JP4469983B2 - Biosensor for simultaneous measurement of multiple items and method of using the biosensor for simultaneous measurement of multiple items - Google Patents

Description

  The present invention relates to a biosensor for simultaneous measurement of multiple items, a method for using the same, and a method for measuring a test compound using the biosensor for simultaneous measurement of multiple items. The present invention also relates to a biosensor device including a multisensor simultaneous measurement biosensor.

  Conventionally, as a disposable sensor, a mechanism is known in which a three-dimensional structure is taken in order to ensure quantitativeness, and a sample liquid is automatically introduced into the sensor using a capillary phenomenon or the like (Patent Document 1). ). The sensor having such a configuration is assembled by stacking a spacer and a cover on an electrically insulating substrate. An electrode pattern is formed on the substrate, and an air hole is formed on the cover for exhausting air necessary for capillary action. A sample introduction port having an air hole on one side and a sample transport path are formed by the substrate, the spacer, and the cover to introduce a predetermined amount of sample liquid into the detection unit by capillary action.

  As a biosensor that can measure many items at the same time with such a biosensor, there is a method (Patent Document 1, FIG. 8) in which sample transport paths of at least two connected biosensors are shared.

Japanese Patent Laid-Open No. 1-291153

  The conventional biosensor for simultaneous measurement of multiple items has a problem that the sample liquid introduced into the sample transport path is affected by the reagent of at least one biosensor.

  In addition, as a method for introducing the sample solution, a method of introducing the sample solution from the through hole communicating with the sample conveyance path on the surface of the biosensor substrate or cover has been adopted. When the sample is introduced, the sample solution also adheres to the edge of the through hole, which is the sample introduction port. As a result, a larger amount of sample solution than the volume in the sample transport path may be used.

  The present invention solves the above-described problem, and a biosensor capable of performing multi-item simultaneous measurement even with a small amount of sample liquid and in which the sample liquid introduced into the sample transport path can eliminate the influence of at least one biosensor reagent It is an object of the present invention to provide a test compound measurement method using the multi-item simultaneous measurement biosensor.

  The present inventors have intensively studied to solve the above problems, and by using a biosensor for multi-item simultaneous measurement with a specific structure, multi-item simultaneous measurement can be performed even with a small amount of sample liquid at the time of use, and introduced into the sample transport path The present invention was completed by finding that the prepared sample solution can eliminate the influence of the reagent of at least one biosensor.

That is, the present invention includes the following.
<Biosensor for multi-item simultaneous measurement>
[1] A biosensor for simultaneous measurement of multiple items according to the present invention includes:
Having a substrate and a cover coupled to the substrate via a spacer layer;
A plurality of biosensor unit-containing substrates including at least one biosensor unit having one reaction detection unit including one electrode system and one reagent layer and one sample transporting path including the reagent layer on the substrate. A biosensor for simultaneous measurement of multiple items including
Each biosensor unit includes one reagent layer on one sample transport path,
A cutting line for dividing each biosensor unit-containing substrate is provided on the substrate or the cover surface,
When the substrate or cover is cut along the cutting line, a sample introduction port for introducing a sample liquid into the sample conveyance path is opened as a cut opening of the sample conveyance path on the cut surface of each biosensor unit-containing substrate. As described above, the cutting line and the sample transport path are arranged.

That is, the biosensor for simultaneous measurement of multiple items of the present invention includes at least two biosensor unit-containing substrates, and a cutting line is provided so as to separate each biosensor unit-containing substrate.
Each biosensor unit-containing substrate includes at least one biosensor unit.

  The biosensor unit includes the one reaction detection unit and one sample transport path. The reaction detection unit has one electrode system and one reagent layer, and the electrode system is connected to an external connection terminal by a lead wire. The reagent layer is preferably disposed at a position where the sample transport path and the electrode system intersect.

  In the biosensor for multi-item simultaneous measurement according to the present invention, one biosensor unit-containing substrate includes one or more biosensor units, and each biosensor unit is one on one sample transport path. Contains a reagent layer. For this reason, since the sample liquid introduced into the sample transport path reaches only one reagent layer and is separated from the other reagent layers, it is not affected by components diffusing from the other reagent layers. .

  The biosensor for multi-item simultaneous measurement of the present invention includes a plurality of the biosensor unit-containing substrates, and the cutting line is provided so as to partition each biosensor unit-containing substrate. When the cover is cut, each biosensor unit-containing substrate is separated.

  When the substrate or the cover is cut along the cutting line, the cutting port of the sample transport path opens on each cutting surface of the biosensor unit-containing substrate formed by cutting. This cutting port is a sample introduction port. In the case where the sample transport path has a structure that intersects the cutting line twice, two cutting ports appear, which can be a sample introduction port and an air discharge port, respectively.

  Since the cutting opening is opened only after being cut during use, the cutting opening can be reliably maintained. Therefore, it is particularly useful when the cutting port has a fine diameter. In addition, when both the sample introduction port and the air discharge port are opened by cutting, the sample transport path can be sealed until it is used, so that the activity of the reagent layer can be maintained. Thereby, the packaging of a biosensor becomes unnecessary and manufacturing cost can be reduced significantly.

  When using the biosensor for multi-item simultaneous measurement of the present invention, the opened sample introduction port can be brought into contact with the sample solution, and the sample introduction port can be introduced into the sample conveyance path by, for example, capillary action. The amount of liquid can be remarkably suppressed. For this reason, even if the amount of the test compound contained in the sample solution is small, it can be detected with high sensitivity.

  The cutting line is provided on at least one of the substrate and the cover. The cutting line is preferably formed by grooves or cuts. When a cutting line is provided in both the substrate and the cover, it is preferable that the groove or the cut is arranged so as to face the same position of the substrate and the cover.

If there are grooves or cuts in the cutting line, cutting becomes easy. In addition, if the cutting lines are arranged so as to face the same position, the outer substrate or cover can be easily cut when folded, and the inner substrate or cover can be easily cut when folded. Or it can be folded.
Here, the cut refers to a case where a cut is made from the outside at a depth that does not reach the inside of the substrate and cover forming the biosensor. Therefore, the substrate and the cover are not penetrated by the cut before cutting.
The cutting method is not particularly limited, and the cutting can be performed by folding, cutting, or cutting along the cutting line.

  The sample transport path in the present invention is patterned on the spacer layer and provided on the substrate. Although the thickness (height) of the sample transport path depends on the thickness of the spacer layer, for example, it is preferably in the range of about 5 to 500 μm, more preferably about 10 to 100 μm. In such a range, capillary action is likely to occur.

  The sample transport path preferably connects at least the sample introduction port and the reaction detection unit with a straight line or a gentle curve. With such a shape, the sample liquid can be moved smoothly. Therefore, it is desirable that there is no portion having an angle, particularly an acute angle, in the interval.

Hereinafter, the substrate, cover, spacer, electrode system, reagent layer, etc. will be described.
Substrate The substrate is not particularly limited as long as it is electrically insulating, but as such a substrate, for example, any one of plastic, biodegradable material, paper, and glass can be preferably used.

Examples of the plastic include rigid polyvinyl chloride, polystyrene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polyester, polyether nitrile, polycarbonate, polyamideimide, phenol resin, epoxy resin, acrylic resin, and ABS resin.
The biodegradable material is preferably polylactic acid.
The substrate may be made of an ultraviolet non-transparent material.

  Although the thickness of a board | substrate is not specifically limited, For example, Preferably it is 10-1000 micrometers, What is necessary is just to exist in the range of about 100-500 micrometers.

Cover The cover may be made of the same material as the substrate. Further, the thickness of the cover is not particularly limited, but it may be, for example, preferably in the range of about 10 to 1000 μm, and more preferably about 100 to 500 μm.

Spacer layer The spacer layer adheres the substrate and the cover and defines the sample transport path.

  The spacer layer can be made of the same material as the substrate. In this case, an adhesive is applied to the spacer surface and bonded to the substrate and the cover by the adhesive layer. Further, the spacer itself may be an adhesive layer formed of an adhesive. The adhesive is not particularly limited as long as it does not react or dissolve with the substrate and the cover, and examples thereof include acrylic resins.

  The spacer itself may be formed of an adhesive and a resist. The resist in this case is not particularly limited as long as it does not react or dissolve with the substrate and the cover, as in the case of the adhesive. For example, UV curable vinyl / acrylic resin, urethane acrylate resin, polyester acrylate resin Etc. The purpose of using the resist is mainly to clarify the electrode pattern, to clarify the definition of the electrode area, and to insulate the sample transport path where the reagent layer does not exist. Therefore, the resist layer may or may not form the same pattern as the adhesive layer. In the latter case, the resist layer is preferably formed on the electrode substrate for insulation.

As the acrylic resin, a thermosetting type or a photocurable type, specifically, an ultraviolet curable type or a visible ray curable type may be used. The surface of the spacer may be coated with the above-described ultraviolet absorbent or ultraviolet non-transparent substance.
The thickness of the spacer is not particularly limited, but it may be, for example, preferably in the range of about 5 to 500 μm, more preferably about 10 to 100 μm.

  The spacer layer can be formed by a screen printing method. The spacer layer may contain reagents such as enzymes, mediators, and surfactants.

  In the present invention, the periphery of the sample inlet and the surface of the sample transport path can be coated with a surfactant and lipid. By coating the surfactant or lipid, the sample solution can be moved smoothly.

Electrode System The electrode system is a set of electrodes and their lead wires in which the + electrode and the-electrode face each other.
Such an electrode system may be composed of two electrodes consisting of + and −, or may be two or more.
The electrode can be composed of any one of carbon, silver, silver / silver chloride, platinum, gold, nickel, copper, palladium, titanium, iridium, lead, tin oxide, and platinum black. Carbon may be carbon nanotubes, carbon microcoils, carbon nanohorns, fullerenes, dendrimers, or derivatives thereof.

The thickness of the electrode is not limited as long as it does not greatly hinder contact with the spacer, but in the case of screen printing, it is usually about 1 to 100 μm, preferably about 3 to 20 μm. Further, in the case of a vapor deposition method, a sputtering method, a foil pasting method, or a plating method, it is usually about 200 to 2000 angstroms, preferably about 500 to 1000 angstroms. In such a range, the edge of the electrode formed on the substrate does not have a saw-like shape, and the electrode is highly accurate. In addition, electrode peeling and disconnection can be prevented.
Such an electrode can be formed on the substrate or the cover by any of screen printing, vapor deposition, sputtering, foil sticking, and plating.

Reagent layer In the biosensor for multi-item simultaneous measurement according to the present invention, the sample liquid sent from the sample introduction port through the sample transport path, for example, by capillary action, comes into contact with the reagent layer on the electrode system serving as the reaction detection unit. The reagent and the sample react. This reaction is monitored as an electrical change at the electrode. One such reagent layer can exist on the electrode system through which the sample transport path passes.
The reagent layer is preferably present on one surface or both surfaces on the + electrode and the − electrode.

  In the present invention, the surface of the reagent layer can be coated with a compound that facilitates movement of the sample liquid such as a surfactant and lipid. If the surface of the reagent layer is coated with a surfactant or the like, deterioration due to air oxidation can be further suppressed. When the sample solution is blood or the like, heparin, prolyxin-S, ethylenediaminetetraacetic acid, a metal salt of citric acid, or the like may be coated as an anticoagulant.

  The reagent layer includes an enzyme, an antibody, a ribosome, a nucleic acid, a primer, a peptide nucleic acid, a nucleic acid probe, a microorganism, an organelle, a receptor, a cell tissue, a molecular identifier such as a crown ether, a mediator, an intercalating agent, a coenzyme, an antibody labeling substance, A substrate, inorganic salts, surfactants, lipids, sugars such as trehalose, humectants such as glycerin, stabilizers such as cysteine, or a combination thereof may be appropriately contained depending on the test object by the biosensor. it can.

  When the sample solution is blood or the like, an anticoagulant may be included. Examples of the anticoagulant include heparin, prolixin-S, ethylenediaminetetraacetic acid, and a metal salt of citric acid.

Examples of the enzyme include enzymes such as oxidase or dehydrogenase, such as glucose oxidase, fructosylamine oxidase, lactate oxidase, urate oxidase, cholesterol oxidase, alcohol oxidase, glutamate oxidase, pyruvate oxidase, pyruvate okinase, acetate kinase, peroxidase, glucose Dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, cholesterol esterase, inorganic pyrophosphatase, acid phosphatase, alkaline phosphatase, nucleotide triphosphatase, nucleotide diphosphatase, nucleotide monophosphatase, inositol phosphatase, protein phosphatase Tase, adenosine triphosphatase, guanosine triphosphatase, adenosine-5'-diphosphatase, casein phosphatase, tyrosine phosphatase, serine phosphatase, threonine phosphatase, maltose phosphorylase, sucrose phosphorylase, purine nucleotide phosphorylase, adenyl cyclase, Examples thereof include guanylate cyclase, glucose isomerase, mutarotase, catalase, protease, NADH oxidase, diaphorase, osmium peroxidase complex, or nucleic acid-linked enzymes such as DNA polymerase, RNA polymerase, DNA ligase, and DNase, and restriction enzymes.
These can be used singly or in combination.

  Further, the reagent layer may be contained not as an enzyme alone but as a combination of mediators.

  As this mediator, potassium ferricyanide, ferrocene, benzoquinone, osmium peroxidase complex, 1-methoxy-5-methylphenazinium methylsulfonate (1-M-PMS), 2,6-dichloroindophenol (DCIP), 9-dimethylaminobenzo-α-phenazoxonium chloride, methylene blue, indigo trisulfonic acid, phenosafranine, thionine, new methylene blue, 2,6-dichlorophenol, indophenol, Azure B, N, N, N ′, N '-Tetramethyl-p-phenylenediamine dihydrochloride, resorufin, safranine, sodium anthraquinone β-sulfonate, dyes such as indigo carmine, riboflavin, L-ascorbic acid, flavin adenine dinucleo Flavin mononucleotide, nicotine adenine dinucleotide, lumichrome, ubiquinone, hydroquinone, 2,6-dichlorobenzoquinone, 2-methylbenzoquinone, 2,5-dihydroxybenzoquinone, 2-hydroxy-1,4-naphthoquinone, glutathione, peroxidase , Cytochrome C, bioredox substances such as ferredoxin or derivatives thereof, other Fe-EDTA, Mn-EDTA, Zn-EDTA, mesosulfate, 2,3,5,6-tetramethyl-p-phenylenediamine, etc. The

  The reagent layer may contain a combination of inorganic salts such as sodium chloride and potassium chloride and quinhydrone.

  The concentration of these mediators is preferably about 40 nM or more.

  Among the above compounds, potassium ferricyanide, ferrocene, benzoquinone, osmium peroxidase complex, DCIP, 1-M-PMS and 9-dimethylaminobenzo-α-phenazoxonium chloride are preferable.

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

  The reagent layer is formed in the vicinity of each set of electrodes or part or all of the electrode surface, and constitutes a reaction detection unit together with the electrodes.

  Such a reagent layer can be formed by a dispenser method in which the reagent layer is dropped by a dispenser or the like and dried, a screen printing method in which the viscosity is adjusted, and the like, and among these, the dispenser method is preferable. The reagent layer can be immobilized on the electrode surface or substrate surface by an adsorption method involving drying or a covalent bonding method.

The reagent layer is installed at one place on one sample conveyance path in each biosensor unit.
In the present invention, after cutting the multi-sensor simultaneous measurement biosensor, each biosensor unit-containing substrate includes at least one biosensor unit. When many different reagent layers are employed, the biosensor unit-containing substrate can include a plurality of biosensor units.

  In the present invention, since one reagent layer is provided on one sample transport path, even if two or more biosensor units are included in one biosensor unit-containing substrate, the sample solution is not mixed. However, in order to more reliably prevent the sample liquid from being mixed, when two or more biosensor units are included in one biosensor unit-containing substrate, a convex shape is formed between the reagent layers in each biosensor unit. A partition part can also be provided. 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.

[2] In the present invention, a sample transport path is provided so that a sample introduction port opens on the cut surface, and an air discharge port is formed on the substrate or the cover surface or on a biosensor unit-containing substrate different from the cut surface. It is preferable to be provided on the side surface.

  In this case, the angle at which the cut surface and the sample transport path intersect is not limited, but is preferably 0 ° or more and less than 180 °, more preferably 0 ° or more and 120 ° or less, and further preferably 0 ° or more and less than 100 °.

[3] In the biosensor for multi-item simultaneous measurement according to the present invention, it is more preferable that the sample transport path is sealed.
For example, as another preferred embodiment of the present invention, a biosensor for simultaneous measurement of multiple items is:
The sample transport path is sealed,
A cutting line (first cutting line) for dividing each biosensor unit-containing substrate, and the first cutting line for exposing the air discharge port by cutting a part of the substrate and the cover are different. A second cutting line is provided on the substrate and the cover surface;
When the substrate or cover is cut along the first cutting line, the sample introduction port opens as a cutting port on the first cutting surface,
When the substrate or cover is cut along the second cutting line, the air discharge port opens as a cutting port on the second cutting surface.
The first and second cutting lines and the sample transport path are arranged.

  In this case, a sample transport path is provided on the cut surface of each biosensor unit-containing substrate so that the sample introduction port and the air discharge port are opened separately, and the sample transport path is arranged in a sealed state before cutting. It may be.

Auxiliary tool [4] Here, on the surface of the substrate or cover, the substrate and the cover are bent along the second cutting line in conjunction with the folding of the substrate and the cover along the first cutting line. You may have the auxiliary tool provided.

  The auxiliary tool is not particularly limited in material, and may or may not be stretchable, but is sufficient for cutting the substrate or cover cutting portion for opening the air outlet along the cutting line. Those having a sufficient strength are preferred. Furthermore, it is desirable that the auxiliary tool is fixed to a part or all of the section that is responsible for opening the air outlet.

  Examples of such auxiliary tools include strap-like stretchable plastics. As shown in FIG. 35, for example, both ends of one strap are respectively fixed to upper outer folds 131 at both ends of the biosensor unit-containing substrate, and a cover (or substrate) is attached along the first cutting line. When bending and cutting, the upper outer folding part 131 is pulled by the strap in conjunction with the folding, and the upper outer folding part 131 is bent and cut along the second cutting line. Thereby, an air exhaust port can be opened automatically.

Connector In order to capture an electrochemical signal from the biosensor for simultaneous measurement of multiple items of the present invention, a dedicated connector is required.

That is, the connector of the present invention is a connector that captures an electrical signal by fixing the second multi-item simultaneous measurement biosensor.
The connector fixes a shape of the folded state of the biosensor unit-containing substrate for opening the sample introduction port, a sensor shape fixing portion (folder), and an electrical signal on the biosensor to the biosensor. It is desirable that the connector is a biosensor connector having an electrical connection portion and wiring for capturing an electrical signal at the electrode.

  The connector has a structure that maintains the shape of the biosensor until the measurement is completed by changing the structure of the flat sensor and introducing the sample solution into the newly opened sensor sample inlet. is doing. Therefore, the shape of the connector part is not particularly limited as long as it satisfies the above conditions and can reliably capture an electrical signal from the sensor. Further, the connector may be either a built-in connector or a type that is used by being connected to an electrochemical measuring device alone.

  For example, the connector is preferably provided with a folder that keeps the folded shape and the folded sensor shape. In this case, the folded portion and the folder may be the same or independent. Further, the folding part is preferably a structure that can be deformed so that the structure of the biosensor takes a shape different from a flat surface. More preferably, the sensor has a structure for deforming into a V-shape. In that case, that is, the angle at which the cut surface and the sample transport path intersect is not limited, but is preferably 0 ° or more and less than 180 °, more preferably 0 ° or more and 120 ° or less, and further preferably 0 ° or more and less than 100 °. It is desirable to design the folds so that The above conditions can also be included when the folding part also has a folder function. In addition, the folding part may be a method of deforming the sensor by sliding the sensor to the connector and connecting it, or a method of deforming the sensor by sandwiching the sensor between the upper and lower folding parts, The method is not particularly limited.

[5] In the present invention, as another preferred embodiment, a sample transport path is provided so that both the sample introduction port and the air discharge port are opened on the cut surface of each of the biosensor unit-containing substrates. Before cutting, the sample transport path may be arranged in a sealed state.

  Such a biosensor for simultaneous measurement of multiple items of the present invention maintains the airtightness in the sample transport path including the reaction detection unit before cutting, and the internal biosensor for simultaneous measurement of multiple items is manufactured after manufacturing. Can be maintained over a long period of time, and the atmosphere inside the biosensor for simultaneous measurement of multiple items, specifically the gas composition (deoxygenated state), atmospheric pressure, humidity (wet state), etc. can be controlled to a desired constant atmosphere. it can.

  For this reason, for example, even if the sample liquid cannot be smoothly introduced when the sample transport path of the biosensor for simultaneous measurement of multiple items is in a dry state, a surfactant or the like is uniformly applied to the inner wall of the sample transport path. By applying and maintaining a constant humidity, the sample solution can be smoothly introduced into the biosensor. When the sample solution is blood or the like, heparin, prolyxin-S, ethylenediaminetetraacetic acid, a metal salt of citric acid, or the like may be coated alone or with a surfactant as an anticoagulant.

[6] In the present invention, it is preferable that the sample transport path is arranged so that one sample introduction port is formed for one biosensor unit. When the sample transport path is arranged in this manner, the influence of components diffusing from other reagent layers can be eliminated to a higher degree when in use.

[7] In the present invention, at least one of the substrate and the cover may have a multilayer structure of two or more layers, and the folding line may be formed in any layer except the innermost layer of the multilayer structure.

  When the substrate and the cover each have a multilayer structure of at least two layers, a cutting line is formed leaving at least the innermost layer of the multilayer structure. Further, it is preferable that the cutting lines are arranged so that the substrate and the cover face each other at the same position. When the multi-layer structure is used, a biosensor for simultaneous measurement of multiple items can be formed without causing damage such as a scratch on the inner layer portion by forming the cutting line portion leaving at least the innermost layer. Therefore, there is a merit that it can withstand a force such as an unexpected bending when in a manufacturing process or a storage state.

  In the present invention, a desiccant and / or an oxygen scavenger may be included in a part of the region sandwiched between the substrate and the cover.

  That is, since the biosensor for simultaneous measurement of multiple items according to the present invention is held in a state in which the reaction detection unit is included, by having such a desiccant and / or oxygen scavenger in the biosensor, A dry state or an oxygen-free state can be maintained for a long time.

  Also, even when the biosensor reaction detector is manufactured and sealed in an atmosphere containing moisture or oxygen during the assembly process of the biosensor, the inside of the biosensor can be brought into a dry state or a deoxygenated state. .

  The desiccant and / or oxygen scavenger is preferably avoided from direct contact with the sample solution. Moreover, it is preferable to arrange | position a desiccant and / or an oxygen scavenger so that it may not cross | intersect a sample conveyance path.

Examples of the desiccant include porous structures such as silica gel, activated alumina, calcium chloride, molecular sieves, and hygroscopic polymers.
Examples of the oxygen scavenger include powders made of metals such as iron and metal halides, hydrosulfite, activated magnesium (for example, JP-A-2001-37457), ascorbic acid (for example, special Kaihei 05-7772), catechol compounds (for example, JP 09-75724 A), and polyhydric alcohols (for example, JP 2003-144113 A). These oxygen scavengers may be carried on, for example, a known carrier (for example, JP-A-2001-37457). Examples of commercially available oxygen scavengers include “AGELESS” (trademark, manufactured by Mitsubishi Gas Chemical Co., Inc.), “Vitalon” (trademark, manufactured by Toagosei Co., Ltd.), and the like.

  The biosensor for simultaneous measurement of multiple items of the present invention may include a humidity indicator and / or an oxygen detector in a part of the region sandwiched between the substrate and the cover.

Before use, use a desiccant and humidity indicator together, or use a deoxidizer and oxygen detector so that you can check the dry and / or deoxygenated state inside the biosensor for multi-item simultaneous measurement. You can also
The humidity indicator is not particularly limited as long as it can be used in the package of the present invention.
Examples of commercially available oxygen detectors include “Ageless Eye” (trademark, manufactured by Mitsubishi Gas Chemical Co., Ltd.), “Vitalon-Oxygen Detector” (trademark, manufactured by Toagosei Co., Ltd.), and the like.

  In this case, it is preferable that a part or the whole of the substrate and / or the cover is made of a material transparent to visible light, and the humidity indicator and / or the oxygen detector is visible.

  Furthermore, it is preferable that a part or the whole of the substrate and / or the cover is made of a material capable of blocking ultraviolet rays. In this case, the entire substrate and / or cover may be an ultraviolet blocking material, or the surface of the substrate and cover may be covered with an ultraviolet blocking material film. Examples of the film include a film containing an organic compound such as a benzotriazole type, a fluorescent agent that converts ultraviolet light into visible light, and the like.

Further, when using a substrate and a cover that are not transparent to visible light, the humidity indicator and / or the oxygen detector may be disposed in the spacer portion of the cross section that newly appears by cutting. . In this case, the humidity indicator and the oxygen detector may be included in the spacer layer or may be configured as a part of the spacer layer.
By doing so, the internal state indicated by the humidity indicating agent and / or the oxygen detecting agent can be confirmed on the cut surface or in the vicinity of the cut surface immediately after use after cutting.

  The substrate and the cover are preferably made of an ultraviolet non-transparent material. In addition, the surfaces of the substrate and the cover may be coated with an ultraviolet absorber or an ultraviolet non-transparent substance.

As described above, when the substrate and the cover are made of an ultraviolet non-transparent material, or are covered with an ultraviolet absorber or an ultraviolet non-transparent material, the transmission of ultraviolet rays can be suppressed or blocked.
Although it does not specifically limit as a ultraviolet absorber, For example, organic compounds, such as metals, such as aluminum, metal halides, such as silver chloride, a fluorescent agent, and a benzotriazole type | system | group, etc. are mentioned.
Although it does not specifically limit as an ultraviolet-ray impermeable substance, For example, vapor deposition films, such as metal halides, such as metals, such as aluminum, or a silver chloride, and organic compound-type films, such as a benzotriazole type | system | group, etc. are mentioned.

  The substrate and the cover may contain a compound having a photocatalytic action, or the surface of the substrate and the surface of the cover may be coated with a coating containing a compound having a photocatalytic action.

Here, the photocatalyst is a compound that is excited by absorption of light and becomes activated, and exerts a strong oxidation / reduction action on an organic substance etc. in contact with the surface of the photocatalyst. It means oxidation / reduction action.
Examples of light include ultraviolet light and / or visible light. Photocatalysis occurs on the surface of a biosensor for simultaneous measurement of multiple items due to the incidence of ultraviolet rays and / or visible light, thereby sterilizing and degrading viruses with capsids and envelopes made of proteins, and degrading dirt attached to the surface. The self-cleaning effect can be obtained, and it can be stored in a hygienic state at all times. For this reason, a biosensor of this form is particularly effective when it is necessary to be brought into direct contact with a biological sample, for example, at a medical site, or at a site where food is handled.

Here, a metal oxide is mentioned as a compound which has a photocatalytic action. Specifically, at least one selected from the group consisting of titanium oxide, titanium dioxide, zinc oxide, strontium titanate, tungsten trioxide, ferric oxide, dibismuth trioxide and tin oxide is used as the metal oxide. However, it is not limited to these.
The spacer layer present in the vicinity of the exposed sample inlet or in the vicinity of the sample inlet and the air outlet may contain a fluorescent agent or a luminescent agent. In particular, it is preferable that a fluorescent agent or a luminescent agent is contained in the vicinity of the sample inlet.

Since the fluorescent agent or the luminescent agent serves as a mark for improving the visibility, it is possible to prevent an error in handling in introducing the sample. When the fluorescent agent or the luminescent agent is used for the cross-sectional portion, the fluorescent agent or the luminescent agent may be included in the spacer material or may be configured as a part of the spacer. Further, when a fluorescent agent or a luminescent agent is used as a mark near the sample inlet of the substrate or cover, the portion can be formed by printing or the like.
Here, as a luminescent agent, the well-known thing which a luminescent reaction starts by contact with the oxygen in air can be used.

[8] In the biosensor for simultaneous measurement of multiple items according to the present invention, the electrode system may form an array.
When the biosensors forming the array are cut along the cutting line, at least one sample introduction port opens on the cross section of each biosensor unit-containing substrate and communicates with the sample introduction port. A reaction detection unit including one electrode system and one reagent layer is present at the tip of one sample transport path.

The one sample introduction port communicates with one sample conveyance path or communicates with at least two sample conveyance paths branched from the sample introduction port, and a reaction including one electrode system at the tip of the sample conveyance path. A detection unit may be present.
Among these, it is preferable to communicate with one sample conveyance path from one sample introduction port. In this case, mixing of the sample solution in the reagent layer can be prevented more reliably.

  In this specification, “array” means an array.

  When at least two sample liquid transport paths branch from one sample introduction port, the sample transport path may be coated with a surfactant so that the sample liquid can reach all the reagent layers in the array, or the sample When the liquid is blood or the like, heparin, prolyxin-S, ethylenediaminetetraacetic acid, a metal salt of citric acid or the like may be coated as an anticoagulant.

  The biosensor for multi-item simultaneous measurement of the present invention includes at least two biosensor unit-containing substrates. When the biosensor unit-containing substrate includes at least three biosensor unit-containing substrates, each biosensor-containing substrate is on a continuous substrate. In addition, it is preferable that the lines are regularly arranged at predetermined intervals while being divided by cutting lines.

  With this configuration, a multi-item simultaneous measurement biosensor can be efficiently manufactured at a time. Further, by connecting each of the individual biosensor units in the connected biosensor-containing substrate to the measurement unit, a large number of specimens can be measured simultaneously.

The biosensor for multi-item simultaneous measurement according to the present invention includes, for example, patterning the electrode system on the substrate, the spacer in advance on the surface of the substrate or cover, further arranging the reagent layer, and bonding the substrate and cover to the adhesive. Can be manufactured by pasting.
For example, specifically, an electrode pattern is formed by screen printing or the like on the inside of the substrate on which cutting lines are previously formed on the outer surface. On the other hand, similarly, a pattern of a pressure-sensitive adhesive layer as a spacer is formed on the inside of the cover, in which cutting lines are formed on the outer surface as necessary, by screen printing or the like.

  A reagent solution containing an enzyme can be dropped on the sample transport path of the substrate by a dispenser method to form a reagent layer. Moreover, the space for placing the adjusting agent and the indicator can be simultaneously formed on the substrate or the cover as a part of the spacer formation pattern formed on the inside of the cover. A biosensor for simultaneous measurement of multiple items can be constructed by bonding the cover and the substrate thus formed.

<How to use biosensor for multi-item simultaneous measurement / Measurement method of test compound>
The method for using the multi-item simultaneous measurement biosensor according to the present invention is a method including the following steps using the multi-item simultaneous measurement biosensor.
(1) Cut the substrate or cover along the cutting line that divides each biosensor unit-containing substrate, and set the cutting port (sample inlet) of the sample transport path on the cut surface of each biosensor unit-containing substrate. A step of opening, (2) a step of bringing the opened sample introduction port into contact with a solution containing the measurement target, and (3) a step of introducing the solution containing the measurement target into the sample transport path.

More preferably, the following steps (1) to (4) are included.
(1) The substrate and the cover are bent along a cutting line that divides the biosensor unit-containing substrate, and the substrate or the cover is cut. On the cut surface of each biosensor unit-containing substrate, Opening the sample inlet),
(2) fixing the shape of the bent biosensor unit-containing substrate in order to maintain the open state of the sample inlet;
(3) A step of bringing the opened sample introduction port into contact with a solution containing the measurement object, and (4) a step of introducing the solution containing the measurement object into the sample conveyance path.

Moreover, the measuring method of the measuring object which concerns on this invention is a method including the following processes using the said biosensor for multi-item simultaneous measurement.
(1) Cut the substrate or cover along the cutting line that divides each biosensor unit-containing substrate, and set the cutting port (sample inlet) of the sample transport path on the cut surface of each biosensor unit-containing substrate. Opening,
(2) a step of bringing the opened sample inlet into contact with a solution containing a measurement target;
(3) A step of introducing a solution containing the measurement target into the sample transport path, and (4) a step of measuring the measurement target with each biosensor.

More preferably, the following steps (1) to (5) are included.
(1) The substrate and the cover are bent along a cutting line that divides the biosensor unit-containing substrate, and the substrate or the cover is cut. On the cut surface of each biosensor unit-containing substrate, Opening the sample inlet),
(2) fixing the shape of the bent biosensor unit-containing substrate in order to maintain the open state of the sample inlet;
(3) a step of bringing the opened sample introduction port into contact with a solution containing a measurement object;
(4) A step of introducing a solution containing the measurement target into the sample conveyance path, and (5) a step of measuring the measurement target with each biosensor.

  In the step 1, both the substrate and the cover are cut to expose the cut surface, and the step 2 can be performed. Further, either of the substrate and the cover is cut, folded while the other is connected, the cut surface is exposed, and the biosensor for simultaneous measurement of multiple items is folded and the step 2 (or the above) is performed. In a more preferred embodiment, step 3) can be performed. Of these, the latter embodiment is preferred.

  In the biosensor for multi-item simultaneous measurement of the present invention, after cutting, in the step 2 (or step 3 in the more preferred embodiment), a cross section of each biosensor unit-containing substrate or a plurality of biosensor unit-containing substrates is cut. Can be simultaneously contacted with the solution.

  When cross sections of a plurality of biosensor unit-containing substrates are simultaneously brought into contact with the solution, the biosensor-containing substrate preferably includes one or more biosensor units. In this case, it is preferable that one of the substrate and the cover is cut and bent while the other is connected to expose the cut surface.

  When the cross section of each biosensor unit-containing substrate is brought into contact with the solution, the biosensor-containing substrate preferably includes a plurality of biosensor units. The substrate and cover are cut to expose the cut surface, and the biosensor unit-containing substrate is used.

  In this way, a biosensor unit having a plurality of reagent layers can be simultaneously used for measurement.

  The measurement object is not limited as long as it can be measured by a biosensor. Examples thereof include compounds such as enzymes, DNA, ion amounts and oxygen amounts, properties of solution such as pH and conductivity.

<Biosensor device>
The biosensor device according to the present invention, the biosensor for simultaneous measurement of multiple items,
A measurement unit for measuring an electrical value in the electrode of the biosensor;
A display unit for displaying a measurement value in the measurement unit;
And a memory unit for storing the measured values.

More preferably, it is as follows.
The biosensor for simultaneous measurement of multiple items;
A connector for capturing an electrical signal at the electrode of the biosensor;
A measurement unit for measuring an electrical value via the connector unit;
A display unit for displaying a measurement value in the measurement unit;
A biosensor device comprising a memory unit for storing the measurement value.

  The connector part deforms the shape of the biosensor unit-containing substrate for opening the sample introduction port, and then fixes the biosensor unit-containing substrate with the shape to capture an electrical signal at the electrode of the biosensor. A structure is preferred.

The measurement method in the measurement unit is preferably a potential step chronoamperometry method, a coulometry method, or a cyclic voltammetry method.
The potential step chronoamperometry method is a method in which a constant potential is externally applied to an electrode and a change in current flowing by electrolysis is measured.
The coulometry method is a method of measuring the amount of electricity that has flowed until the target substance is completely electrolyzed, and determining the amount of substance or the number of reaction electrons based on Faraday's law.
The cyclic voltammetry method is a method for obtaining a current-voltage curve when the electrode potential is scanned positively and negatively at a constant speed within a certain range, and is also called a potential scanning method.
<Application>

  The biosensor for simultaneous measurement of multiple items according to the present invention can measure the following measurement object by changing the type of reagent layer.

  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 oxidase or glucose dehydrogenase when the measurement target is blood sugar (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 Pyruvate oxidase in the case of acid or phosphate, maltose phosphorylase, alkaline or acid phosphatase, and / or if the measurement object is maltose or phosphate Mutarotase, we used a combination of glucose oxidase, sucrose phosphorylase if the measurement target is sucrose or phosphoric acid, alkaline or acid phosphatase, mutarotase, and combinations of glucose oxidase.

  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 microbial sensors, for example, Pseudomonas fluorescence (measuring object: glucose or BOD; biochemical oxygen demand, soil), Trichosporon cutaneum , Pseudomonas putida (measuring object: BOD), Trichosporon brassicae (measuring object: ethanol) microorganisms or soil microorganisms such as, Acetobacter genus, Actinomaaura spp., Agrobacterium sp., Alcaligenes sp., Aphanomyces spp., Armillaria spp, Aspergillus spp., Bacillus sp., Burkholderia spp., Candida spp., Cephalosporium spp., Ceratocystis spp., Cladosporium spp., Clavibacter spp., Corticium spp., Corynebacterium spp., Cylindrocarpon genus, Cylindrocladium spp., Enterobacter spp., Erwinia sp., Flavobacterium spp., Fusarium sp., Gaeumannomyces spp., Ganoderma sp., Gibberella spp., Gliocladium spp., Gluconobacter genus, Glycomyces genus, Helicobasidium genus, Actobacillus spp., Leptosphaeria species, Micobacterium spp., Micrococcus sp., Monosporascus spp., Mucor spp. Genus Nocardia, Olpidium genus, Pasteuria spp., Penicillium sp., Phoma spp., Plasmodiophora spp., Phytophthora spp., Polymyxa spp, Proteus spp., Pseudomonas spp., Pyrenochaeta spp., Pythium spp., Ralstonia spp., Rhizobium sp., Rhizoctonia spp., Rhizopus spp., Rhodococcus sp. , Rosellinia genus Saccharomonospora sp, Sclerotina spp, Scletotium genus Serratia genus Sphingomonas sp, Spongospora genus Streptococcus genus Streptomyces genus, Streptoverticiliu m genus Synchytrium genus Talaromyces genus Thanatephorus spp, Thielaviopsis genus Torula sp, Trichoderma sp, Use the genus Typhula , Verticillium , Zymomonas , and Xanthomonas .

  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 nerve transomics. 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. Examples of animal and plant tissues that can be used include frog skin, animal liver slices, cucumbers, and banana peels. 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 application of the biosensor described here is a DNA chip. Many kinds of single-stranded nucleic acid probes having complementarity to many kinds of target genes to be detected are immobilized on the electrode array. 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.
<Manufacturing method>

The biosensor of the present invention is manufactured, for example, by patterning electrodes on a substrate and spacers on the surface of the substrate or cover in advance, arranging a reagent layer, and bonding the substrate and cover with an adhesive. can do.
For example, more specifically, it can be produced as follows. An electrode pattern is formed by screen printing or the like on the inside of the substrate on which cutting lines are previously formed on the outer surface. On the other hand, the pattern of the pressure-sensitive adhesive layer as a spacer is also formed on the inside of the cover in which a cutting line is previously formed on the outer surface by screen printing or the like. At this time, the part where the adhesive layer on the cover does not exist becomes a space for placing the sample conveyance path, the adjusting agent, the indicator and the like.

  On the sample transport path of the substrate, a reagent layer can be formed by dropping a reagent solution containing an enzyme into at least a part of the sample introduction port and the air exhaust port formed by separating the sealing cap by the dispenser method. Moreover, the space for placing the adjusting agent and the indicator, which will be described later, can be simultaneously formed on the cover as a part of the formation pattern of the pressure-sensitive adhesive layer formed inside the cover. A biosensor can be constructed by bonding the cover and the substrate thus formed.

As described above, in the biosensor assembly process after the development of the reagent layer, the substrate can be manufactured by simply bonding the substrate to the cover via the spacer without using a packaging method involving heat such as thermocompression bonding.
The biosensor manufactured in this way can include a sample transport path serving as a sample introduction port and an air discharge port inside the biosensor. In the case of a sealed biosensor, the inside is in a highly airtight state. Can hold.

  Since the biosensor for simultaneous measurement of multiple items according to the present invention has a specific structure, it can simultaneously measure multiple items even with a small amount of sample liquid at the time of use, and the sample liquid introduced into the sample transport path is at least one of the biosensors. The influence of the reagent of the sensor can be eliminated.

  EXAMPLES Hereinafter, although this invention is demonstrated using an Example, this invention is not limited to a present Example at all.

  FIG. 1 shows a representative example of a multi-sensor simultaneous measurement biosensor according to the present invention, and two biosensor unit-containing substrates arranged in parallel (an example in which each biosensor unit-containing substrate includes one biosensor unit). It is an example of the biosensor of this invention by which the cross section of a sample conveyance path is formed as a sample inlet of each biosensor by folding along the V-shaped groove | channel provided in the boundary line.

  FIG. 1 a shows the outside of a rectangular substrate 1 of a typical multi-item simultaneous measurement biosensor. A cutting line extending vertically from the upper end side to the lower end side is provided in the shape of a V-shaped groove 7 at the center of the substrate 1. The groove 7 is for folding the multi-item simultaneous measurement biosensor along a broken line 12 into a V-shape when using the multi-item simultaneous measurement biosensor.

  FIG. 1 b shows the inside of the substrate 1. Inside the substrate 1, a pattern 4 including two sets of electrodes is arranged in parallel from the upper end side to the lower end side with respect to the central broken line 12 of the substrate. A reagent layer 6 is formed on a part of each set of electrode patterns. Although not shown in FIG. 1b, in order to clarify the section of the pattern to be the reagent layer 6, between the adhesive layer forming the spacer 3 shown in FIG. 1d and the substrate 1 including the electrode pattern 4 of FIG. 1b, A resist layer may be provided in the same pattern as the adhesive layer. In this case, the resist layer becomes the spacer 3 like the adhesive layer, but for example, the resist layer may not form the same pattern as the adhesive layer. In that case, the resist layer can also be provided as an insulating layer for preventing the electrode pattern 4 other than the reaction layer 6 from crossing the sample transport path 5. Further, the adhesive layer 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1.

  FIG. 1 c shows the outer part of the cover 2. A V-groove 7 extending vertically from the upper end side to the lower end side is provided at the center of the cover 2 in the same manner as the substrate 1. FIG. 1 d shows the inside of the cover 2. An adhesive layer as a spacer layer 3 is formed on the inner surface of the cover 2. A portion 5 where no spacer is present is provided on the cover 2, and the reagent transport path 5 is formed by bonding to the substrate.

  FIG. 1e is a configuration diagram when the insides of the substrate 1 and the cover 2 are overlapped in accordance with the upper ends, and shows the biosensor 15 for simultaneous measurement of multiple items. By making the length of the cover 2 shorter than that of the substrate 1, the lower end portion of the electrode pattern 4 is exposed when the cover 2 is overlapped with the upper ends of both. This is the terminal 8 shown in FIG. Further, there are two biosensor unit-containing substrates 28 each including one biosensor unit 27 with the groove 7 as a boundary.

  FIG. 1f is a cross-sectional view taken along the line AA ′ of the sample transport path portion above the biosensor for simultaneous measurement of multiple items shown in FIG. 1e. Two sets of two electrodes 4 are arranged on the substrate 1 and there is an adhesive layer between the substrate and the cover, but the cross-sectional portion of FIG. To do. On the outside of the substrate 1 and the cover 2 between the two sets of electrodes, V-shaped grooves 7 are provided so as to overlap each other. FIG. 1g shows a BB ′ cross-sectional view on the electrode pattern of the biosensor for multi-item simultaneous measurement shown in FIG. 1e. An electrode 4 is formed on the substrate 1, and one spacer 3 and one sample transport path 5 are provided between the substrate 1 and the cover 2.

  FIG. 1h shows an example of use of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 1 h shows a case where the biosensor for simultaneous measurement of multiple items is vertically folded along the V groove 7 in the cover portion 2. Thereby, the substrate 1 of the multi-sensor simultaneous measurement biosensor is divided into two. On the other hand, the cover portion 2 is folded along the V-groove 7 but is not divided. As a result, the two biosensor unit-containing substrates can take a V-shape as shown in the figure. At this time, the sample transport path 5 is divided along the V-groove at the boundary between the two biosensor unit-containing substrates, and the sample inlet 9 for each biosensor unit is formed in a shape adjacent to one place.

  In this state, the two sample introduction ports 9 adjacent to the sample solution 11 are brought into contact with each other, whereby the sample solution 11 is independently introduced into the adjacent sample transport path 5 by capillary action. At this time, if the sample solution 11 is rounded due to surface tension as shown in the figure, the sample solution is because the two biosensor unit-containing substrates are bonded to each other as shown in FIG. 1h. 11 is efficiently introduced into the sample transport path 5. In order to smoothly introduce the sample liquid 11 into the sample transport path 5, the air discharge port 10 is provided on the opposite side of the sample introduction port 9. FIG. 1i shows a front view when two biosensor unit-containing substrates have a V-shape.

In the case of the structure of FIG. 1, it can be mentioned that one sample solution can be measured in a completely independent system without interference of two adjacent biosensor units that are present in each reagent layer. It is done. Here, the multi-item simultaneous measurement biosensor illustrated in FIG. 1 has a crack on the substrate side, but the cover 2 side may be divided. Furthermore, it is not limited to a biosensor for simultaneous measurement of multiple items deformed into a V-shape, and even if it is completely folded along either the V-groove of the substrate 1 or the cover 2, it cracks at less than 180 degrees by using a hard substrate. May be used.
In addition to the features of the multi-item simultaneous measurement biosensor common to the present invention described above, the features of the multi-item simultaneous measurement biosensor proposed in the present invention will be described below.

  FIG. 2 shows the structure of the biosensor for simultaneous measurement of multiple items shown in FIG. 1, in which a total of four biosensor units 27 are included in the biosensor unit-containing substrate 28, each of which is an independent type along the V-groove. This is a biosensor 15 for simultaneous measurement of items (FIG. 2 (e)).

  FIG. 2a shows the outside of the rectangular substrate 1 of the biosensor for multi-item simultaneous measurement. A cutting line 12 is formed in the shape of a V-shaped groove 7 at the center of the substrate 1 extending vertically from the upper end side to the lower end side. The groove 7 is for folding the multi-item simultaneous measurement biosensor into a V shape along the broken line 12 when the multi-item simultaneous measurement biosensor is used.

  FIG. 2 b shows the inside of the substrate 1. Inside the substrate 1, the pattern 4 including four sets of electrodes is arranged on the substrate 1 symmetrically with respect to the central broken line 12 of the substrate. Four reagent layers 6 are formed on a part of each set of electrode patterns. In the structure inside the substrate 1 in FIG. 2 b, an insulating resist 13 is applied except for the electrode portions around the reaction vessel 6 and the terminals 8. Furthermore, the use of the resist 13 has a feature that the area of the electrode area in the reaction vessel is clearer than that of the adhesive layer.

  FIG. 2 c shows the outer part of the cover 2. In the center of the cover 2, a cutting line 12 is formed in the shape of a V-shaped groove 7 which is formed vertically like the substrate 1 and extends from the upper end side to the lower end side. FIG. 2 d shows the inside of the cover 2. An adhesive layer as a spacer layer 3 is formed on the inner upper surface of the cover 2. A portion 5 where no spacer is present is provided on the cover 2, and the reagent transport path 5 is formed by bonding to the substrate. In this case, the adhesive layer 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1.

  FIG. 2e shows a configuration diagram of the biosensor 15 for simultaneous measurement of multiple items when the inside of the substrate 1 and the cover 2 are overlapped with each other at the upper end. By making the length of the cover 2 shorter than that of the substrate 1, the lower end portion of the electrode pattern 4 is exposed when the cover 2 is overlapped with the upper ends of both. This is the terminal 8 shown in FIG. Further, four biosensor units 27 are divided into two with the groove 7 as a boundary, and there are two sets of biosensor unit-containing substrates 28 each including two biosensor units.

  FIG. 2f is a cross-sectional view taken along the line AA ′ of the sample transport path portion above the multi-item simultaneous measurement biosensor shown in FIG. 2e. Four sets of two electrodes 4 are arranged on the substrate 1, a sample transport path 5 branched in two directions with the adhesive layer 3 and the V groove 7 between the substrate 1 and the cover 2, and a reaction layer There is a layer of resist 13 for covering the intersection with the sample transport path 5 other than 6. The V-grooves 7 outside the substrate 1 and the cover 2 are provided so as to overlap each other. FIG. 2g shows a BB ′ cross-sectional view on the electrode pattern of the multi-item simultaneous measurement biosensor 15 shown in FIG. 2e. Two electrodes 4 are formed on the substrate 1 on the upper and lower sides. Between the substrate 1 and the cover 2, there are a resist layer 13 and an adhesive layer 3 as a spacer 3, and a sample transport path 5 branched into two.

  FIG. 2h shows an example of use of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 2 h shows a case where the biosensor for multi-item simultaneous measurement is vertically folded along the V groove 7 in the cover portion 2. Thereby, the board | substrate 1 which has four biosensor units is divided | segmented into two, and becomes two biosensor unit containing board | substrates containing two biosensor units. On the other hand, the cover portion is folded along the V-groove, but is not divided.

  As a result, the two biosensor unit-containing substrates can take a V-shape as shown in the figure. At this time, the sample transport path 5 is divided along the V-groove at the boundary between the two biosensor unit-containing substrates, and the sample introduction ports 9 of all four biosensor units are formed in a shape adjacent to one place.

  In this state, the four sample introduction ports 9 are brought into contact with the sample solution 11, whereby the sample solution is independently introduced into the sample transport path 5 by capillary action. In order to smoothly introduce the sample liquid 11 into the sample transport path 5, a total of four air discharge ports 10 are provided on the opposite side of the sample introduction port 9. FIG. 2i shows a front view when two biosensor unit-containing substrates have a V-shape.

  In the case of the structure of FIG. 2, it is characterized in that measurement can be performed in a completely independent system without receiving interference of a reagent in which four biosensor units adjacent to one sample solution are present in each reagent layer tank. Can be mentioned.

  The biosensor for simultaneous measurement of multiple items shown in FIG. 3 is similar in structure to that shown in FIG. 1, but is characterized in that both the air discharge port 10 and the sample introduction port 9 are formed for the first time when folded during use.

  FIG. 3a shows the outside of the rectangular substrate 1 of the biosensor for multi-item simultaneous measurement. A cutting line 12 is formed in the shape of a V-shaped groove 7 at the center of the substrate 1 extending vertically from the upper end side to the lower end side. The groove 7 is for folding the multi-item simultaneous measurement biosensor into a V shape along the broken line 12 when the multi-item simultaneous measurement biosensor is used.

  FIG. 3 b shows the inside of the substrate 1. Inside the substrate 1, a pattern 4 including two sets of electrodes is arranged on the substrate 1 symmetrically with a slight distance from the upper end portion, with the central broken line 12 of the substrate as a boundary. Two reagent layers 6 are formed on a part of each set of electrode patterns. Although not shown in FIG. 3b, a resist layer having a pattern similar to the adhesive layer may be provided between the adhesive layer forming the spacer 3 shown in FIG. 3d and the substrate 1 including the electrode pattern 4 of FIG. 1b. Good. Further, as shown in the figure, the adhesive layer 3 may be formed either on the cover 2 in advance or on the resist layer on the substrate 1.

  FIG. 3 c shows the outer part of the cover 2. In the center of the cover 2, a cutting line 12 is formed in the shape of a V-shaped groove 7 which is formed vertically like the substrate 1 and extends from the upper end side to the lower end side. FIG. 3 d shows the inside of the cover 2. An adhesive layer as a spacer layer 3 is formed on the inner upper surface of the cover 2. A portion 5 where no spacer is present is provided on the cover 2, and the reagent transport path 5 is formed by bonding to the substrate. In this case, the adhesive layer 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1. 3D is different from FIGS. 1 and 2 in that the sample transport path 5 is formed inside the adhesive layer as shown in FIG. 3D.

  FIG. 3e shows a configuration diagram of the biosensor 15 for simultaneous measurement of multiple items when the inside of the substrate 1 and the cover 2 are overlapped with each other at the upper end. By making the length of the cover 2 shorter than that of the substrate 1, the lower end portion of the electrode pattern 4 is exposed when the cover 2 is overlapped with the upper ends of both. This becomes the terminal 8 shown in FIG. Further, two biosensor units 27 are divided into two with the groove 7 as a boundary, and there are two biosensor unit-containing substrates 28 each including one biosensor unit. Further, in this embodiment, the sample transport path 5 is completely contained in the structure of the multi-sensor simultaneous measurement biosensor and does not come into direct contact with the outside air, so that the inside can be kept secret. .

  FIG. 3f is a cross-sectional view taken along line AA ′ of the sample transport path portion above the multi-item simultaneous measurement biosensor 15 shown in FIG. 3e. Two sets of electrodes 4 are arranged on the substrate 1, and there are an adhesive layer 3, a sample transport path 5, and a reaction layer 6 between the substrate 1 and the cover 2. The V-grooves 7 outside the substrate 1 and the cover 2 are provided so as to overlap each other. FIG. 3g shows a BB ′ cross-sectional view on the electrode pattern in the multi-item simultaneous measurement biosensor shown in FIG. 3e. Between the substrate 1 and the cover 2, there are two adhesive layers 3 and sample transport paths 5 as spacers 3, and the electrode 4 on the surface of the substrate 1 has a structure that intersects only the lower part of the two sample transport paths 5. I'm taking it.

  FIG. 3h shows an example of use of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 3 h shows a case where the biosensor for simultaneous measurement of multiple items is vertically folded along the V groove 7 in the cover portion 2. Thus, the substrate 1 having two biosensor units is divided into two, and there are two biosensor unit-containing substrates each including one biosensor unit. On the other hand, the cover portion is folded along the V-groove, but is not divided. As a result, the two biosensor unit-containing substrates can take a V-shape as shown in the figure.

At this time, the sample transport path 5 is divided along the V-groove at the boundary between the two biosensor unit-containing substrates, and the sample introduction port 9 and the air discharge port 10 of the two biosensor units are each formed in a shape adjacent to one place. Is done. In this state, the two sample introduction ports 9 are brought into contact with the sample solution 11, whereby the sample solution is independently introduced into the semicircular sample transport path 5 by capillary action. In order to smoothly introduce the sample liquid 11 into the sample transport path 5, the air discharge port 10 is provided on the same cut surface of the biosensor unit-containing substrate as the sample introduction port 9. FIG. 3i shows a front view when two biosensor unit-containing substrates divide the substrate into two and take a V shape.
In the case of the biosensor for simultaneous measurement of multiple items illustrated in FIG. 3, it can take a completely sealed structure that does not require packaging.

  FIG. 4 is the same as the structure of the multi-item simultaneous measurement biosensor of FIG. 3 on the outside, but the internal structure is slightly different. In FIG. 4, the sample introduction port 9 formed at the time of use is formed at the position of the air discharge port 10 of FIG.

  FIG. 4a shows the outside of the rectangular substrate 1 of the biosensor for multi-item simultaneous measurement. A cutting line 12 is formed in the shape of a V-shaped groove 7 at the center of the substrate 1 extending vertically from the upper end side to the lower end side. The groove 7 is for folding the multi-item simultaneous measurement biosensor into a V shape along the broken line 12 when the multi-item simultaneous measurement biosensor is used.

  FIG. 4 b shows the inside of the substrate 1. Inside the substrate 1, a pattern 4 and two reagent layers 6 including two sets of electrodes from the upper end portion to the lower end portion of the substrate with the central broken line 12 of the substrate as a boundary are formed on a part of each electrode pattern. Yes. A resist layer is provided on a part of the two sets of electrode patterns. As a result, the intersection of the lower part of the substrate 1 can be insulated by the resist 13 among the intersections of the electrode patterns existing at two places per set of sample transport paths, so that the reagent layer 6 is provided at one point of intersection of the upper part of the substrate 1. Can do.

  FIG. 4 c shows the outer part of the cover 2. In the center of the cover 2, a cutting line 12 is formed in the shape of a V-shaped groove 7 which is formed vertically like the substrate 1 and extends from the upper end side to the lower end side. FIG. 4d shows the same inside of the cover 2 as FIG. 3d. An adhesive layer as a spacer layer 3 is formed on the inner upper surface of the cover 2. A portion 5 where no spacer is present is provided on the cover 2, and the reagent transport path 5 is formed by bonding to the substrate. In this case, the adhesive layer 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1. 4D is different from FIGS. 1 and 2 in that the sample transport path 5 is formed inside the adhesive layer as shown in FIG. 4D.

  FIG. 4e shows a configuration diagram of the multi-item simultaneous measurement biosensor 15 when the insides of the substrate 1 and the cover 2 are overlapped with each other at the upper end. By making the length of the cover 2 shorter than that of the substrate 1, the lower end portion of the electrode pattern 4 is exposed when the cover 2 is overlapped with the upper ends of both. This is the terminal 8 shown in FIG. 4e. Further, two biosensor units 27 are divided into two with the groove 7 as a boundary, and there are two biosensor unit-containing substrates 28 each including one biosensor unit. Further, in this embodiment, the sample transport path 5 is completely contained in the structure of the multi-sensor simultaneous measurement biosensor and does not come into direct contact with the outside air, so that the inside can be kept secret. .

  FIG. 4f shows an AA ′ cross-sectional view of the sample transport path portion above the multi-item simultaneous measurement biosensor shown in FIG. 4e. Two sets of electrodes 4 are arranged on the substrate 1, and there are an adhesive layer 3, a sample transport path 5, and a reaction layer 6 between the substrate 1 and the cover 2. The V-grooves 7 outside the substrate 1 and the cover 2 are provided so as to overlap each other.

  FIG. 4g shows a BB ′ cross-sectional view on the electrode pattern in the multi-item simultaneous measurement biosensor shown in FIG. 4e. Between the substrate 1 and the cover 2, a resist 3 is used as a spacer 3 so as to insulate the lower part of the adhesive layer and the sample transport path 5 at two locations and the two locations where the electrode pattern 4 and the sample transport path 5 intersect. Thirteen layers partially cover the electrode pattern 4.

  FIG. 4h shows an example of use of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 4 h shows a case where the multi-item simultaneous measurement biosensor is vertically folded along the V groove 7 in the cover portion 2. Thus, the substrate 1 having two biosensor units is divided into two biosensor unit-containing substrates each including one biosensor unit. On the other hand, the cover portion is folded along the V-groove, but is not divided. As a result, the two biosensor unit-containing substrates can take a V-shape as illustrated.

  At this time, the sample transport path 5 is divided along the V-groove at the boundary between the two biosensor unit-containing substrates, and the sample introduction port 9 and the air discharge port 10 of each of the two biosensor units are each in a shape adjacent to one place. Formed on top.

  In the case of the structure of FIG. 4, the sample introduction port 9 is provided near the upper end of the multi-item simultaneous measurement biosensor, and the air discharge port 10 is provided near the center of the multi-item simultaneous measurement biosensor. . In this state, the two sample introduction ports 9 are brought into contact with the sample solution 11, whereby the sample solution is independently introduced into the semicircular sample transport path 5 by capillary action. In order to smoothly introduce the sample liquid 11 into the sample transport path 5, air discharge ports 10 are respectively provided on the same cross section of the biosensor unit-containing substrate as the sample introduction port 9. FIG. 4i shows a front view when two biosensor unit-containing substrates divide the substrate 1 into two and take a V shape.

  In the case of the biosensor for multi-item simultaneous measurement illustrated in FIG. 4, it has a completely sealed structure that does not require packaging as in FIG. 3, and the sample inlet 9 is close to the upper end of the biosensor for multi-item simultaneous measurement. Since the multi-item simultaneous measurement biosensor is connected to the measuring device and the sample liquid 11 is introduced into the multi-item simultaneous measurement biosensor, the operation shown in FIG. This is easier than in the case of a biosensor for simultaneous measurement.

  FIG. 5 is similar in structure to the biosensor for simultaneous measurement of multiple items in FIG. 3, but with an adjusting agent such as a desiccant for adjusting the atmosphere in the contained sample transport path. There are features.

  FIG. 5a shows the outside of the rectangular substrate 1 of the biosensor for multi-item simultaneous measurement. A cutting line 12 is formed in the shape of a V-shaped groove 7 at the center of the substrate 1 extending vertically from the upper end side to the lower end side. The groove 7 is for folding the multi-item simultaneous measurement biosensor into a V shape along the broken line 12 when the multi-item simultaneous measurement biosensor is used.

  FIG. 5 b shows the inside of the substrate 1. Inside the substrate 1, a pattern 4 including two sets of electrodes is arranged on the substrate 1 symmetrically with a slight distance from the upper end portion, with the central broken line 12 of the substrate as a boundary. Two reagent layers 6 are formed on a part of each set of electrode patterns. Although not shown in FIG. 5b, a resist layer having a pattern similar to the adhesive layer may be provided between the adhesive layer forming the spacer 3 shown in FIG. 5d and the substrate 1 including the electrode pattern 4 of FIG. 1b. Good. Further, the adhesive layer 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1.

  FIG. 5 c shows the outer part of the cover 2. In the center of the cover 2, a cutting line 12 is formed in the shape of a V-shaped groove 7 which is formed vertically like the substrate 1 and extends from the upper end side to the lower end side. FIG. 5 d shows the inside of the cover 2. An adhesive layer as the spacer layer 3 is disposed on the surface of the cover 2, and an internal atmosphere adjusting agent such as a desiccant 14 is disposed on the cover 2. A portion 5 where no spacer is present is provided on the surface of the cover 2 so as to be connected to the adjusting agent, and the reagent transport path 5 is formed by bonding to the substrate. In this case, the adhesive layer 3 may be either formed in advance on the surface of the cover 2 as shown in the figure, or may be formed on the resist layer on the substrate 1. Similar to the multi-item simultaneous measurement biosensor illustrated in FIG. 3, the multi-item simultaneous measurement biosensor of FIG. 5 is characterized in that the sample transport path 5 is encapsulated inside the adhesive layer as shown in FIG. 5d. It is that it is formed with.

  FIG. 5e shows a configuration diagram of the biosensor 15 for simultaneous measurement of multiple items when the inside of the substrate 1 and the cover 2 are overlapped with each other at the upper end. By making the length of the cover 2 shorter than that of the substrate 1, the lower end portion of the electrode pattern 4 is exposed when the cover 2 is overlapped with the upper ends of both. This is the terminal 8 shown in FIG. 5e. Further, two biosensor units 27 are divided into two with the groove 7 as a boundary, and there are two sets of biosensor unit-containing substrates 28 each including one biosensor unit. Further, in this embodiment, like the multi-item simultaneous measurement biosensor illustrated in FIG. 3, the sample transport path 5 is completely included in the structure of the multi-item simultaneous measurement biosensor, and does not directly contact the outside air. Therefore, the inside can be kept secret.

  FIG. 5f shows a cross-sectional view taken along line AA ′ of the sample transport path portion above the biosensor for simultaneous measurement of multiple items shown in FIG. 5e. Two sets of electrodes 4 are arranged on the substrate 1, and there are an adhesive layer 3, a sample transport path 5, and a reaction layer 6 between the substrate 1 and the cover 2. The V-grooves 7 outside the substrate 1 and the cover 2 are provided so as to overlap each other. FIG. 5g shows a BB ′ cross-sectional view on the electrode pattern in the multi-item simultaneous measurement biosensor shown in FIG. 5e. Between the substrate 1 and the cover 2, a structure in which an adhesive layer 3, a sample transport path 5, and an internal atmosphere adjusting agent 14, which is a feature of the multi-item simultaneous measurement biosensor of FIG. ing.

  FIG. 5h shows an example of use of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 5 h shows a case where the multi-item simultaneous measurement biosensor is vertically folded along the V-groove 7 in the cover portion 2. Thus, the substrate 1 having two biosensor units is divided into two biosensor unit-containing substrates each including one biosensor unit. On the other hand, the cover portion is folded along the V-groove, but is not divided. As a result, the two biosensor unit-containing substrates can take a V-shape as illustrated. At this time, the sample transport path 5 is divided along the V-groove at the boundary between the two biosensor unit-containing substrates, and each of the two biosensor unit sample inlets 9 and the air outlet 10 adjacent to the adjusting agent layer is provided at one location. Is formed in a shape adjacent to.

In this state, by bringing the two sample introduction ports 9 into contact with the sample solution 11, the sample solution is independently introduced into the semicircular sample transport path 5 by capillary action, and is adjacent to the adjusting agent layer. The sample solution stops near the conveyance path 5. In order to smoothly introduce the sample liquid 11 into the sample transport path 5, air discharge ports 10 are respectively provided on the same cross section of the biosensor unit-containing substrate as the sample introduction port 9.
FIG. 5i shows a front view when two biosensor unit-containing substrates divide the substrate into two and take a V-shape.
In the case of the biosensor for simultaneous measurement of multiple items illustrated in FIG. 5, no packaging is required and a completely sealed structure in which a regulator is held inside can be taken.

  FIG. 6 shows an example of use of the biosensor for multi-item simultaneous measurement illustrated in FIG. 4 together with a dedicated measuring device.

  6a-i show a case where the biosensor 15 for multi-item simultaneous measurement and the measuring device 16 are viewed from directly above. A terminal 8 is disposed at the lower end of the multi-item simultaneous measurement biosensor 15. The introduction part 17 of the measuring device 16 is for a horizontal movement part 18 and a horizontal movement guide part 19 for sliding the biosensor 15 for simultaneous measurement of multiple items and a parallel movement part for simultaneous measurement of multiple items in the introduction part of the measurement instrument. When inserting the biosensor, the multi-item simultaneous measurement biosensor 15 is constituted by a folded portion upper portion 20 for folding along the V-groove 7.

  FIG. 6a-ii shows the state when the multi-item simultaneous measurement biosensor is connected to the measuring device. When the multi-item simultaneous measurement biosensor 15 is connected to the measuring device 16, the multi-item simultaneous measurement biosensor is deformed into a V shape in the shape shown in FIG. FIGS. 6b-i and 6b-ii show AA ′ and BB ′ cross-sectional views of the biosensor 15 for multi-item simultaneous measurement and the measuring device 16. FIG. FIG. 6 c shows a case where the biosensor 15 for multi-item simultaneous measurement and the measuring device 16 are viewed from the side. FIG. 6 c-ii shows a state where the multi-item simultaneous measurement biosensor 15 is connected to the measuring device 16 and the sample solution 11 is introduced.

  FIG. 7 shows a front view of the multi-item simultaneous measurement biosensor 15 and the measuring device 16 shown in FIG. FIG. 7 a shows a front view before introducing the multi-sensor simultaneous measurement biosensor 15 into the measuring device 16. FIG. 7 b shows a front view when the multi-item simultaneous measurement biosensor 15 is introduced into the measuring device 16. Here, from the state in which the V-groove 7 in the center of the cover of the multi-item simultaneous measurement biosensor 15 is in contact with the folded portion 20 of the measuring device introduction portion (FIG. 7a), the multi-item simultaneous measurement biosensor 15 Is horizontally pushed into the horizontal moving part 18 of the measuring instrument, so that the multi-item simultaneous measurement biosensor 15 takes the shape shown in FIG. 7b. In FIG. 7b, the two biosensor unit-containing substrates 28 folded in a V shape are connected to the sample introduction port 9 and the air discharge port 10 so as to be adjacent to each other, and the shape is changed to take in the sample liquid. It shows a state.

  FIG. 8 shows a state in which the sample solution is introduced in a state where the multi-item simultaneous measurement biosensor 15 and the measuring device 16 shown in FIG. 6 are connected. FIG. 8a shows a state in which the multi-item simultaneous measurement biosensor 15 and the measuring device 16 are viewed from the side. In this manner, the sample liquid 11 is introduced from the sample introduction port 9 of the multi-sensor simultaneous measurement biosensor 15 by tilting the measuring instrument 16 by about 30 degrees. FIG. 8 b shows the state at that time when the multi-item simultaneous measurement biosensor having a V-shape is viewed from the front. As apparent from FIG. 8b, two sample inlets 9 adjacent to each other of the openings of the V-shaped multi-item simultaneous measurement biosensor can be formed, and the sample liquid 11 can be introduced there. As shown in the figure, the multi-item simultaneous measurement biosensor 15 is easily deformed into a V shape so that the sample liquid 11 in a droplet (sphere) shape can be easily taken in.

  FIG. 9 shows an array-like biosensor for simultaneous measurement of multiple items.

  FIG. 9a is a perspective view. The electrode pattern 4 is arranged in two rows and ten columns symmetrically about the V groove 7 of the substrate 1 and the cover 2, and the sample transport path 5 intersects each electrode. FIG. 9b shows an example of use of the biosensor for simultaneous measurement of multiple items when it is folded along the V-groove 7 existing in the vertical direction. In the multi-item simultaneous measurement biosensor of FIG. 9, 20 biosensor units are divided into two by the V-groove 7 to form two sets of biosensor unit-containing substrates each including 10 biosensor units. Since one sample introduction port is formed for each biosensor unit, 10 sample introduction ports 9 are formed on the cut surface of the biosensor unit-containing substrate, for a total of 20 sample introduction ports 9. Further, in order to smoothly introduce the sample liquid 11 into the sample transport path 5, the air discharge port 10 is provided on the opposite side of the sample introduction port 9.

9c is a cross-sectional view taken along the line AA ′, and FIG. 9d is a cross-sectional view taken along the line BB ′. The arrangement of the resist 13 is shown in the AA ′ cross-sectional view shown in FIG. 9c. The purpose of using the resist layer 13 in this array-shaped biosensor for simultaneous measurement of multiple items is to insulate the wiring other than the electrodes and to make the pattern of the insulating layer clearer than when only the adhesive layer 3 is used. Because. Therefore, the size of the pattern of this resist in FIG. 9a is the same as that of the cover 2 and covers the portion other than the electrode 23 forming the reaction layer.
In the array-type multi-item simultaneous measurement biosensor of FIG. 9, a plurality of biosensor units are regularly arranged so that not only a large number of sample solutions can be measured simultaneously, With the biosensor unit-containing substrate, more items can be measured.

  FIG. 10 shows a multi-sensor simultaneous measurement biosensor in which a plurality of multi-item simultaneous measurement biosensors 15 in the form of an array are connected. In FIG. 10, there are 10 biosensor unit-containing substrates, and each biosensor unit-containing substrate has 20 biosensor units.

  FIG. 10a is a perspective view. FIG. 10 b shows an example of use of the biosensor for simultaneous measurement of multiple items when it is folded along the V groove 7 in the vertical direction so as to be V-shaped.

  For example, in this array-shaped multi-item simultaneous measurement biosensor, since 20 biosensor units are included in one biosensor unit-containing substrate, a total of 200 sample inlets 9 are formed.

FIG. 10c is a cross-sectional view taken along line AA ′, and FIG. 10d is a cross-sectional view taken along line BB ′.
In the array-type biosensor for simultaneous measurement of multiple items shown in FIG. 10, by arranging a plurality of biosensors regularly than shown in FIG. 9, not only a large number of sample solutions can be measured simultaneously, but also one sample solution is opposed. With the two disposed biosensor unit-containing substrates, a larger number of items (reagent layers) can be measured. In actual use, the sample liquid 11 previously arranged on a flat substrate so as to have a certain contact angle can be used for measurement. Further, by directing the sample inlet 9 upward, the sample liquid can be directly introduced using a sample dispenser such as a spotter.

  FIG. 11 shows an array-like multi-item simultaneous measurement biosensor in which the air discharge port 24 of each biosensor unit is provided between the electrode pattern 4 and the wiring 22 in the array-like multi-item simultaneous measurement biosensor of FIG. Show.

  In the case of this array-shaped multi-item simultaneous measurement biosensor, unlike the array-shaped multi-item simultaneous measurement biosensor shown in FIGS. 9 and 10, the wiring 22 orthogonal to the sample transport path is not necessarily insulated by the resist layer 13. There is no need to do. Furthermore, compared to the array-shaped biosensor for multi-item simultaneous measurement shown in FIGS. 9 and 10, since the air outlet 24 is provided in the vicinity of the electrode 23, the length of the sample transport path 5 is shortened. In addition, the amount of the sample solution necessary for the measurement is small as a whole. FIG. 11c is a cross-sectional view taken along line AA ′, and FIG. 11d is a cross-sectional view taken along line BB ′.

  FIG. 12 shows a multi-item simultaneous measurement biosensor in which a plurality of multi-item simultaneous measurement biosensors of FIG. 11 are further connected. In FIG. 12, there are 10 sets of biosensor unit-containing substrates, and each biosensor unit-containing substrate has 20 biosensor units.

  FIG. 12a is a perspective view. FIG. 12 b shows an example of use of the biosensor for simultaneous measurement of multiple items when folded into a V shape along the V groove 7 existing in the vertical direction. For example, in this array-shaped multi-item simultaneous measurement biosensor, since 20 biosensor units are included in one biosensor unit-containing substrate, a total of 200 sample inlets 9 are formed.

12c is a cross-sectional view taken along the line AA ′, and FIG. 12d is a cross-sectional view taken along the line BB ′.
In the array type multi-item simultaneous measurement biosensor of FIG. 12, by arranging a plurality of biosensors regularly than shown in FIG. 11, not only a large number of sample solutions can be measured simultaneously, but also one sample solution is opposed. A large number of items (reagent layers) can be measured by the two sets of biosensor unit-containing substrates arranged.

  In actual use, as in Example 10, the sample liquid 11 previously arranged on the flat substrate so as to have a certain contact angle can be used for the measurement. Further, by directing the sample inlet 9 upward, the sample liquid can be directly introduced using a sample dispenser such as a spotter. In these cases, for the reason described in Example 11, measurement can be performed with a smaller amount of sample solution used than in Example 10.

  FIG. 13 shows a V array in which two biosensor units are arranged on a single substrate so as to face each other vertically and are provided on two biosensor unit-containing substrates each including one biosensor unit. A biosensor for simultaneous measurement of multiple items that can be used for measurement by folding along a groove is shown.

  FIG. 13a shows the outside of the rectangular substrate 1 of the multi-item simultaneous measurement biosensor. A horizontal V-groove 7 is provided at the center of the substrate 1.

  FIG. 13 b shows the inside of the substrate 1. Inside the substrate 1, a pattern 4 including two sets of electrodes is disposed so as to face each other with the central broken line 12 of the substrate as a boundary. A reagent layer 6 is formed on a part of each set of electrode patterns. Although not shown in FIG. 13b, in order to clarify the section of the pattern to be the reagent layer 6, between the adhesive layer forming the spacer 3 shown in FIG. 13d and the substrate 1 including the electrode pattern 4 of FIG. A resist layer may be provided in the same pattern as the adhesive layer. In this case, the resist layer becomes the spacer 3 like the adhesive layer. For example, the resist layer may not form the same pattern as the adhesive layer. In this case, the resist layer is an electrode other than the reaction layer 6. It can also be provided as an insulating layer for preventing the pattern 4 from crossing the sample transport path 5. Further, the adhesive layer 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1.

  FIG. 13 c shows the outer part of the cover 2. A cutting line that is horizontally formed in the center of the cover 2 is provided in the form of a V-groove 7 in the same manner as the substrate 1. FIG. 13 d shows the inside of the cover 2. An adhesive layer as a spacer layer 3 is formed on the inner surface of the cover 2. A portion 5 without a spacer is provided on the cover 2 so as to divide the adhesive layer 3 into two vertically, and the reagent transport path 5 is formed by bonding to the substrate 1. In this case, both ends of the sample transport path extend from the upper end to the lower end of the cover.

  FIG. 13e shows a configuration diagram of the biosensor 15 for simultaneous measurement of multiple items when the insides of the substrate 1 and the cover 2 are superimposed on the horizontal line 12 at the center. By making the length of the cover 2 shorter than that of the substrate 1, terminals 8 are formed at both ends. There are also two biosensor unit-containing substrates 28 each including one biosensor unit 27 with the groove 7 as a boundary.

  FIG. 13f shows a cross-sectional view along the line AA ′ of the sample transport path portion above the biosensor for simultaneous measurement of multiple items shown in FIG. 13e. An electrode 4 is disposed on the substrate 1, and a sample transport path 5 is formed between the substrate 1 and the cover 2 as an empty portion of the adhesive layer 3 and the spacer. FIG. 13g shows a BB ′ cross-sectional view on the electrode pattern of the biosensor for multi-item simultaneous measurement shown in FIG. 13e. On the outside of the substrate 1 and the cover 2 between the two sets of electrodes, a V-shaped groove 7 is provided so as to overlap each other. On the substrate 1, two electrodes 4 extend from the upper end and the lower end to the vicinity of the center, respectively.

  FIG. 13h shows an example of use of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 13 h shows a case where the multi-item simultaneous measurement biosensor is vertically folded along the V-groove 7 in the cover portion 2. Thus, the substrate 1 is folded in two so as to separate the two biosensor unit-containing substrates, but is not divided. On the other hand, the cover part is divided along the V-groove. As a result, the two biosensor unit-containing substrates can have a folded shape as illustrated.

  At this time, the sample transport path 5 is divided along the V-groove at the boundary between the two biosensor unit-containing substrates, and the sample inlet 9 for each biosensor unit is formed in a shape adjacent to one place. In this state, the two sample introduction ports 9 adjacent to the sample solution 11 are brought into contact with each other, whereby the sample solution is introduced into the sample transport path 5 of the adjacent biosensor unit independently of each other by capillary action. In order to smoothly introduce the sample liquid 11 into the sample transport path 5, the air discharge port 10 is provided on the opposite side of the sample introduction port 9.

  In the case of the biosensor structure for multi-item simultaneous measurement in FIG. 13, one sample solution is measured in a completely independent system without being interfered by a reagent in which two adjacent biosensor units are present in each reagent layer. It can be mentioned as a feature. Here, the biosensor for multi-item simultaneous measurement illustrated in FIG. 13 is folded in such a manner that the two biosensor unit-containing substrates 1 face each other and the terminals 8 face each other. In addition, the cover 2 of the two biosensor unit-containing substrates may be folded so that they face each other, and the terminals 8 may face each other.

  FIG. 14 is substantially the same as the multi-item simultaneous measurement biosensor of FIG. 13 with respect to the outside, but the internal structure is different.

  FIG. 14a shows the outside of the rectangular substrate 1 of the multi-item simultaneous measurement biosensor. A horizontal V-groove 7 is provided at the center of the substrate 1.

  FIG. 14 b shows the inside of the substrate 1. Inside the substrate 1, a pattern 4 including two sets of electrodes is disposed so as to face each other with the central broken line 12 of the substrate as a boundary. A reagent layer 6 is formed on a part of each set of electrode patterns. Although not shown in FIG. 14b, in order to clarify the section of the pattern that becomes the reaction vessel 6, between the adhesive layer forming the spacer 3 shown in FIG. 14d and the substrate 1 including the electrode pattern 4 of FIG. 14b, A resist layer may be provided in the same pattern as the adhesive layer. In this case, the resist layer becomes the spacer 3 like the adhesive layer. For example, the resist layer may not form the same pattern as the adhesive layer. In this case, the resist layer is an electrode other than the reaction layer 6. It can also be provided as an insulating layer for preventing the pattern 4 from crossing the sample transport path 5. Further, the adhesive layer 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1.

  FIG. 14 c shows the outer part of the cover 2. A cutting line that is horizontally formed in the center of the cover 2 is provided in the form of a V-groove 7 in the same manner as the substrate 1. Furthermore, unlike FIG. 13c, the air discharge port 24 is provided in two places. FIG. 14 d shows the inside of the cover 2. An adhesive layer as a spacer layer 3 is formed on the inner surface of the cover 2. A portion 5 where the adhesive layer 3 does not exist is formed between the air discharge ports 24 of the two sets of biosensor unit-containing substrates on the cover 2.

  FIG. 14e shows a configuration diagram of the biosensor 15 for simultaneous measurement of multiple items when the insides of the substrate 1 and the cover 2 are overlapped with the horizontal line 12 at the center. By making the length of the cover 2 shorter than that of the substrate 1, terminals 8 are formed at both ends. Two biosensor units 27 are included in two biosensor unit-containing substrates 28 one by one with the groove 7 as a boundary, and one air exhaust port is provided in each biosensor unit-containing substrate 28. 24 is provided.

  FIG. 14f shows an AA ′ cross-sectional view of the sample transport path portion above the multi-item simultaneous measurement biosensor shown in FIG. 14e. An electrode 4 is disposed on the substrate 1, and a sample transport path 5 is formed between the substrate 1 and the cover 2 as an empty portion of the adhesive layer 3 and the spacer. FIG. 1g shows a BB ′ cross-sectional view on the electrode pattern of the biosensor for multi-item simultaneous measurement shown in FIG. 1e. On the outside of the substrate 1 and the cover 2 between the two sets of electrodes, a V-shaped groove 7 is provided so as to overlap each other. On the substrate 1, two electrodes 4 extend from the upper end and the lower end to the vicinity of the center, respectively.

  FIG. 14h shows a usage example of the biosensor for multi-item simultaneous measurement of the present invention. FIG. 14 h shows a case where the multi-item simultaneous measurement biosensor is vertically folded along the V-groove 7 in the cover portion 2. Thus, the substrate 1 is folded in two so as to separate the two biosensor unit-containing substrates, but is not divided. On the other hand, the cover part is divided along the V-groove. As a result, the two biosensor unit-containing substrates can take a V-shape as illustrated. At this time, the sample transport path 5 is divided along the V-groove at the boundary between the two biosensor unit-containing substrates, and the sample inlet 9 for each biosensor unit is formed in a shape adjacent to one place. In this state, the two sample introduction ports 9 adjacent to the sample solution 11 are brought into contact with each other, whereby the sample solution is introduced into the sample transport path 5 of the adjacent biosensor unit independently of each other by capillary action. In order to smoothly introduce the sample liquid 11 into the sample transport path 5, the air discharge port 24 exists in a shape penetrating the cover 2 after passing through the reagent layer 6 where the electrode is present.

  Unlike the multi-item simultaneous measurement biosensor illustrated in FIG. 13, the multi-item simultaneous measurement biosensor illustrated in FIG. 14 has a small volume of the sample transport path 5 due to the air discharge port 24 provided through the cover 2. As a result, it is characterized in that the amount of sample solution required for measurement can be reduced. Other features and application examples of use are the same as the biosensor for simultaneous measurement of multiple items illustrated in FIG. 13, the two biosensor unit-containing substrates 1 are folded so as to face each other, and the respective terminals 8 face back. In addition to the form in use in such a state, the cover 2 of the two biosensor unit-containing substrates may be folded so that they face each other, and each terminal 8 faces each other.

  FIG. 15 is substantially the same as the multi-item simultaneous measurement biosensor in FIG. 13 with respect to the outside, but the internal structure is different.

  FIG. 15a shows the outside of the rectangular substrate 1 of the biosensor for multi-item simultaneous measurement. A horizontal V-groove 7 is provided at the center of the substrate 1.

  FIG. 15 b shows the inside of the substrate 1. Inside the substrate 1, two sets of patterns 4 including four sets of electrodes are arranged so as to face each other with the center broken line 12 of the substrate as a boundary. A reagent layer 6 is formed on a part of each set of electrode patterns. Although not shown in FIG. 15b, in order to clarify the section of the pattern to be the reaction vessel 6, between the adhesive layer forming the spacer 3 shown in FIG. 15d and the substrate 1 including the electrode pattern 4 of FIG. 15b, A resist layer may be provided in the same pattern as the adhesive layer. In this case, the resist layer becomes the spacer 3 like the adhesive layer. For example, the resist layer may not form the same pattern as the adhesive layer. In this case, the resist layer is an electrode other than the reaction layer 6. It can also be provided as an insulating layer for preventing the pattern 4 from crossing the sample transport path 5. Further, the adhesive layer 3 may be formed first on the surface of the cover 2 as shown in the drawing, or may be formed on the resist layer on the substrate 1.

  FIG. 15 c shows the outer part of the cover 2. A cutting line that is horizontally formed in the center of the cover 2 is provided in the form of a V-groove 7 in the same manner as the substrate 1. FIG. 15 d shows the inside of the cover 2. An adhesive layer as a spacer layer 3 is formed on the inner surface of the cover 2. A portion 5 where no spacer is present is provided in the adhesive layer 3 at the top of the cover 2 in an X shape, and the reagent transport path 5 is formed by bonding to the substrate 1. In this case, both ends of the sample transport path have a shape that appears at four places from the side surface different from the cross section of the multi-item simultaneous measurement biosensor.

  FIG. 15e shows a configuration diagram of the biosensor 15 for simultaneous measurement of multiple items when the insides of the substrate 1 and the cover 2 are overlapped with the central horizontal line 12, respectively. By making the length of the cover 2 shorter than that of the substrate 1, terminals 8 are formed at both ends. In addition, two biosensor units 27 exist in two biosensor unit-containing substrates 28, each having two grooves, with the groove 7 as a boundary. Furthermore, the air outlet 10 derived from each biosensor unit is provided on a side surface different from the cross section of the biosensor unit-containing substrate 28.

  FIG. 15f shows a cross-sectional view taken along line AA ′ of the sample transport path portion above the biosensor for simultaneous measurement of multiple items shown in FIG. 15e. An electrode 4 is disposed on the substrate 1, and a sample transport path 5 is formed between the substrate 1 and the cover 2 as an empty portion of the adhesive layer 3 and the spacer. FIG. 15g shows a BB ′ cross-sectional view on the electrode pattern of the biosensor for multi-item simultaneous measurement shown in FIG. 15e. On the outside of the substrate 1 and the cover 2 between the two sets of electrodes, a V-shaped groove 7 is provided so as to overlap each other. Two electrodes 4 extend from the upper end and the lower end to the vicinity of the center on the substrate 1, and a sample transport path 5 is formed in the vicinity of the V groove 7.

  FIG. 15h shows a usage example of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 15 h shows a case where the biosensor for simultaneous measurement of multiple items is vertically folded along the V groove 7 in the cover portion 2. Thereby, the substrate 1 including four biosensor units is folded so as to be divided into two biosensor unit-containing substrates each including two biosensor units, but is not divided. On the other hand, the cover part is divided along the V-groove. As a result, the two biosensor unit-containing substrates can have a folded shape as illustrated.

  At this time, the sample transport path 5 is divided along the V-groove by two of the four biosensor units, and the sample inlet 9 of each biosensor unit is formed in a shape adjacent to one place. In this state, by bringing the four sample inlets 9 adjacent to the sample solution 11 into contact, the sample solution is independently introduced into the sample transport path 5 of the adjacent biosensor unit by capillary action. In order to smoothly introduce the sample solution 11 into the sample transport path 5, the air discharge port 10 is provided on the side surface of the biosensor unit-containing substrate located behind the reagent layer 6 where the electrodes are present.

  Unlike the multi-item simultaneous measurement biosensor illustrated in FIG. 13, the multi-item simultaneous measurement biosensor illustrated in FIG. 15 is configured such that two biosensor units are back-to-back by folding along the central V-groove. It becomes a state. As a result, a maximum of four items can be measured from one sample solution. Other features and application examples of use are the same as the biosensor for simultaneous measurement of multiple items illustrated in FIG. 13, the two biosensor unit-containing substrates 1 are folded so as to face each other, and the respective terminals 8 face back. In addition to the form in use in such a state, the cover 2 of the two biosensor unit-containing substrates may be folded so that they face each other, and each terminal 8 faces each other.

  FIG. 16 is substantially the same as the multi-item simultaneous measurement biosensor of FIG. 13 with respect to the outside, but the internal structure is different.

  FIG. 16a shows the outside of the rectangular substrate 1 of the biosensor for multi-item simultaneous measurement. A horizontal V-groove 7 is provided at the center of the substrate 1.

  FIG. 16 b shows the inside of the substrate 1. Inside the substrate 1, two sets of patterns 4 including four sets of electrodes are arranged so as to face each other with the center broken line 12 of the substrate as a boundary. A reagent layer 6 is formed on a part of each set of electrode patterns. Although not shown in FIG. 16b, in order to clarify the section of the pattern to be the reaction vessel 6, between the adhesive layer forming the spacer 3 shown in FIG. 16d and the substrate 1 including the electrode pattern 4 of FIG. 16b, A resist layer may be provided in the same pattern as the adhesive layer. In this case, the resist layer becomes the spacer 3 like the adhesive layer. For example, the resist layer may not form the same pattern as the adhesive layer. In this case, the resist layer is an electrode other than the reagent layer 6. It can also be provided as an insulating layer for preventing the pattern 4 from crossing the sample transport path 5. Further, the adhesive layer 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1.

  FIG. 16 c shows the outer part of the cover 2. In the center of the cover 2, a cutting line formed in the horizontal direction in the same manner as the substrate 1 is provided in the form of a V groove 7. Furthermore, unlike FIG. 15c, the air discharge port 24 is provided in four places. FIG. 16 d shows the inside of the cover 2. An adhesive layer as a spacer layer 3 is formed on the inner surface of the cover 2. Further, on the surface of the cover 2, a portion 5 where no spacer exists is provided in the adhesive layer 3 in an X shape, and through holes 24 are provided at four ends thereof. Reagent transport path 5 is formed by bonding to substrate 1.

  FIG. 16e shows a configuration diagram of the biosensor 15 for simultaneous measurement of multiple items when the insides of the substrate 1 and the cover 2 are superimposed on the central horizontal line 12, respectively. By making the length of the cover 2 shorter than that of the substrate 1, terminals 8 are formed at both ends. In addition, four biosensor units 27 are divided into two and are included in two biosensor unit-containing substrates 28 with the groove 7 as a boundary, and each biosensor unit has one air outlet. 24.

  FIG. 16f shows a cross-sectional view along the line AA ′ of the sample transport path portion above the biosensor for simultaneous measurement of multiple items shown in FIG. 16e. An electrode 4 is disposed on the substrate 1, and between the substrate 1 and the cover 2, an adhesive layer 3, a sample transport path 5 as a blank portion of a spacer, and two air discharge ports 24 are formed. FIG. 16g shows a BB ′ cross-sectional view on the electrode pattern of the biosensor shown in FIG. 16e. On the outside of the substrate 1 and the cover 2 between the two sets of electrodes, a V-shaped groove 7 is provided so as to overlap each other. Two electrodes 4 extend from the upper end and the lower end to the vicinity of the center on the substrate 1, and a sample transport path 5 is formed in the vicinity of the V groove 7.

  FIG. 16h shows an example of use of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 16 h shows a case where the multi-item simultaneous measurement biosensor is vertically folded along the V-groove 7 in the cover portion 2. Accordingly, the substrate 1 including four biosensor units is divided so as to include two biosensor units and folded into two biosensor unit-containing substrates, but is not divided. On the other hand, the cover part is divided along the V-groove.

  As a result, the two biosensor unit-containing substrates can have a folded shape as illustrated. At this time, the sample transport path 5 is divided along the V-groove by two of the four biosensor units, and the sample inlet 9 of each biosensor unit is formed in a shape adjacent to one place. In this state, by bringing the four sample inlets 9 adjacent to the sample solution 11 into contact, the sample solution is independently introduced into the sample transport path 5 of the adjacent biosensor unit by capillary action. In order to smoothly introduce the sample solution 11 into the sample transport path 5, an air outlet 24 is provided in the state of penetrating the cover 2 behind the reagent layer 6 where the electrodes are present.

  Unlike the biosensor for multi-item simultaneous measurement illustrated in FIG. 13, the biosensor for multi-item simultaneous measurement illustrated in FIG. 16 has two biosensor unit-containing substrates by folding along a central V-groove. Back to back. As a result, a maximum of four items can be measured from one sample solution. Furthermore, as in the example of the multi-item simultaneous measurement biosensor illustrated in FIGS. 13 and 14, the measurement with a smaller number of sample liquids is possible as compared with the multi-item simultaneous measurement biosensor illustrated in FIG. Other features and application examples of use are the same as the biosensor for simultaneous measurement of multiple items illustrated in FIG. 13, the two biosensor unit-containing substrates 1 are folded so as to face each other, and the respective terminals 8 face back. In addition to the form in use in such a state, the cover 2 of the two biosensor unit-containing substrates may be folded so that they face each other, and each terminal 8 faces each other.

  FIG. 17 illustrates the multi-item simultaneous measurement biosensor having a structure in which the terminals of the multi-item simultaneous measurement biosensor of FIG. 13 are protected by the cover until they are used. Here, only the multi-item simultaneous measurement biosensor of FIG. 13 is taken as an example, but the shape is not limited at all as long as it is a multi-item simultaneous measurement biosensor according to the present invention.

  17a, b and f are the same as FIGS. 13a, b and f. 17c, d, e, g, and h, the cover 2 shown in FIGS. 13c, d, e, g, and h covers the entire substrate 1, and the adhesive layer constituting the spacer 3 is shown in 13c. The pattern is the same. Two perforations 25 are provided in the cover portion 2 that covers the upper and lower terminals 8.

FIG. 18 shows a usage example of the terminal portion 8 of the biosensor for simultaneous measurement of multiple items illustrated in FIG. FIG. 18a shows the state before use. 18B, the cover 2 covering the top of the terminal 8 is removed along the perforation 25, FIG. 18C is the cover 2 folded along the perforation 25, and FIG. A folded state along the eyes 25 is shown.
Thus, by covering the terminal portion 8 of the multi-item simultaneous measurement biosensor with the cover 2, the terminal 8 can be protected until it is used.

  FIG. 19 shows an example in which the terminal protection type multi-item simultaneous measurement biosensor 15 illustrated in FIG. 17 is connected. Here, the multi-item simultaneous measurement biosensor 15 that can be connected can be applied to the multi-item simultaneous measurement biosensor illustrated in FIGS. 13 to 16. That is, the multi-item simultaneous measurement biosensor is not particularly limited as long as the individual biosensor unit-containing substrate 28 is oriented around the V-grooves provided in the substrate 1 and the cover 2. As shown in the figure, the multi-item simultaneous measurement biosensor 15 can be used by being separated by a perforation 25 provided vertically.

  FIG. 20 shows an example in which the multi-item simultaneous measurement biosensor 15 exemplified in FIGS. 1, 3, 4 and 5 is connected. In this case, the multi-item simultaneous measurement biosensor 15 is not particularly limited as long as the individual biosensor unit-containing substrates 28 are oriented around the V grooves provided in the substrate 1 and the cover 2. As shown in the drawing, each multi-item simultaneous measurement biosensor 15 can be used by being separated by a perforation 25 provided vertically.

  FIG. 21 shows an example in which the multi-item simultaneous measurement biosensor 15 exemplified in FIGS. 1, 3, 4 and 5 is connected by a soft sheet. Also in this case, as in Example 20, the multi-item simultaneous measurement biosensor 15 is not particularly limited as long as the individual biosensor unit-containing substrates 28 are oriented around the V-grooves provided in the substrate 1 and the cover 2. Not. As shown in the drawing, the individual multi-item simultaneous measurement biosensor 15 is connected by a soft sheet and can be used by being separated by a perforation 25 provided vertically. If it is this form, unlike Example 20, each multi-item simultaneous measurement biosensor 15 can be folded and put together. Accordingly, the multi-item simultaneous measurement biosensor 15 in a connected state can be accommodated in a dedicated container or the like.

  FIG. 22 shows the structure of the biosensor for multi-item simultaneous measurement shown in FIG. 1, in which an air outlet opening 31 is provided on each biosensor unit-containing substrate 28, and the air outlet opening is interlocked with the opening of the sample inlet. This is a multi-sensor simultaneous measurement biosensor 15 of a type that can be bent by an auxiliary tool 29 provided in the section 31 (FIG. 22 (e)).

  FIG. 22a shows the outside of the rectangular substrate 1 of the multi-item simultaneous measurement biosensor. In the central portion of the substrate 1, a cutting line 12 formed vertically from the upper end side to the lower end side and an upper outer folding portion (air outlet opening portion) 31 of the substrate 1 are formed so as to form a small rectangle, A cutting line 12 is provided in the form of a V-shaped groove 7. These grooves 7 fold the multi-item simultaneous measurement biosensor into a V shape along the broken line 12 at the center of the substrate 1 when using the multi-item simultaneous measurement biosensor. 12 to fold in the direction of the center broken line 12. Further, the upper outer fold 31 is provided with an auxiliary tool 29 for opening the air outlet opening 31 in conjunction with the opening of the sample inlet. Two auxiliary tools 29 are provided so as to connect both ends of the upper outer folding part 31. The auxiliary tool 29 is provided with a fixing portion for fixing to the upper outer folding part 31.

  FIG. 22 b shows the inside of the substrate 1. Inside the substrate 1, a pattern 4 including two sets of electrodes is arranged from the upper end side to the lower end side in parallel with the central broken line 12 of the substrate as a boundary. Similarly, the broken lines 12 indicating the air outlet openings 31 are arranged at both ends on the inner upper side of the substrate 1 in parallel with the outer side of the pattern 4 including two sets of electrodes. A reagent layer 6 is formed on a part of each set of electrode patterns. Although not shown in FIG. 22b, in order to clarify the section of the pattern to be the reagent layer 6, between the adhesive layer forming the spacer 3 shown in FIG. 22d and the substrate 1 including the electrode pattern 4 of FIG. 22b, A resist layer may be provided in the same pattern as the adhesive layer. In this case, the resist layer becomes the spacer 3 like the adhesive layer, but for example, the resist layer may not form the same pattern as the adhesive layer. In that case, the resist layer can also be provided as an insulating layer for preventing the electrode pattern 4 other than the reagent layer 6 from crossing the sample transport path 5. The spacer layer (adhesive layer) 3 may be formed on the cover 2 in advance as shown in the drawing, or may be formed on the resist layer on the substrate 1.

  FIG. 22 c shows the outer part of the cover 2. At the center of the cover 2, the V-groove 7 extending vertically from the upper end side to the lower end side in the same manner as the substrate 1 and the upper outer folded portion 31 of the substrate 1 are formed so as to form a small rectangle. 12 is provided in the form of a V-shaped groove 7. FIG. 22 d shows the inside of the cover 2. An adhesive layer as a spacer layer 3 is formed on the inner surface of the cover 2. A portion 5 where no spacer is present is provided on the cover 2, and the reagent transport path 5 is formed by bonding to the substrate.

  FIG. 22e is a configuration diagram when the inner sides of the substrate 1 and the cover 2 are overlapped with each other at the upper end, and shows the biosensor 15 for simultaneous measurement of multiple items. By making the length of the cover 2 shorter than that of the substrate 1, the lower end portion of the electrode pattern 4 is exposed when the cover 2 is overlapped with the upper ends of both. This is the terminal 8 shown in FIG. In addition, two biosensor unit-containing substrates 28 each including one biosensor unit 27 with the groove 7 as a boundary, and air outlet openings 31 respectively exist on the upper outer portions of the biosensor unit-containing substrates 28. To do.

  FIG. 22f shows an AA ′ cross-sectional view of the sample transport path portion above the multi-item simultaneous measurement biosensor shown in FIG. 22e. Two sets of two electrodes 4 are arranged on the substrate 1 and there is an adhesive layer between the substrate and the cover. The central portion of the cross section in FIG. Form. From this structure, it can be seen that the sample transport path 5 is sealed in an empty portion of the spacer sandwiched between the substrate and the cover. V-shaped grooves 7 are provided between the two sets of electrodes and on the outside of the substrate 1 and the cover 2 of the air discharge port openings 31 at two locations so as to overlap each other. FIG. 22g shows a BB ′ cross-sectional view on the electrode pattern of the biosensor for multi-item simultaneous measurement shown in FIG. 22e. An electrode 4 is formed on the substrate 1, and one spacer 3 and one sample transport path 5 are provided between the substrate 1 and the cover 2. Further, two auxiliary tools 29 are arranged on the outer upper surface of the substrate 1 so as to sandwich the sample transport path 5.

  FIG. 22h shows an example of use of the biosensor for simultaneous measurement of multiple items of the present invention. FIG. 22 h shows a case where the multi-item simultaneous measurement biosensor is vertically folded along the V-groove 7 in the cover portion 2. Thereby, the substrate 1 of the multi-sensor simultaneous measurement biosensor is divided into two. On the other hand, the cover portion 2 is folded along the V-groove 7 but is not divided. As a result, the two biosensor unit-containing substrates can take a V-shape as shown in the figure. At this time, the sample transport path 5 is divided along the V-groove at the boundary between the two biosensor unit-containing substrates, and the sample inlet 9 for each biosensor unit is formed in a shape adjacent to one place. On the other hand, by taking the V-shaped shape, the two auxiliary tools 29 are pulled, the upper outer folds 31 on the two biosensor unit-containing substrates are bent, and the two air discharge ports 10 are formed. . By passing through the above process, the sample conveyance path 5 changes from a sealed state to an open state.

  In this state, the two sample introduction ports 9 adjacent to the sample solution 11 are brought into contact with each other, whereby the sample solution 11 is independently introduced into the adjacent sample transport path 5 by capillary action. At this time, if the sample liquid 11 is rounded due to surface tension as shown in the figure, the two biosensor unit-containing substrates are V-shaped while being bonded to each other as shown in FIG. 11 is efficiently introduced into the sample transport path 5. In order to smoothly introduce the sample liquid 11 into the sample transport path 5, the air discharge port 10 is provided on the opposite side of the sample introduction port 9. FIG. 22i shows a front view when two biosensor unit-containing substrates have a V-shape. From this figure, it can be seen that the auxiliary tool 29 is pulled by the V-shaped formation of the biosensor unit-containing substrate, and each air outlet opening 31 is bent from the biosensor unit-containing substrate.

  In the case of the structure of FIG. 22, similarly to the biosensor for simultaneous measurement of multiple items shown in FIG. 1, one sample solution is not affected by the interference of two adjacent biosensor units in the reagent layer. The characteristic is that the measurement can be performed in a completely independent system. Here, the multi-item simultaneous measurement biosensor illustrated in FIG. 22 has a crack on the substrate side, but the cover 2 side may be divided. Furthermore, it is not limited to a biosensor for simultaneous measurement of multiple items deformed into a V-shape, and even if it is completely folded along either the V-groove of the substrate 1 or the cover 2, it cracks at less than 180 degrees by using a hard substrate. May be used. In addition, the multi-item simultaneous measurement biosensor illustrated in FIG. 22 has two auxiliary tools 29 for opening the air discharge port, but it may be one or three or more. By using the auxiliary tool 29, the sample introduction port 9 and the air discharge port 10 can be formed simultaneously by simply deforming the multi-item simultaneous measurement biosensor into a V shape. As described above, in FIG. 22, the multi-item simultaneous measurement biosensor using the auxiliary tool 29 is illustrated, but the auxiliary tool may not be particularly provided. In this case, each air outlet opening 31 may be bent by hand to open the air outlet.

  FIG. 23 is a diagram showing an operation process in which the two-item simultaneous measurement type biosensor according to the embodiment of the present invention is applied to simultaneous measurement of glucose or two-item simultaneous measurement of glucose and lactic acid. Further, this sensor is an example to which the sensor shown in FIG. 1 is applied.

  FIG. 23a is a biosensor before being used for measurement, and an example in which the reagent layer 6 is developed in the sample transport path 5 for each biosensor unit, and b is a dedicated connector 32 for the two-item simultaneous measurement type biosensor 15. And c is an example of a sensor after sample introduction, and d is an example of a sensor after measurement.

  FIG. 24 shows an example of a connector 32 for a two-item simultaneous measurement type biosensor according to an embodiment of the present invention. FIG. 24a shows an example in which the connector 32 is opened. The connector includes a base portion 33 and a lid 34, a folder portion 35 and a presser 36 for setting a biosensor, a terminal 8 for capturing an electric signal from the biosensor, and a wiring 22. A terminal 8 and a pressing tool 36 are provided on the surface of the folder 35 on the base 33. Therefore, when connecting the biosensor with this connector, the biosensor cover portion 2 is on the lower side so that the terminal 8 of the biosensor is connected to the terminal 8 on the base, and a V-shaped structure is used. Must be placed outside. Further, the folder part 35 of the connector, that is, the upper part 20 and the lower part 21 of the folding part, was designed so that the angle when the biosensor was connected to the connector with a V-shaped structure was 90 degrees. The connection to the connector may be performed after the planar biosensor is previously formed into a V-shaped structure, or may be deformed into a V-shaped structure by setting the planar biosensor in a folder.

  FIG. 24b shows an example in which the two-item simultaneous measurement type biosensor 15 is connected to the connector 32, and the sample introduction port of each biosensor unit is opened downward near the tip of the V-shaped biosensor. Yes.

  In one application example of this sensor, glucose oxidase and potassium ferricyanide were used in the reagent layer. In this sensor, the amount of glucose in whole blood was electrochemically measured by introducing the sample separately from the sample inlet of each biosensor unit 27 by capillary action. The measurement principle of such a glucose sensor is as follows.

This sensor introduces a sample into the inside through a sample introduction port 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 biosensor manufacturing method and measurement method will be described.
The sensor substrate used was PET having a length of 35 mm, a width of 12 mm, and a thickness of 188 μm, and a cover of 30 mm, a width of 12 mm, and a thickness of 188 μm. Two sets of biosensor units 27 are arranged on the sensor substrate with the groove 7 as a boundary. The groove was formed so that the depth was more than half of the thickness of the substrate and the cover. On the sensor substrate of each sensor unit 27, two carbon electrodes having a width of 1.3 mm were formed by a screen printing apparatus with an interval of 0.5 mm. A resist and an adhesive were also formed as a spacer layer by screen printing.

  The total sample amount of the two sensors was about 0.90 μl in the theoretical design, but when the actual sample amount was measured with whole blood (specific gravity: 1.05), 1.13 ± 0.064 mg (n = 10 and the coefficient of variation CV = 5.7%). The difference in the sample amount between the two may be that the spacer layer was actually thick, or that the sample liquid adhered to the vicinity of the sample inlet.

  Enzyme and mediator reagent layers are dissolved in distilled water to form 9 units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator), applied to the electrode surface of each sensor, and vacuum dried to form on both electrodes did.

  The results of measuring blood glucose (blood glucose) using this glucose sensor will be described. Blood glucose measurement using this glucose sensor uses whole blood with a hematocrit value of 40% prepared so that the glucose concentration is 0, 100, 300, or 500 mg / dl as the sample liquid sample, Compared. As the measuring device, an electrochemical measuring device (ALS / CHI-1220, BAS) capable of simultaneous measurement of two items was used. The potential step chromoamperometry was used as the measurement method. Twenty seconds after about 1.1 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 10 seconds after the application was taken as the measured value.

  FIG. 25 shows changes in current value due to blood glucose concentration of the sensor of the present invention for each result obtained by the left and right sensors. Referring to FIG. 25, changes in the current value in the vicinity of 1 to 11 μA were observed by the left and right sensors in the blood glucose range of 0, 100, 300, and 500 mg / dl (n = 3). From the above results, it was found that there is no significant difference in the response values between the left and right sensors.

  Subsequently, the storage stability test of this sensor of FIG. 23a was performed on the 0th day, the second week, the first month, the second month, and the third month under the same conditions. The sensor was stored in a drawer (room temperature) of a laboratory table in the test room. The result is shown in FIG. From this figure, the correlation coefficient (r = 0.951 ± 0.053) and slope (0.0139 ± 0.0047) and the slope (0.0139 ± 0.0047) between the blood glucose level and the output current value during storage in the room for 3 months, Correlation coefficient (r = 0.979 ± 0.013) and slope (0.0175 ± 0.0074) for the right sensor, and correlation coefficient (r = 0.965 ± 0.020) and slope (0.0157 ± 0.0025) for both are maintained, and the response of the left and right sensors Were also observed in each. However, when the same experiment is performed on a non-sealed sensor such as this sensor in a normal living environment, in addition to the effects of temperature and humidity, it is affected by the growth of mold and microorganisms in the reagent layer. I can easily imagine that. Therefore, it is desirable that the biosensor for simultaneous measurement of multiple items is a sealed type that does not require packaging.

Next, two items of glucose and lactic acid were simultaneously measured using this sensor.
The reagent layers of the enzyme and mediator are different between the two left and right biosensors. The reagent layer of one biosensor is dissolved in distilled water to be 1.65 units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator), applied to the electrode surface of each sensor, vacuum-dried and both Formed on the electrode. The reagent layer of the biosensor on the other side is dissolved in distilled water to 3.7 units lactate oxidase (LOD) and 0.1 mg potassium ferricyanide (mediator), applied to the electrode surface of each sensor, and vacuum dried. And formed on both electrodes.

  Sample solution is 0.1 M containing 100 mg / dl lactic acid, pH 7.4 phosphate buffer, phosphate buffer containing 100 mg / dl lactic acid and 300 mg / dl glucose, phosphate buffer containing 300 mg / dl glucose It was used.

  The results of simultaneous measurement of glucose and lactic acid using this multi-item simultaneous measurement biosensor will be described. The measurement of glucose and lactic acid using a two-item simultaneous measurement sensor is performed by using four types of mixed solutions (glucose + lactic acid; 0 + 0 mg / dl, 100 + 50 mg / dl, 300 + 100 mg / dl) prepared as a sample solution. 500 + 140 mg / dl). Further, 0.1 M, pH 7.4 phosphate buffer was used for the preparation of this mixed solution. As the measuring device, an electrochemical measuring device (ALS / CHI-1220, BAS) capable of simultaneous measurement of two items was used. The potential step chromoamperometry was used as the measurement method. 20 seconds after introducing about 1.1 μl of the sample solution 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 10 seconds after the application was taken as the measured value. .

  FIG. 27 shows the results of simultaneous measurement of glucose and lactic acid using this sensor. In this way, this sensor has a linear relationship between the concentration of each substance to be measured and the output current value without being affected by the reagent layer of each adjacent sensor in the measurement of a mixed solution containing glucose and lactic acid. Could get.

  Subsequently, in order to solve the problems of the open-type multi-item simultaneous measurement biosensor in Example 23, the following example relates to a sealed multi-item simultaneous measurement biosensor that does not require packaging in the present invention. Describe the results of the study.

  FIG. 28 is a diagram showing an operation process in which the sealed two-item simultaneous measurement biosensor according to the embodiment of the present invention is applied to the simultaneous measurement of glucose. Further, this sensor is an example applied to the sensor shown in FIG. 22 with the auxiliary tool 29 removed.

  FIG. 28 a is a biosensor before being used for measurement, and is an example in which the reagent layer 6 is developed in the sample transport path 5 for each biosensor unit. The sample transport path is cut off from the outside by the upper outer folding portion 31. In a sealed state. b is an example in which a two-item simultaneous measurement type biosensor is deformed into a V shape and the sample introduction port is opened, and then whole blood is introduced as a sample. , D also shows an example of the biosensor after introduction of the sample solution viewed from the side. Thus, the biosensor may be bent while being inserted into the shape fixing portion (folder) of the connector, or may be inserted into the connector after being bent.

Next, a biosensor manufacturing method and measurement method will be described.
The sensor substrate used was PET having a length of 35 mm, a width of 15 mm, and a thickness of 188 μm, and a cover of 30 mm, a width of 15 mm, and a thickness of 188 μm. Two sets of biosensor units 27 are arranged on the sensor substrate with the groove 7 as a boundary. Similarly, the air discharge opening 31 is formed on the outer upper portion of each sensor unit by the groove 7. The groove was formed so that the depth was more than half of the thickness of the substrate and the cover. Two carbon electrodes having a width of 1.3 mm were formed on the sensor substrate of each sensor unit 27 by a screen printing apparatus with an interval of 0.8 mm. A resist and an adhesive were also formed as a spacer layer by screen printing.

  The total sample amount of the two sensors was about 0.90 μl by design, but when the actual sample amount was measured with whole blood (specific gravity: 1.05), 1.12 ± 0.077 mg (n = 10 The coefficient of variation was CV = 6.9%). The difference in the sample amount between the two may be that the spacer layer was actually thick, or that the sample liquid adhered to the vicinity of the sample inlet.

  Enzyme and mediator reagent layers are dissolved in distilled water to form 9 units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator), applied to the electrode surface of each sensor, and vacuum dried to form on both electrodes did.

  The results of measuring blood glucose (blood glucose) using this glucose sensor will be described. Blood glucose measurement using this glucose sensor uses whole blood with a hematocrit value of 40% prepared so that the glucose concentration is 0, 100, 300, or 500 mg / dl as the sample liquid sample, Compared. As the measuring device, an electrochemical measuring device (ALS / CHI-1220, BAS) capable of simultaneous measurement of two items was used. The potential step chromoamperometry was used as the measurement method. Twenty seconds after about 1.1 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 10 seconds after the application was taken as the measured value.

  FIG. 29 shows the change in the current value according to the blood glucose concentration of the sensor of the present invention for each result obtained by the left and right sensors. Referring to FIG. 29, changes in the current value in the vicinity of 0.5 to 14 μA were observed by the left and right sensors in the blood glucose range of 0, 100, 300, and 500 mg / dl (n = 3). From the above results, it was found that there is no significant difference in the response values between the left and right sensors.

Subsequently, the storage stability test of this sensor was performed on the 0th day, the second week, the first month, the second month, and the third month under the same conditions. The sensor was stored in a drawer (room temperature) of a laboratory table in the test room. The result is shown in FIG. From this figure, the response slightly fluctuated depending on the day while stored in the room for 3 months. However, the correlation coefficient (r = 0.996 ± 0.0040) was obtained between the blood glucose level and the output current value. ) And slope (0.0179 ± 0.0039), correlation coefficient (r = 0.994 ± 0.0050) and slope (0.0190 ± 0.0048) for the right sensor, both correlation coefficient (r = 0.995 ± 0.0013) and slope (0.0184 ± 0.0008) The relationship was maintained, and left and right sensor responses were also observed for each.
From the above, this sensor obtained a good result in the correlation between the blood glucose concentration and the output current value. It was found that this sensor is a sealed sensor and can be measured for at least three months without packaging.

  FIG. 31 is a diagram showing an operation process in which the sealed two-item simultaneous measurement biosensor according to the embodiment of the present invention is applied to simultaneous measurement of glucose or two-item simultaneous measurement of glucose and lactic acid. Further, this sensor is an example to which the sensor shown in FIG. 3 is applied.

  FIG. 31a is a biosensor before being used for measurement, and is an example in which the reagent layer 6 is developed in each biosensor unit sample transport path 5. The sample transport path is shut off from the outside and is in a sealed state. b is an example in which a two-item simultaneous measurement type biosensor is set in a dedicated connector and whole blood is introduced as a sample, c is an example of a sensor after sample introduction, and d is an example of a sensor after measurement.

Next, a biosensor manufacturing method and measurement method will be described.
The sensor substrate used was PET having a length of 35 mm, a width of 12 mm, and a thickness of 188 μm, and a cover of 30 mm, a width of 12 mm, and a thickness of 188 μm. Two sets of biosensor units 27 are arranged on the sensor substrate with the groove 7 as a boundary. The groove was formed so that the depth was more than half of the thickness of the substrate and the cover. Two carbon electrodes having a width of 1.3 mm were formed on the sensor substrate of each sensor unit 27 by a screen printing apparatus with a spacing of 0.5 mm. A resist and an adhesive were also formed as a spacer layer by screen printing.

  The total sample amount of the two sensors was about 2.0 μl by design, but when the actual sample amount was measured with whole blood (specific gravity: 1.05), it was 2.71 ± 0.097 mg (n = 10 The coefficient of variation was CV = 3.6%), and a highly reproducible result was obtained. The difference in the sample amount between the two may be that the spacer layer was actually thick, or that the sample liquid adhered to the vicinity of the sample inlet.

  Enzyme and mediator reagent layers are dissolved in distilled water to 20 units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator), applied to the electrode surface of each sensor, and vacuum dried to form on both electrodes did.

  The results of measuring blood glucose (blood glucose) using this glucose sensor will be described. Blood glucose measurement using this glucose sensor uses whole blood with a hematocrit value of 40% prepared so that the glucose concentration is 0, 100, 300, or 500 mg / dl as the sample liquid sample, Compared. As the measuring device, an electrochemical measuring device (ALS / CHI-1220, BAS) capable of simultaneous measurement of two items was used. The potential step chromoamperometry was used as the measurement method. Twenty seconds after about 3 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 10 seconds after the application was taken as the measured value.

  FIG. 32 shows the change in the current value according to the blood glucose concentration of the sensor of the present invention for each result obtained by the left and right sensors. Referring to FIG. 32, current values in the vicinity of 1.5 to 19 μA are observed by the left and right sensors in the blood glucose range of 0, 100, 300, and 500 mg / dl (n = 3). Responses matched very well.

Subsequently, the storage stability test of this sensor was performed on the 0th day, the second week, the first month, the second month, and the third month under the same conditions. The sensor was stored in a drawer (room temperature) of a laboratory table in the test room. The result is shown in FIG. From this figure, the response fluctuated depending on the day while stored in the room for 3 months, but the correlation coefficient (r = 0.986 ± 0.020) between the blood glucose level and the output current value is shown in the left sensor. And slope (0.0260 ± 0.0082), correlation coefficient (r = 0.985 ± 0.0080) and slope (0.0254 ± 0.010) for right sensor, correlation coefficient (r = 0.985 ± 0.00) and slope (0.0257 ± 0.0004) for both The left and right sensor responses were also observed for each.
From the above, this sensor obtained a relatively good result in the correlation between the blood glucose concentration and the output current value. It was found that this sensor is a sealed sensor and can be measured for at least three months without packaging.

Next, simultaneous measurement of glucose and lactic acid was performed using this sensor.
The reagent layers of the enzyme and mediator are different between the two left and right biosensors. The reagent layer of one biosensor is dissolved in distilled water so as to be 9 units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator), applied to the electrode surface of each sensor, and vacuum-dried on both electrodes. Formed. The reagent layer of the biosensor on the other side is dissolved in distilled water so as to be 20 units lactate oxidase (LOD) and 0.1 mg potassium ferricyanide (mediator), applied to the electrode surface of each sensor, and vacuum dried. Formed on both electrodes.

  Sample solution is 0.1 M containing 100 mg / dl lactic acid, pH 7.4 phosphate buffer, phosphate buffer containing 100 mg / dl lactic acid and 300 mg / dl glucose, phosphate buffer containing 300 mg / dl glucose It was used.

  The results of simultaneous measurement of two items of glucose and lactic acid using this sealed multi-item simultaneous measurement biosensor will be described. The measurement of glucose and lactic acid using a two-item simultaneous measurement sensor is performed by using four types of mixed solutions (glucose + lactic acid; 0 + 0 mg / dl, 100 + 50 mg / dl, 300 + 100 mg / dl) prepared as a sample solution. 500 + 140 mg / dl). Further, 0.1 M, pH 7.4 phosphate buffer was used for the preparation of this mixed solution. As the measuring device, an electrochemical measuring device (ALS / CHI-1220, BAS) capable of simultaneous measurement of two items was used. The potential step chromoamperometry was used as the measurement method. Twenty seconds after introducing about 3 μl of the sample solution 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 10 seconds after the application was taken as the measured value.

  FIG. 34 shows the results of simultaneous measurement of glucose and lactic acid using this sensor. In this way, this sensor has a linear relationship between the concentration of each substance to be measured and the output current value without being affected by the reagent layer of each adjacent sensor in the measurement of a mixed solution containing glucose and lactic acid. Could get.

FIG. 1 shows an example of a biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor is viewed from the side. i shows a usage example of a biosensor for simultaneous measurement of multiple items as seen from the front. FIG. 2 shows another example of the biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor in which and are attached, f is an example of an enlarged cross-sectional view taken along line AA ′ of e, and g is an example of an enlarged enlarged view of a cross-sectional view taken along line BB ′. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. i shows a usage example of a biosensor for simultaneous measurement of multiple items as seen from the front. FIG. 3 shows another example of the biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. i shows a usage example of a biosensor for simultaneous measurement of multiple items as seen from the front. FIG. 4 shows another example of the biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. i shows a usage example of a biosensor for simultaneous measurement of multiple items as seen from the front. FIG. 5 shows another example of the biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. i shows a usage example of a biosensor for simultaneous measurement of multiple items as seen from the front. FIG. 6 shows an example of use of the biosensor for multi-item simultaneous measurement according to the present invention together with a measuring instrument (connector). i shows an example of a state before the multi-item simultaneous measurement biosensor is connected to the measuring device. ii shows an example of the state when the multi-item simultaneous measurement biosensor is connected to the measuring device. a shows an example of a state seen from directly above. b shows an example of AA 'and BB' sectional views. c shows an example of the situation seen from the side. FIG. 7 shows a use example of the biosensor for simultaneous measurement of multiple items according to the present invention when viewed from another angle together with a measuring instrument. a shows an example when the state before the biosensor for simultaneous measurement of multiple items is connected to the measuring device is viewed from the front. b shows an example of the state when the biosensor for simultaneous measurement of multiple items is connected to the measuring device viewed from the front. FIG. 8 shows an example of using the biosensor for multi-item simultaneous measurement according to the present invention when viewed from another angle together with the measuring device. a shows an example in which a biosensor for simultaneous measurement of multiple items is connected to a measuring instrument, and the measuring instrument is tilted in order to introduce a sample solution when viewed from the side. b shows an example when the state of the biosensor for simultaneous measurement of multiple items in the state of a and the state of the measuring device are viewed from the front. FIG. 9 shows an example of an array-type multi-item simultaneous measurement biosensor according to the present invention. a shows an example of a state in which the array type multi-item simultaneous measurement biosensor is seen through from above. b shows an example of using an array-type multi-item biosensor for simultaneous measurement. c is an example of an AA ′ cross-sectional enlarged view of a, and d is an example of an BB ′ cross-sectional enlarged view of a. FIG. 10 shows an example in which the array type multi-item simultaneous measurement biosensor according to the present invention is connected. a shows an example of a state when the biosensor for simultaneous measurement of multi-item simultaneous measurement is seen through from above. b shows an example of use of a biosensor for simultaneous measurement of multiple items in a connected array type. c is an example of an AA ′ cross-sectional enlarged view of a, and d is an example of an BB ′ cross-sectional enlarged view of a. FIG. 11 shows another example of an array type multi-item simultaneous measurement biosensor according to the present invention. a shows an example of a state in which the array type multi-item simultaneous measurement biosensor is seen through from above. b shows an example of using an array-type multi-item biosensor for simultaneous measurement. c is an example of an AA ′ cross-sectional enlarged view of a, and d is an example of an BB ′ cross-sectional enlarged view of a. FIG. 12 shows another example in which the array-type multi-item simultaneous measurement biosensor according to the present invention is connected. a shows an example of a state when the biosensor for simultaneous measurement of multi-item simultaneous measurement is seen through from above. b shows an example of use of a biosensor for simultaneous measurement of multiple items in a connected array type. c is an example of an AA ′ cross-sectional enlarged view of a, and d is an example of an BB ′ cross-sectional enlarged view of a. FIG. 13 shows another example of the biosensor for multi-item simultaneous measurement according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. FIG. 14 shows another example of the biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. FIG. 15 shows another example of the biosensor for multi-item simultaneous measurement according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. FIG. 16 shows another example of the biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. FIG. 17 shows another example of the biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor for multi-item simultaneous measurement is viewed from the side. FIG. 18 shows a usage example of the biosensor for simultaneous measurement of multiple items according to the present invention. a is an example of a biosensor for multi-item simultaneous measurement in a folded state before use, and b is a use when the terminal cover of the biosensor for multi-item simultaneous measurement in a folded state is removed along the perforation Example, c is an example of use when the terminal cover of the biosensor for simultaneous measurement of multiple items in the folded state is folded along the perforation, and d is for the biosensor for simultaneous measurement of multiple items in the folded state. An example of use when the terminal cover is folded along the perforation is shown. FIG. 19 shows an example of a biosensor for simultaneous multi-item simultaneous measurement according to the present invention. a shows an example in which the multi-item simultaneous measurement biosensor is cut off from the connection sheet along the perforation, and b shows an example of the connection sheet made up of the multi-item simultaneous measurement biosensor. FIG. 20 shows another example of the biosensor for simultaneous multi-item simultaneous measurement according to the present invention. a shows an example in which the multi-item simultaneous measurement biosensor is cut off from the connection sheet along the perforation, and b shows an example of the connection sheet made up of the multi-item simultaneous measurement biosensor. FIG. 21 shows an example of a multi-item simultaneous measurement biosensor using a flexible sheet according to the present invention. a is an example in which a multi-item simultaneous measurement biosensor is cut along a perforation provided on a soft sheet, and b is an example of a multi-item simultaneous measurement biosensor connected by a soft sheet. FIG. 22 shows an example of a sealed multi-item simultaneous measurement biosensor according to the present invention. a is an example of the outside of the substrate, b is an example of the bonding surface side of the substrate having the wiring pattern, c is an example of the outside of the cover, d is an example of the bonding surface side of the cover having the spacer, e is the substrate and the cover Is an example of a plan view of a biosensor for simultaneous measurement of multiple items, wherein f is an example of an enlarged sectional view taken along line AA ′ of e, and g is an example of an enlarged sectional view taken along line BB ′ of e. h shows how the biosensor is viewed from the side. i shows a usage example of a biosensor for simultaneous measurement of multiple items as seen from the front. FIG. 23 shows an example of the operation of the multi-item simultaneous measurement biosensor according to the present invention. a is an example of a biosensor before being used for measurement, b is an example of introducing a sample solution into the biosensor after the biosensor is connected to the connector, c is an example of a biosensor after introduction of the sample solution, and d is after measurement An example of a biosensor is shown. FIG. 24 shows an example of the operation of the connector for connecting the multi-item simultaneous measurement biosensor according to the present invention. a is an example of the connector in an open state before connecting the biosensor, and b is an example of the connector after connecting the biosensor. FIG. 25 is a graph showing the result of measuring the glucose concentration in whole blood by the biosensor for simultaneous measurement of two items according to the present invention, divided into left and right biosensor responses. FIG. 26 is a graph showing the results of examining the storage stability of the biosensor for two-item simultaneous measurement according to the present invention separately for the left and right biosensor responses. FIG. 27 is a graph showing the results of examining the response of the biosensor for two-item simultaneous measurement according to the present invention with a mixed solution of glucose and lactic acid. FIG. 28 shows another example of the operation of the sealed multi-item simultaneous measurement biosensor according to the present invention. a is an example of a biosensor before being used for measurement, b is an example of introducing a sample solution into the biosensor, c is an example of the biosensor after introduction of the sample solution, and d is a biosensor after introduction of the sample solution The example which looked at from diagonally forward is shown. FIG. 29 is a graph showing the results of measuring the glucose concentration in whole blood by the sealed two-item simultaneous measurement biosensor according to the present invention, divided into left and right biosensor responses. FIG. 30 is a graph showing the results of examining the storage stability of the sealed two-item simultaneous measurement biosensor according to the present invention divided into left and right biosensor responses. FIG. 31 shows another example of the operation of the multi-item simultaneous measurement biosensor according to the present invention. a is an example of a biosensor before being used for measurement, b is an example of introducing a sample solution into the biosensor after the biosensor is connected to the connector, c is an example of a biosensor after introduction of the sample solution, and d is after measurement An example of a biosensor is shown. FIG. 32 is a graph showing the results of measuring the glucose concentration in whole blood by the sealed two-item simultaneous measurement biosensor according to the present invention, divided into left and right biosensor responses. FIG. 33 is a graph showing the results of examining the storage stability of the sealed two-item simultaneous measurement biosensor according to the present invention separately for the left and right biosensor responses. FIG. 34 is a graph showing the results of examining the response of the sealed two-item simultaneous measurement biosensor according to the present invention with a mixed solution of glucose and lactic acid.

Explanation of symbols

1 Substrate 2 Cover 3 Spacer (adhesive layer)
4 Pattern including electrodes 5 Spacer space (sample transport path)
6 Reagent layer (reaction layer)
7 Groove 8 Terminal 9 Sample inlet 10 Air outlet 11 Sample liquid 12 Broken line 13 showing fold part Resist 14 Desiccant 15 Biosensor 16 for simultaneous measurement of multiple items Measuring instrument (connector)
17 Introduction part 18 Horizontal movement part 19 Guide 20 Folding part upper part 21 Folding part lower part 22 Wiring 23 Electrode 24 Air discharge port 25 Perforation 26 Soft sheet for connection
27 Biosensor unit 28 Biosensor unit-containing substrate 29 Auxiliary tool 30 Auxiliary tool fixing part 31 Upper outer fold (air outlet opening)
32 Two-item simultaneous connector 33 Base 34 Lid 35 Folder 36 Presser 37 Wiring

Claims (16)

Having a substrate and a cover coupled to the substrate via a spacer layer;
A plurality of biosensor unit-containing substrates including at least one biosensor unit having one reaction detection unit including one electrode system and one reagent layer and one sample transport path including the reagent layer on the substrate. A biosensor for simultaneous measurement of multiple items including
Each biosensor unit includes one reagent layer on one sample transport path,
A cutting line for dividing each biosensor unit-containing substrate is provided on the substrate or the cover surface,
When the substrate or cover is cut along the cutting line, a sample introduction port for introducing a sample liquid into the sample conveyance path is opened as a cut opening of the sample conveyance path on the cut surface of each biosensor unit-containing substrate. As described above, the multi-item simultaneous measurement biosensor is characterized in that a cutting line and a sample transport path are arranged.   A sample conveyance path is provided so that a sample introduction port opens on the cut surface, and an air discharge port is provided on the substrate or cover surface, or on a side surface of the biosensor unit-containing substrate different from the cut surface. The biosensor for simultaneous measurement of multiple items according to claim 1. The sample transport path is sealed,
A first cutting line that separates each of the biosensor unit-containing substrates, and a second cutting that is different from the first cutting line for exposing the air discharge port by cutting a part of the substrate and the cover Lines are provided on the substrate and the cover surface;
When the substrate or cover is cut along the first cutting line, the sample introduction port opens as a cutting port on the first cutting surface,
When the substrate or cover is cut along the second cutting line, the air discharge port opens as a cutting port on the second cutting surface.
The biosensor according to claim 1, wherein the first and second cutting lines and the sample transport path are arranged.   The substrate or cover surface has an auxiliary tool provided to bend the substrate and the cover along the second cutting line in conjunction with the folding of the substrate and the cover along the first cutting line. The biosensor according to claim 3.   A sample transport path is provided so that both the sample introduction port and the air discharge port are opened on the cut surface of each biosensor unit-containing substrate, and the sample transport path is arranged in a sealed state before cutting. The biosensor for simultaneous measurement of multiple items according to claim 1.   The multi-item simultaneous measurement according to any one of claims 1 to 5, wherein a sample transport path is arranged so that one sample inlet is formed for one biosensor unit. Biosensor. The multi-item simultaneous measurement biosensor according to claim 1 , wherein the electrode system forms an array. The method for using the biosensor for multi-item simultaneous measurement according to any one of claims 1 to 7 , comprising the following steps:
(1) The substrate and the cover are bent along a cutting line that divides the biosensor unit-containing substrate, the substrate or the cover is cut, and a sample transport serving as a sample introduction port is formed on the cut surface of each biosensor unit-containing substrate. Opening the road cut-off ,
(2) fixing the shape of the bent biosensor unit-containing substrate in order to maintain the open state of the sample inlet;
(3) A step of bringing the opened sample introduction port into contact with a solution containing the measurement object, and (4) a step of introducing the solution containing the measurement object into the sample conveyance path. In step 1, either one of the substrate and the cover is cut and folded while the other is connected to expose the cut surface.
The method according to claim 8 , wherein the step 3 is performed while the multi-item simultaneous measurement biosensor is bent. The method according to claim 8 or 9 , wherein in the step 3, sample introduction ports of two or more biosensor unit-containing substrates are brought into contact with the solution at a time. Wherein comprises a biosensor unit-containing substrate is 2 or more biosensor unit, in the step 3, the sample inlet of a biosensor unit containing substrate, to claim 8, characterized in that contacting the solution at a time The method described. The measuring method of the measuring object including the following steps using the biosensor for multi-item simultaneous measurement according to any one of claims 1 to 7 ;
(1) The substrate and the cover are bent along a cutting line that divides the biosensor unit-containing substrate, the substrate or the cover is cut, and a sample transport serving as a sample introduction port is formed on the cut surface of each biosensor unit-containing substrate. Opening the road cut-off ,
(2) fixing the shape of the bent biosensor unit-containing substrate in order to maintain the open state of the sample inlet;
(3) a step of bringing the opened sample introduction port into contact with a solution containing a measurement object;
(4) A step of introducing a solution containing the measurement target into the sample conveyance path, and (5) a step of measuring the measurement target with each biosensor. The biosensor according to any one of claims 1 to 7 ,
A connector for capturing an electrical signal at the electrode of the biosensor;
A measurement unit for measuring an electrical value via the connector unit;
A display unit for displaying a measurement value in the measurement unit;
A biosensor device comprising a memory unit for storing the measurement value. The connector part deforms the shape of the biosensor unit-containing substrate for opening the sample introduction port, and then fixes the biosensor unit-containing substrate with the shape to capture an electrical signal at the electrode of the biosensor. 14. The biosensor device according to claim 13 , which is a structure. The biosensor device according to claim 13 or 14 , wherein a measurement method in the measurement unit is a potential step chronoamperometry method, a coulometry method, or a cyclic voltammetry method. A connector to capture the electrical signal by fixing the biosensor according to any one of claims 1 to 7,
The connector is a sensor shape fixing part for fixing a shape of the biosensor unit-containing substrate folded for opening the sample introduction port,
A biosensor connector having an electrical connection portion and wiring for capturing an electrical signal on an electrode of the biosensor from an electrical signal on the biosensor.