 which is a method of hardening all or one part of an adhesive bond layer.   The biosensor plate member is made of a light-transmitting material, the adhesive layer contains a photocurable resin, and the denaturing process irradiates the biosensor with light to cure the adhesive layer. The method of claim 4, wherein   The plate member of the biosensor includes a thermosetting resin, and the modification process is a method of heating all or part of the plate member to cure all or part of the plate member. The method described in 1.   The method according to claim 4, wherein the plate member of the biosensor includes a photocurable resin, and the modification process is a method of curing the plate member by irradiating the plate member with light.   5. The method according to claim 4, wherein the denaturing process is a method of applying a solvent to the folded portion of the biosensor or a surface of the folded portion and the surrounding surface, and allowing the solvent to penetrate into the folded portion.   Application of the curing agent is a method in which the thermosetting resin is cured by applying a thermosetting resin to the folded portion of the biosensor, or the folding portion and the periphery thereof, and further heating the thermosetting resin. The method of claim 4, wherein   The curing agent is applied by applying a photocurable resin to the folded portion of the biosensor, or the folded portion and the periphery thereof, and further irradiating the photocurable resin with light to cure the photocurable resin. The method of claim 4, which is a method.   Application of the heat shrink agent is a method in which a heat shrink agent is applied to a folded portion of the biosensor, or a fold portion and the periphery thereof, and the heat shrink agent is further heated to semi-cure the heat shrink agent. The method of claim 4, wherein:   The method according to claim 4, wherein the mounting of the fixing device is pinching processing, surrounding processing, mounting of a cap, mounting of a tightening tool using an elastic body, processing using a heat shrink agent, and mounting of an adhesive tape. The method according to claim 1 , wherein the plate member is bent so that the electrode is provided only on the substrate surface .