JP4399581B2 - Biosensor - Google Patents

Description

  The present invention relates to a biosensor. More specifically, the present invention relates to a biosensor having a structure capable of maintaining the airtightness inside the reaction detection unit until it is used. The present invention also relates to a biosensor package. Furthermore, the present invention relates to a method for using a biosensor and a biosensor device.

  In the conventional disposable sensor packaging form, as a method of using a container, a method of storing a plurality of biosensors in a bottle container, storing one biosensor in one container one by one, and opening the container entrance There is a method of thermocompression bonding with a film (Patent Document 1). In these systems, it is possible to keep a dry state by putting a desiccant or the like in the container.

  However, in the case of the former method, the number of times of opening and closing increases each time it is used, and the dehumidifying ability of the desiccant decreases due to moisture at the time of opening. Such a packaging form is not suitable for use under humid climatic conditions as in Japan. Further, in order to keep the storage state of the biosensor in good condition, it is necessary to block ultraviolet rays and oxygen, but such measures are not taken.

  In the case of the latter method, since biosensors are stored one by one in the container, a large amount of materials must be used for packaging. From the viewpoints of effective use of limited resources and waste disposal, It is not a friendly packaging form.

  In addition, an attempt has been made to wrap the biosensor by sandwiching the biosensor with a desiccant between two films that are coated with a UV absorber or UV opaque material on the surface and then bonding them by thermocompression bonding from the outside of the film. (Patent Document 2).

  However, in this method, heat is applied at the time of packaging. Therefore, there is a risk of deformation of the biosensor main body, deterioration of the chemical substance developed in the reagent layer, and denaturation of the biomaterial due to the heat during processing and the accompanying thermal oxidation. In addition, in order to maintain a certain wet state, it is necessary to consider the influence of heat vapor pressure. Moreover, since the oxygen removing agent is not included in the packaging form, there is an influence of air oxidation during the storage period due to packaging in the air. Furthermore, in a package formed by thermocompression bonding, the bonding portion is strongly bonded with two films, so that it cannot be easily opened. For this reason, when a person with a physical disability, an elderly person, a low-aged person, etc. open such a package, it is anticipated that difficulty will be forced depending on the case.

JP 2000-314711 A JP 2003-72861 A

  In the conventional method, first, the bottle container method has a problem that the internal dry state cannot be maintained by repeated opening and closing. Further, in the method of packaging a single biosensor in a container, there is a problem that a packaging process is complicated and a large amount of material is required because a container that is bulky with respect to the size of the biosensor is used. Furthermore, in the method of packaging a single biosensor by thermocompression bonding with two films, it is impossible to eliminate the effects of heat and oxidation even if UV rays are blocked and the dry state can be maintained by using a desiccant. Also, there was a problem in operability when opening the package.

  That is, the present invention realizes a mild manufacturing process suitable for the use of biomaterials, such as the manufacturing process after the development of the reagent layer in the reaction detection section and the case where simple packaging is required, such as no heat. Another object of the present invention is to provide a biosensor capable of reliably blocking the inside of the reaction detection unit from the outside world until it is used.

The present invention has been made in view of the above problems, and the inventors of the present invention are a biosensor in which a reaction detection unit including at least one pair of electrodes and a sample transport path are sealed, and the biosensor is used when the biosensor is used. By cutting a part of the structure and adopting a structure in which the sample inlet and air outlet are exposed as a cross section of the sample transport path, the biosensor before use can completely contain the reaction detection unit. The present invention has been completed by finding that it has excellent sealing properties and can be stored in a desired state by using a desiccant or the like as required.
In such a biosensor, since the reaction detection unit and the like can be sealed by a process of joining the substrate and the cover via a spacer, the biosensor manufacturing process and packaging after forming the reaction layer on the reaction detection unit No heat treatment is involved in the process.
That is, the present invention includes the following.

[1] A biosensor according to the present invention comprises an electrically insulating substrate;
An electrically insulating cover bonded to the substrate via a spacer layer;
A reaction detection unit including at least one set of electrodes formed on the substrate in a region sandwiched between the substrate and the cover, and an external terminal connected to the reaction detection unit;
A biosensor having a sealed sample transport path defined by a spacer layer between the substrate and the cover,
The sample transport path has a portion intersecting with the electrode,
The outermost surface of the substrate and the cover is provided with a cutting line that borders a sensor part including an electrode and a sealing cap part not including an electrode,
A sample introduction port derived from the sample transport path on the cut surface, the cut surface does not cross the electrode when the hermetic cap portion is cut along the cutting line, and the cut surface crosses the sample transport path; The cutting line is present at a position where the air discharge port is exposed.

  That is, in the biosensor of the present invention, since the cut surface crosses the sample transport path, the two sections of the sample transport path in the sensor section are cut by cutting the sealing cap portion along the cutting line before use. One of the two exposed cross sections is a sample introduction port, and the other cross section is an air discharge port. The cutting method is not particularly limited, and the cutting can be performed by folding, cutting, or cutting along the cutting line.

  The sensor unit is a reaction including the substrate, the cover coupled to the substrate through a spacer layer, and at least one set of electrodes formed on the substrate in a region sandwiched between the substrate and the cover. A main body part of a biosensor having a sample conveying path, a sample introduction port, and an air discharge port defined by a spacer layer between the detection unit and the external detection unit connected to the reaction detection unit, and the substrate and the cover. is there. The sealing cap portion is a portion that does not include an electrode and is a portion that can be discarded by cutting.

  In the biosensor, the opening directions of the sample introduction port and the air discharge port are not particularly limited as long as they exist on the same cross section when the biosensor is used. Further, the sample introduction port and the air discharge port may be formed anywhere as long as they are inside the cross section that appears at the time of use at a position where the sample liquid can be injected into the sample transport path.

  In this specification, “on the same cross section” means that all of the sample introduction port and the air discharge port appear on the same cross section due to deformation of the biosensor during use. Moreover, although the shape of the side which comprises the cross section of a sensor part is dependent on the shape of the corresponding sealing cap part, it may be linear form or curvilinear form. In the shape of the cut surface, if the side near the sample inlet is curved, the risk of damaging the human body can be reduced, especially in blood glucose measurement where blood is collected from the body and used. it can.

  Furthermore, the shape of the said sealing cap part is not specifically limited, Preferably a rectangle, trapezoid, a triangle, etc. are mentioned.

The sample transport path is patterned by the spacer layer. Examples of the spacer layer include an adhesive layer or a spacer layer having an adhesive layer in which an adhesive is applied to both sides of the spacer. Therefore, the spacer layer adheres the substrate and the cover and defines the sample transport path.
The electrodes are at least one set of electrodes in which a + electrode and a − electrode face each other.
Such an electrode may be composed of two electrodes consisting of + and −, or may be two or more.

  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.

  In such a biosensor of the present invention, the airtightness in the sample transport path including the reaction detection unit is maintained before the sealing cap unit is cut, and the state inside the biosensor has been maintained for a long time since the time of manufacture. In addition, the atmosphere inside the biosensor, specifically the gas composition (deoxygenated state), atmospheric pressure, humidity (wet state), and the like can be controlled to a desired constant atmosphere.

  For this reason, for example, even if the sample liquid cannot be smoothly introduced when the sample transport path of the biosensor is in a dry state, a surfactant or the like is uniformly applied to the inner wall of the sample transport path to maintain a constant humidity. By keeping this, 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.

  Furthermore, it is preferable that the sample transport path is connected at least between 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.

The substrate is not particularly limited as long as it is electrically insulating, and as such a substrate, for example, any one of plastic, biodegradable material, and paper 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. When used in the form of a sheet, polycarbonate, hard polyvinyl chloride, polyethylene terephthalate, acrylic resin, ABS resin and the like can be preferably used.
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.

  The cover can 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.

The spacer can be made of the same material as that of the substrate. In this case, an adhesive is applied to the spacer surface and bonded to the substrate and the cover with an 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.
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 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.

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

  [2] Preferably, the cutting line is formed by a groove or a cut, and the groove or the cut is disposed 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. Further, when the cutting lines are arranged so as to face the same position, cutting can be easily performed.
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.
In the present specification, “A and / or B” means at least one of A and B.

  [3] The substrate and the cover may each have a multilayer structure of at least two layers, and the cutting line may be formed leaving at least the innermost layer of the multilayer structure.

  When the substrate and the cover each have a multilayer structure of at least two layers, cutting lines, grooves, perforations, etc. are formed leaving at least the innermost layer of the multilayer structure. Furthermore, it is preferable that these grooves and perforations 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 can be formed without damaging the inner layer portion by damaging 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.

  [4] It is preferable that a reagent layer is provided in a region where the sample transport path and the electrode intersect.

In the biosensor according to the present invention, the sample sent from the sample introduction port through the sample conveyance path by capillary action comes into contact with the reagent layer on the electrode serving as the reaction detection unit, whereby the reagent and the sample react. This reaction is monitored as an electrical change at the electrode. One or a plurality of such reagent layers can be present on the electrode 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.

  Since the biosensor of the present invention is excellent in hermeticity before use, the reagent layer can also maintain a constant humidity. Therefore, even when oxygen is present in the atmosphere inside the biosensor, the reagents protected by moisture Deterioration or denaturation due to air oxidation can be suppressed.

  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 oxidase, peroxidase, glucose dehydrogenase, lactate Dehydrogenase, alcohol dehydrogenase, cholesterol esterase, inorgano pyrophosphatase, acid phosphatase, alkaline phosphatase, nucleotide triphosphatase, nucleotide diphosphatase, nucleotide monophosphatase, inositol phosphatase, protein phosphatase, adenosine・ Triphosphatase, guanosine triphosphatase, adenosine-5′-diphosphatase, casein phosphatase, tyrosine phosphatase, serine phosphatase, threonine phosphatase, maltose phosphorylase, sucrose phosphorylase, purine nucleotide phosphorylase, adenyl cyclase, guanylate cyclase Glucose isomerase, mutarotase, catalase, protease, DNA polymerase, RNA polymerase, DNA ligase, DNase, NADH oxidase, diaphorase, osmium-peroxidase complex and other nucleic acid-linked enzymes, restriction enzymes and the like.
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. This mediator is selected from potassium ferricyanide, ferrocene, and benzoquinone. The reagent layer may contain a combination of inorganic salts such as sodium chloride and potassium chloride and quinhydrone.

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

  When 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.
A convex partition can be provided between them. The reagent layer is not limited to one place, and two or more places can be provided. In that case, two or more kinds of different reagent layers may be provided.
The reagent layer may be mixed with an adhesive or the like used as a spacer material.

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

[5] A part of a region sandwiched between the substrate and the cover may contain a desiccant and / or an oxygen scavenger.
[6] It is preferable that the desiccant and / or oxygen scavenger is included in the sealing cap part.

  That is, since the biosensor of the present invention is hermetically sealed with the reaction detector included therein, such a desiccant and / or oxygen scavenger is contained in the biosensor, so that the inside of the biosensor is in a dry state or no state. The oxygen 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 present inside the hermetic cap portion that becomes unnecessary after cutting in order to avoid direct contact with the sample solution. Moreover, even if it exists in the sealing cap part, since the sealing cap part and the sensor part are connected inside via the spacer or the reagent transport path, in the structure of the biosensor in the storage state before use, the substrate and The entire internal space of the spacer existing between the covers can be dried or deoxygenated. In particular, by arranging the desiccant and / or oxygen scavenger so as to intersect the sample transport path existing in the hermetic cap part, the internal space of the biosensor is dried and / or deoxygenated through the sample transport path. Can be kept in.

  In addition, when the biosensor is used, the sealing cap portion provided with the desiccant and / or oxygen scavenger is cut and detached, so that the sample liquid comes into contact with the desiccant and / or oxygen scavenger when using the biosensor. There is nothing to do.

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.

  [7] Furthermore, the biosensor of the present invention may contain a humidity indicator and / or an oxygen detector in a part of the region sandwiched between the substrate and the cover.

Before use, a desiccant and a humidity indicator may be used together, or a deoxygenator and an oxygen detector may be used in combination so that the dry state and / or deoxygenated state inside the biosensor can be confirmed.
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.

  [8] 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 is present in the spacer part of the cross section that newly appears by cutting the sealing cap part. It can also be arranged. 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.

[9] It is preferable that the substrate and the cover are made of an ultraviolet opaque material.
[10] 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.

  [11] 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 film 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 biosensor surface by the incidence of ultraviolet rays and / or visible light, thereby obtaining self-cleaning actions such as sterilization and degradation of proteins with capsids and envelopes made of proteins, and degradation of dirt attached to the surface. Can always be stored in a hygienic state. 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.
[12] The spacer layer existing in the vicinity of the exposed sample inlet and 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.

[13] The electrodes may form an array.
[14] The biosensor forming the array is:
When the sealing cap part is cut along the cutting line, at least one sample introduction port is exposed,
It is preferable that a reaction detection unit including at least one pair of electrodes is present at the tip of the sample transport path communicating with the sample introduction port.
[15] The at least one sample introduction port communicates with at least two sample conveyance paths branched from the sample introduction port, and a reaction detection unit including at least one pair of electrodes exists at the tip of the sample conveyance path. You may do it.

  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.

[16] The substrate and / or cover having a material transparent to visible light may be covered with a protective film.
In this case, visibility can be ensured with respect to an oxygen detection agent, a humidity display agent, and the like arranged in the spacer layer of the hermetic cap. The protective film can help to prevent the influence of visible light and ultraviolet light on the reagent layer of the biosensor in that case.
[17] The external terminal may be covered with a protective film.

  The protective film covers an electrode terminal, a connecting portion of a measuring instrument, and the like that are exposed on the same surface of the biosensor with a protective film as needed until use. Furthermore, the protective film may be a single layer or a multilayer structure.

  Such a protective film may have a part having a peelable adhesive layer and a non-adhesive part. This non-adhesion part can be used for peeling of a protective film as a knob part.

  The part having the peelable pressure-sensitive adhesive layer is usually called a peel seal or a weak seal part, and can be easily peeled off by a certain pulling force.

The protective film preferably has a characteristic of blocking at least one of moisture, ultraviolet rays, and oxygen.
Examples of the material for such a protective film include plastic films, and among the plastic films, polyvinylidene chloride, polyethylene, polyester, nylon, ethylene-vinyl alcohol copolymer, fluorine-based resin, and the like are preferable. Such plastic has flexibility and excellent moisture barrier.
Further, it is preferable to use a protective film containing the ultraviolet absorber or the ultraviolet non-transparent substance for blocking ultraviolet rays.
For blocking oxygen, it is preferable to use a protective film containing the desiccant, oxygen scavenger and the like.

  [18] The external terminal may be covered with the cover, and the cover may have a folding line that can be folded back so that the external terminal is exposed.

In this case, in the structure before use of the biosensor of the present invention, since the terminals are also stored in the cover, that is, inside the biosensor main body, packaging of the main body and simple packaging with a protective film are unnecessary.
Examples of the folding line include a perforation, a groove, a cut, and a dent. Furthermore, the operation can be further facilitated by providing two perforations or the like of the folded or folded portion in parallel.
Further, in order to facilitate folding or folding, it is desirable that the adhesive layer used for forming the spacer is not present in the folded or folded portion.

An adhesive that can be attached to and detached from the substrate can be provided in at least one place on the inner surface of the folded or folded portion of the cover. By having a detachable adhesive, the cover can be adhesively fixed to such an extent that the terminal portion of the biosensor in a stored state is not exposed by the folding of the cover or the force naturally applied to the folded portion. Thereby, the shape of the biosensor in the storage state can be kept stable.
In this case, it is preferable to arrange the removable adhesive so as not to contact the terminal so as not to affect the function of the terminal as a conductive material, such as the terminal coming into contact with the removable adhesive and peeling off from the substrate.

  Furthermore, by providing two perforations or the like in parallel on the folded portion or folded portion of the cover, the two cover pieces can be folded one on top of the other. In that case, it is desirable to arrange the removable adhesive inside the cover piece that covers the terminal end portion of the two cover pieces. Thereby, the shape of the biosensor in a preserved state can be kept stable, and the removable adhesive can be used to bond and fix the two cover pieces when folded together.

  [19] A biosensor package according to the present invention houses a plurality of the biosensors.

  More specifically, a plurality of the simply packaged biosensors of the present invention can be packaged together by a bottle container method or a box container method.

  Further, the plurality of biosensors of the present invention can be accommodated in a state in which they are arranged in a container by a box-type container method or the like, and when the biosensor is sequentially removed from the container, the main body or the protective film is individually The biosensor serial number or the remaining number of biosensors in the container can be printed.

  [20] A plurality of the biosensors may be regularly arranged at predetermined intervals, and perforations for separation may be provided on the connected biosensor substrates.

  With this configuration, a large number of biosensors can be efficiently manufactured at one time. Further, by connecting each of the connected biosensors to the measurement unit, it is possible to provide a measurement apparatus that can measure a large number of specimens simultaneously. Furthermore, in this configuration, since a plurality of biosensors are regularly arranged at predetermined intervals, individual biosensors can be rotated and moved sequentially to be connected to a measuring instrument. With this type of measuring apparatus, it is possible to continuously and automatically measure a large number of specimens. Further, by providing perforations or the like on the connected substrates, the storage space can be reduced, the connected biosensors can be bent, and individual electrodes can be separated.

  [21] The method of using the biosensor according to the present invention is characterized in that, in the biosensor, a sealing cap is cut to form a sample introduction port and an air discharge port.

[22] More preferably, the method of using the biosensor according to the present invention is a method including the following steps using the biosensor;
(1) cutting the biosensor sealing cap to open the sample inlet and the air outlet;
(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.

  Since the sample introduction port and the air discharge port are included in the biosensor as a sample transport path during manufacture, the biosensor is shipped in an airtight state and does not include the biosensor electrode when used. By separating a part, the sample introduction port and the air discharge port are formed and exposed for the first time as a cross section of the sample transport path, and can be used.

[23] The biosensor device according to the present invention comprises the biosensor,
A measurement unit for measuring an electrical value in the reaction detection unit of the biosensor;
A display unit for displaying a measurement value in the measurement unit;
And a memory unit for storing the measured values.

  Usually, the measurement unit is connected to the biosensor via a connector unit that captures an electrical signal at the electrode of the biosensor, and an electrical value is measured by the measurement unit via the connector unit.

  The connector is a connector for fixing the biosensor and capturing an electrical signal, a sensor fixing portion (folder) for inserting and fixing the biosensor, an electrical connection portion for capturing an electrical signal at an electrode of the biosensor, And having wiring. The connector may be either a built-in connector in the measuring section or a type that is used as a single unit connected to an electrochemical measuring instrument.

[24] It is preferable to use any one of potential step chronoamperometry, coulometry, and cyclic voltammetry as a measurement method in the measurement unit.
[25] Furthermore, it is preferable that the biosensor includes a wireless means for sending measurement data to the measurement unit, and the wireless means is a non-contact IC card or Bluetooth.

  The biosensor according to the present invention can be used as follows by changing the type of the 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 the microbial sensor, 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 Fusarium oxysporum f. Sp. Lycopersici, Fusarium oxysporum f. Sp. Lactucum, Fusarium oxysporum f. Sp. (Measurement target: soil) is used. Since these microorganisms breathe oxygen (aerobic) or produce metabolites in an oxygen-free environment, they will capture oxygen respiration or metabolites electrically.

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

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

  Tissue sensors use animal and plant tissues as molecular identification elements. 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 example of the application of the biosensor described here is a DNA chip (Japanese Patent Laid-Open No. 5-199898). Many kinds of single-stranded nucleic acid probes having complementarity to many kinds of target genes to be detected are immobilized on an array of electrodes (US 4225410) as shown in FIGS. 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 can be made. In addition, as said double strand recognition body, metallointercalators, such as intercalators (inserting agent), such as acridine orange, a tris (phenanthroline) cobalt complex, etc. can be used.

The biosensor 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, 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, 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 adjusting agent and the indicator inside the sample transport path and the sealing cap.

  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 having a boundary line with the sealing cap 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 enclose a sample conveyance path serving as a sample introduction port and an air discharge port inside the biosensor, and can keep the inside highly airtight.

  The biosensor of the present invention does not need to be manufactured by a method such as thermocompression bonding, is easy to manufacture, and can improve the yield, so that the influence of the oxidation of the reagent layer can be eliminated and long-term storage stability can be achieved. Are better. In addition, the main parts of the biosensor such as the sample introduction port, the sample transport path, and the reaction detection unit are completely sealed after the manufacturing stage, and the atmosphere inside the biosensor is ensured to be highly sealed. Therefore, a desired internal atmosphere can be created by the control during manufacturing, and the state can be maintained for a long time. Furthermore, if necessary, it is possible not only to maintain the internal atmosphere over a long period of time in a better state by including a regulator for adjusting the internal atmosphere, such as a desiccant, as well as the biosensor structure of the present invention. If so, the internal atmosphere modifier contained therein is separated from the biosensor main body at the time of use, so that contact with the sample solution can be completely eliminated.

  In addition, when evaluating the configuration, materials, series of manufacturing methods, etc., the biosensor of the present invention can significantly reduce the environmental burden during manufacturing and after use, compared to conventional disposable biosensors. is there.

  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 the biosensor of the present invention. In FIG. 1, the cross section of the sensor portion 10 appears by cutting the sealing cap portion that does not include an electrode. At the same time, the cross section of the sample transport path is formed as a sample introduction port and an air discharge port. It is an example of the biosensor of the present invention exposed for the first time.

  FIG. 1a shows the outside of a rectangular substrate 1 of a typical biosensor. In the upper part of the substrate 1, a V-shaped groove 7 serving as a cutting line formed horizontally is provided. The groove 7 is for cutting the biosensor sealing cap portion 10 along the broken line 14 by bending or the like when the biosensor is used.

  FIG. 1 b shows the inside of the substrate 1. On the inner upper surface of the substrate 1, a pattern 4 and a reagent layer 6 including a set of electrodes are formed along the center line of the substrate. In the pattern 4 including electrodes, two electrode members are arranged in parallel from the lower end side to the vicinity of the upper broken line 14.

  FIG. 1 c shows the outer part of the cover 2. Similar to the substrate 1, a groove 7 formed horizontally is present along the broken line 14 at the top of the cover 2. FIG. 1 d shows the inside of the cover 2. An adhesive layer as a spacer layer is formed on the inner upper surface of the cover 2. A circular portion 5 having no spacer layer is provided on the cover 2, and a reagent transport path is formed by bonding to the substrate.

  FIG. 1e shows a configuration diagram when the inside of the substrate 1 and the cover 2 are overlapped with each other at the upper end. By bonding the substrate 1 and the cover 2, a portion without the spacer formed on the cover 2 becomes a space enclosed in the biosensor, which becomes the sample transport path 5. Further, by making the length of the cover 2 shorter than that of the substrate 1, the lower end portion of the pattern 4 including the electrodes can be exposed when the cover 2 is overlapped with the upper ends of both. This is the terminal 8 shown in FIG. Further, the sensor portion 9 and the sealing cap portion 10 exist with the groove 7 as a boundary.

  FIG. 8a shows the internal structure of the biosensor development of FIG. 1e with the cover 2 only removed. A broken line 14 indicating the cut portion of the biosensor is shown on the circular sample transport path 5 so as to divide it into two. That is, the broken line 14 shown in this figure overlaps with the groove 7 (see FIG. 1e) of the substrate 1 and the cover 2. Therefore, as shown in FIG. 1h, by cutting the biosensor sealing cap 10 along the groove 7 (broken line 14), the openings are exposed at two locations in the space provided as the sample transport path 5. . That is, two openings are exposed as the sample introduction port 11 and the air discharge port 12 in the cross section of the sensor unit 9. When the sample solution 13 is introduced from the sample introduction port 11, the sample solution 13 is transported in the sample transport path by capillary action, and reaches the reaction detection unit composed of the pattern 4 including two electrodes and the reagent layer 6. At this time, air corresponding to the volume of the sample liquid transferred to the sample transfer path is discharged from the air discharge port 12.

  FIG. 1f shows an AA ′ cross-sectional view of a portion slightly below the groove 7 in the sensor portion 9 of the biosensor shown in FIG. 1e. Two patterns 4 including electrodes are arranged on the substrate 1, and an adhesive layer exists as a spacer 3 between the substrate and the cover, covering the periphery of the electrodes. Sample transport paths 5 are provided on both sides of the electrode spacer layer. FIG. 1g shows a BB ′ cross-sectional view on the electrode pattern of the biosensor shown in FIG. 1e. V-shaped grooves 7 are provided on the outer portions of the substrate 1 and the cover 2 so as to face each other and overlap each other. On the substrate 1, a pattern 4 including electrodes extends to the front of the V-shaped groove 7, and there are two spacers 3 and two sample transport paths 5 between the substrate 1 and the cover 2. The lower sample transport path 5 is on the electrode, and the upper sample transport path 5 is above the V-shaped groove 7, that is, at a portion to be cut when the biosensor is used.

In the case of the biosensor structure of FIG. 1, the reaction detection unit including the space that forms the sample transport path 5 and the pattern 4 including the two electrodes is completely included in the biosensor, and the internal airtightness is maintained. I understand that I can do it.
In addition, when the biosensor of the present invention is used, the sample transport path 5 forms a semicircular shape when viewed from above (FIG. 8a), so that the reagent layer on the two electrodes, that is, the reaction detection unit It is possible to smooth the transportation up to.

Furthermore, if the biosensor has such a structure, the sample solution may be in a very small amount, the structure is simple, the manufacture is easy, and the reaction detection unit is included in the biosensor so that it is airtight. Therefore, it can be maintained until it is used in a state of being processed at the time of manufacture.
In addition to the features of the biosensor common to the present invention described above, features of each shape of the biosensor proposed in the present invention will be described below.

2 is substantially the same as the biosensor structure of FIG. 1 on the outside, but the internal structure is different.
In FIG. 2a, the wiring portion including the terminal of the pattern 4 including the electrode is provided so as to be slightly on the right side from the center on the substrate, and the electrode portion for detecting the reaction is disposed obliquely upward on the left side. 6 is the same as the position shown in FIG. 1a, and is formed just below the broken line 14 on the center line of the substrate.

  FIG. 2 b shows the inside of the cover 2. On the inner upper surface of the cover 2, a spacer layer and a portion 5 in which no spacer exists are provided in a trapezoidal shape with an acute angle facing downward. FIG. 2c is a configuration diagram when the inside of the substrate 1 and the cover 2 are overlapped with each other at the upper end, and is an example in which the terminals on the substrate 1 are exposed at the lower end. FIG. 8b shows the internal structure excluding only the cover 2 in FIG. 2c. In FIG. 8b, the sample transport path 5 extends obliquely upward from an acute angle of the trapezoid, and a portion perpendicular to the electrode in the meantime overlaps with the broken line 14 in the upper part. That is, the portion where the broken line 14 and the obliquely extending sample transport path 5 intersect (the portion 26 serving as the sample introduction port) becomes the sample introduction port 11 of the sample liquid 13 as shown in the usage example of FIG.

  Therefore, from the sample introduction port 11 through the sample conveyance path 5 to the air discharge port 12, the sample conveyance path 5 has an acute angle of less than 90 degrees (inner angle connecting the sample introduction port 11, the bent portion 25, and the air discharge port 12). ) Bend. The reagent transport path from the sample introduction port to the bent portion 25 is linear (the sealed sample transport path is triangular or trapezoidal).

Incidentally, in this structure, the cross-sectional views shown in FIGS. 2d and e are the same as FIGS. 1f and 1g.
In this biosensor, after the sample liquid 13 passes through the electrode portion intersecting the sample conveyance path, the wall surface necessary for capillary action is partially interrupted at the portion that bends at an acute angle. Can be stopped. For this reason, measurement with a smaller amount of sample solution can be performed.
If not stopped, a material with high water repellency can be provided as a stopper at a portion to be stopped by printing or the like, or a material with high water repellency can also be applied to the wall of the sample transport path from an acute angle.

FIG. 3 shows an example in which the reagent transport path from the portion 26 serving as the sample introduction port of the sample transport path to the bent portion 25 is curved (the sealed sample transport path is fan-shaped) in the biosensor structure of FIG. Show.
Since it is curved, the sample liquid is smoothly introduced up to the point where the electrode portion intersecting the sample transport path passes, and stops at the bent portion of the sample transport path ahead, so that the amount of the sample liquid can be reduced.

  In the case of this structure as well as in FIG. 2, the portion that becomes the fan-shaped sample transport path 5 shown in FIG. 3b is arranged in two directions extending vertically by drawing an arc of one fan at the lower end and drawing an arc. (See also FIG. 8c). Among them, it has a structure that intersects with the reaction detection part with the electrode around the center of the arc. Therefore, as shown in the usage example of FIG. 3f and FIG. 8c, after the sample liquid 13 passes through the electrode portion 4 intersecting with the sample transport path 5, the wall portion necessary for the capillary phenomenon is one at the bent portion 25 that bends substantially at a right angle. In order to cut off the part, the transport of the sample liquid almost stops near this acute angle.

  As shown in FIG. 4b, the biosensor shown in FIG. 4 is not a pattern in which the blank portion of the spacer forms a capillary tube, but as shown in FIG. As an example of a structure in which the sample inlet 11 (FIG. 4f) appears widely in the center. As shown in FIG. 8d, the spacer 3 is formed on both sides of the sample inlet so as not to come into contact with surrounding spacers. Thereby, the opening part formed on both sides of the sample introduction port with the spacer interposed therebetween can be used as the air discharge port 12.

  In the biosensor shown in FIG. 4, the sample solution 13 is taken in from the central portion of the cross section that appears when the hermetic cap portion is cut, and is developed on the reaction detection portion in the electrode 4. At this time, the sample liquid 13 introduced from the sample introduction port 11 can be evenly spread on the reaction detection portion of the sample transport path 5 by the air discharge ports 12 provided on both sides of the sample introduction port 11. By adopting such a structure, the sample solution can be reliably introduced into the reaction detection unit.

  FIG. 5 shows an example in which the V-shaped groove 7 serving as the cutting line is not perpendicular to the direction of the pattern 4 including the electrodes of the reaction detection section but obliquely enters as shown in FIG. 5a or 5c. Except for the direction, it is the same as the structure of FIG. In this case, the difference from FIG. 1 is that, as shown in FIGS. 5h and 8e, the distance from the sample introduction port 11 to the portion of the sample transport path 5 where the electrode 4 intersects is short. As a result, for example, when using a sample solution having individual differences in viscosity, such as blood, for measurement, there is a possibility that a temporal variation occurs in the transport of the sample solution due to a capillary phenomenon. This is an effective means for improving the reproducibility in such a case.

  FIG. 6 shows an example of a biosensor having a curved cutting line. 6 also has the same structure as FIG. 1 except for the shape of the cutting line. In this case, as shown in FIG. 6h, since the sample introduction port 11 exists in a curved cross section, for example, in the case where the sample introduction port 11 is used in direct contact with the human body, for example, the structure is taken into consideration for the user. . Further, as can be seen from FIG. 8f, since the distance from the sample introduction port 11 to the reaction detection unit including the electrode 4 is shorter than that of FIG. 1 (FIG. 8a), the same effect as FIG. 5 can be expected.

FIG. 7 shows an example in which a desiccant 15 is built in the sealing cap 10. As shown in FIG. 7d, it is preferable to dispose the desiccant 15 above the broken line 14 that crosses the circular flow path serving as the sample transport path 5 so as to intersect the flow path. By arranging in this way, as shown in FIG. 7h, a desiccant is built into the sealing cap unit 10, and the desiccant further dries the atmosphere around the reagent layer through the sample transport path until use. Can keep. At the time of use, the desiccant 15 is detached together with the sealing cap 10, so that the sample solution 13 and the desiccant 15 do not come into contact with each other.
Here, an example in which the desiccant 15 is built in is shown, but other agents may be included in addition to or in place of the desiccant. Therefore, it may be a deoxygenating agent, a combined use with a deoxygenating agent, or a single use of a humidity indicator or an oxygen detecting agent, or a combined use with the above agent.

  FIGS. 8a to 8f show development views in which only the cover 2 of the biosensor shown in FIGS. 1 to 6 is removed as described above. FIGS. 8g to l show the case where the desiccant 15 is provided in the developed view with the biosensor cover 2 shown in FIGS. 1 to 6 removed. In the biosensor shown in FIGS. 8a to 1l, including the biosensor incorporating the desiccant, the inside of the sample transport path is completely cut off from the outside before use, and the internal airtightness is maintained. Further, a part of the sample transport path intersects with the electrode, and the sample transport path is cut at at least two places by cutting the sealing cap portion 10 not including the pattern 4 including the electrode from the broken line 14 portion, and a new sample is obtained. The introduction port and the air exhaust port are exposed in the cross section of the sensor unit 10.

FIG. 9 shows a case where the pattern of the sample transport path 5 changes to a straight line. In this shape, the sample liquid 13 is introduced from the sample introduction port 11, and the sample transport path 5 is linear.
In FIG. 9, the pattern of the sample transport path 5 is a straight line, but the pattern of the sample transport path 5 is square, and a cutting line is provided so as to cross two opposing flow paths of the square sample transport path 5. (Not shown).

  FIG. 10 shows an example of an array type biosensor. FIG. 10 a shows the outside of the substrate 1, a rectangular substrate 1, a groove 7 that is a V-shaped cutting line formed horizontally on the outer surface of the substrate 1, and a broken line 14 that shows the cutting position of the biosensor across the groove 7. It is shown. FIG. 10 b shows the inside of the substrate 1. On the inner upper surface of the substrate 1, two sets of electrodes 16 are arranged in an array along the vertical direction of the substrate, and wirings 17 from the respective electrodes are wired to the lower end portion of the substrate 1. A reagent layer 6 (not shown) is formed on at least one electrode of each set.

FIG. 10 c shows the outer part of the cover 2. Similar to the substrate 1, a groove 7 formed horizontally is present along the broken line 14 at the top of the cover 2. FIG. 10 d shows the inside of the cover 2. An adhesive layer as a spacer 3 is formed on the inner upper surface of the cover 2. On the cover 2, there is a trapezoidal portion 5 in which no spacer exists. FIG. 10E shows a configuration diagram when the insides of the substrate 1 and the cover 2 are overlapped with each other at the upper end, and the sensor portion 9 and the sealing cap portion 10 are separated by the groove 7 as a boundary. By bonding the substrate 1 and the cover 2, a portion without the spacer formed on the cover 2 becomes a space enclosed in the biosensor, which becomes the sample transport path 5. Further, by making the length of the cover 2 shorter than that of the substrate 1, the lower end portion of the electrode pattern 4 can be exposed when the cover 2 is overlapped with the upper end of both. This becomes the terminal 8 shown in FIG.
FIG. 10f shows an example of using the biosensor, and FIG. 10g shows a development view when the cover 2 is removed.

  In an array-shaped biosensor, different types of DNA sequences in the same sample solution can be detected simultaneously by immobilizing different reagents, particularly DNAs having different base sequences, as probes on each set of electrodes. . For example, in the case of single nucleotide polymorphism (SNPs) sensors (for example, A. Ahmadian et al., Biotechniques, 32, 748, 2002), a mixture of primers, DNA polymerase, deoxyribonucleotide triphosphate, etc. is used as a reagent. The change in pH in the vicinity of the electrode when the sample DNA and the primer complement each other is measured with the electrode.

  Similarly, by immobilizing a plurality of types of antibodies on each set of electrodes, it is possible to simultaneously measure a plurality of types of measurement objects in the same sample solution as an immunosensor. 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 addition to the array-shaped biosensor structure shown above, there are at least two sample introduction ports, and at the end of the sample transport path communicating with each sample introduction port, there is a reaction detection unit with at least one set of electrodes, Similar to the biosensor, there is a structure in which the sample introduction port appears by cutting the hermetic cap portion, or at least two sample solution conveyance paths branched from at least one sample introduction port. A structure in which a reaction detection unit including at least one pair of electrodes is present may be used.

  FIG. 11 shows a case where a protective film 18 for protecting the terminal 8 is attached when the terminal 8 is exposed to the front surface in the biosensor structure of the present invention. FIG. 11 a shows an example in which a protective film 18 is wrapped on a part of the biosensor cover 2 and the terminals 8. The protective film 18 in FIG. 11a is composed of a removable adhesive layer 19 and a knob 20 on which no adhesive layer is formed. A removable adhesive layer 19 is provided on a part of the cover 2, and the terminal 8 is an adhesive. It is covered with a knob 20 where no layer is formed. FIG. 11 b shows a use example of FIG. 11 a, showing a state where the protective film 18 is peeled off from the sensor unit 9 during use.

  In FIG. 11c, the upper end portion of the protective film is fixed to the upper surface of the biosensor cover 2 with a strong adhesive (protective film fixing portion 21), and the other portion is a protective film having no removable adhesive layer. 9 shows an example in which 9 terminals 8 are simply packaged. FIG. 11d is a usage example of FIG. 11c and shows the protective film in a state where the protective film fixing part 21 is peeled off.

  FIG. 12 shows the structure of a biosensor that protects the terminal 8 by using the cover portion 2 without using a protective film. In FIG. 12a, the terminal 8 is covered with a terminal protective cover 22 having no spacer, and a perforation 23 is provided at the boundary with the cover 2 fixed by the adhesive layer portion. FIG. 12 b shows an example of use of the biosensor shown in FIG. 12 a, in which the terminal 8 appears when the terminal protective cover 22 is turned over.

  FIG. 12c shows an example of a biosensor in which a perforation 23 is provided between portions of the terminal protection cover 22, and FIGS. 12d and 12e show usage examples of the biosensor of FIG. 12c. In FIG. 12d, the perforation of the terminal protection cover 22 is facilitated by providing a new perforation 23. In FIG. 12e, the terminal protection cover 22 is provided with a perforation 23, so that the perforation 23 can be folded at the boundary. In this case, it is possible to make it difficult to return the cover once folded by partially providing a removable adhesive layer inside the terminal protective cover 22.

FIG. 13 shows an example of a biosensor assembly sheet in which a plurality of biosensors 24 that do not require packaging are regularly arranged at a predetermined interval. A perforation 23 is provided as a boundary between the individual biosensors 24. With the articulated biosensor arranged in this way, simultaneous or continuous measurement is possible by introducing the test solution from each sample inlet. The number of sensor units disposed in the articulated biosensor is not particularly limited, but is preferably 20-30. As shown in FIG. 13, the sensor units can be arranged horizontally, or although not shown in the figure, the sensor units can be arranged vertically.
In addition, since the individual biosensors 24 can be folded by the perforation 23, the storage space is saved, and bending between the connected electrodes and separation of the individual electrodes are facilitated.

  FIG. 14 is a diagram showing an operation process in which a sealed biosensor for single item measurement according to an embodiment of the present invention is applied to glucose measurement. Further, this sensor is an example in which the terminal protection cover shown in FIG. 12C is provided on the sensor shown in FIG.

  FIG. 14a shows a biosensor before use for measurement, in which the reagent layer 6 is developed in the sample transport path 5, and b shows the sensor part 9 and the sealing cap part 10 with the groove 7 in a as a boundary. As shown in FIG. 12d, the biosensor with the sample transport path 11 and the air discharge port 12 opened by cutting is connected to the connector 27 after folding the terminal protective cover 22, and then the whole blood is sampled as the sample 13 into the sensor. An example of introduction, c is an example of a sensor after sample introduction, and d is an example of a sensor after measurement.

  In this sensor, glucose oxidase and potassium ferricyanide were used as the reagent layer. The measurement principle of the glucose sensor shown in FIG. 14a 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.
PET having a length of 40 mm, a width of 6 mm, and a thickness of 188 μm was used as the sensor substrate and the cover. Two carbon electrodes having a width of 1.3 mm were formed on the sensor substrate by a screen printing apparatus with an interval of 0.5 mm. A resist and an adhesive were also formed as a spacer layer by screen printing. The groove for cutting the sensor part and the sealing cap part was formed at a depth of 10 mm or more from the upper part of the sensor substrate so that the depth was more than half the thickness of the substrate and the cover. In the bent portion of the terminal protective cover, two perforations were made at 5 mm and 10 mm from the bottom of the sensor substrate.

  Although the sample amount was about 0.28 μl by design, when the actual sample amount was measured with whole blood (specific gravity: 1.05), 0.34 ± 0.023 mg (n = 10, coefficient of variation CV = 6.7%). 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.

  The reagent layer of enzyme and mediator was dissolved in distilled water to be 5.5 units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator), applied to the electrode surface, and vacuum dried to form on both electrodes. .

  The results of measuring blood glucose (blood glucose) using this glucose sensor will be described. Blood glucose measurement using this glucose sensor used whole blood with a hematocrit value of 40% prepared so that the glucose concentration was 0, 100, 300, or 500 mg / dl as the sample liquid. An electrochemical measuring instrument (ALS / CHI-1220, BAS) was used for the measurement, and the potential step chromoamperometry was used for the measurement. Twenty seconds after about 0.3 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 application was taken as the measured value.

  FIG. 15 shows a change in current value according to blood glucose concentration of the sensor of the present invention. Referring to FIG. 15, a current value change of 0.5 to 9 μA was observed in the range of blood glucose 0, 100, 300, 500 mg / dl (n = 3).

  Subsequently, the storage stability test of the present sensor in the state of FIG. 14a 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 relationship between the blood glucose concentration and the output current value is maintained as a correlation coefficient (r = 0.976 ± 0.0230) and slope (0.0107 ± 0.00134) even when stored indoors for 3 months. , Not much change.

FIG. 1 shows an example of a 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 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 an example of using the biosensor. FIG. 2 shows another example of the biosensor according to the present invention. a is an example of a bonding surface side of a substrate having a wiring pattern, b is an example of a bonding surface side of a cover having a spacer, c is an example of a plan view of a biosensor in which a substrate and a cover are bonded, and d is c E is an example of an enlarged sectional view of AA ′, e is an example of an enlarged sectional view of BB ′ of c, and f is an example of using a biosensor. FIG. 3 shows another example of the biosensor according to the present invention. a is an example of a bonding surface side of a substrate having a wiring pattern, b is an example of a bonding surface side of a cover having a spacer, c is an example of a plan view of a biosensor in which the substrate and the cover are bonded, d is a figure 3c shows an example of an AA ′ cross-sectional enlarged view, and e shows an example of an BB ′ cross-sectional enlarged view of c. f shows an example of using a biosensor. FIG. 4 shows another example of the biosensor according to the present invention. a is an example of a bonding surface side of a substrate having a wiring pattern, b is an example of a bonding surface side of a cover having a spacer, c is an example of a plan view of a biosensor in which the substrate and the cover are bonded, d is a figure 3c shows an example of an AA ′ cross-sectional enlarged view, e shows an example of an BB ′ cross-sectional enlarged view of c, and f shows an example of use of a biosensor. FIG. 5 shows another example of a 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 An example of a plan view of a biosensor in which e is attached, f is an example of an enlarged cross-sectional view of AA ′ of e, g is an example of an enlarged cross-sectional view of BB ′ of e, and h is an example of use of the biosensor Indicates. FIG. 6 shows another example of a 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 An example of a plan view of a biosensor in which e is attached, f is an example of an enlarged cross-sectional view of AA ′ of e, g is an example of an enlarged cross-sectional view of BB ′ of e, and h is an example of use of the biosensor Indicates. FIG. 7 shows another example of the 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 An example of a plan view of a biosensor in which e is attached, f is an example of an enlarged cross-sectional view of AA ′ of e, g is an example of an enlarged cross-sectional view of BB ′ of e, and h is an example of use of the biosensor Indicates. FIG. 8 shows an example of a configuration diagram when only the cover of the biosensor of the present embodiment is removed. However, in order to increase the visibility of the illustrated biosensor configuration diagram, all the configuration diagrams shown in FIG. 8 are shown as having an electrode disposed on the upper portion of the spacer layer. Covers the top surface. In addition, the configuration diagrams shown in FIGS. 9i and 10g are the same as those in FIG. 1 to 6 show the cases of FIGS. 1 to 6 in order, and g to i show the cases of the structure of FIGS. 1 to 6 in which the desiccant is included in the sealing cap. FIG. 9 shows another example of a 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 An example of a plan view of a biosensor in which e is attached, f is an example of an enlarged cross-sectional view of AA ′ of e, g is an example of an enlarged cross-sectional view of BB ′ of e, and h is an example of use of the biosensor Indicates. i shows an example of a configuration diagram when only the cover of the biosensor is removed. FIG. 10 shows an example of an arrayed biosensor as another example of the 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 The example of the top view of the biosensor which stuck together, f shows the usage example of a biosensor. g shows an example of a configuration diagram when only the cover of the biosensor is removed. FIG. 11 shows an example of packaging with a protective film in the biosensor according to the present invention. a is an example of packaging with a protective film comprising a removable adhesive layer and a knob portion where no adhesive layer is formed, b is an example of use of a, c is partially fixed with a strong adhesive, etc. Example of packaging with protective film of d, d shows an example of use of c. FIG. 12 shows an example of a biosensor without a package in the biosensor of the present invention. a and c are examples of biosensors with a terminal protection cover, b is an example of use where there is one perforation between the cover and the terminal protection cover, and d and e are perforations in the terminal protection cover. In the example of use when there is one, d indicates an example in which the terminal protective cover is folded, and e indicates an example in which the terminal protective cover is folded. FIG. 13 shows an example of an articulated biosensor that is a biosensor assembly sheet according to the present invention and has no packaging. FIG. 14 shows an example of the operation of the sealed single-item measuring 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. 15 is a graph showing the results of measuring the glucose concentration in whole blood using a sealed single-item measurement biosensor according to the present invention. FIG. 16 is a graph showing the results of examining the storage stability of a sealed single-item measurement biosensor according to the present invention.

Explanation of symbols

1 Substrate 2 Cover 3 Spacer 4 Pattern including electrode 5 Spacer space (sample transport path)
6 Reagent layer (reaction layer)
7 Groove 8 Terminal 9 Sensor part 10 Sealing cap part 11 Sample inlet 12 Air outlet 13 Sample liquid 14 Broken line 15 indicating the detached part Desiccant 16 Electrode 17 Wiring 18 Protective film 19 Peel seal part (adhesive layer)
20 Picking part (non-adhesive layer)
21 Protective film adhering portion 22 Terminal protective cover 23 Perforation 24 Fully packaged biosensor 25 Bent portion 26 Sample introduction port 27 Connector

Claims (25)

An electrically insulating substrate;
An electrically insulating cover bonded to the substrate via a spacer layer;
A reaction detection unit including at least one set of electrodes formed on the substrate in a region sandwiched between the substrate and the cover, and an external terminal connected to the reaction detection unit;
A biosensor having a sealed sample transport path defined by a spacer layer between the substrate and the cover,
The sample transport path has a portion intersecting with the electrode,
The outermost surface of the substrate and the cover is provided with a cutting line that borders a sensor part including an electrode and a sealing cap part not including an electrode,
A sample introduction port derived from the sample transport path on the cut surface, the cut surface does not cross the electrode when the hermetic cap portion is cut along the cutting line, and the cut surface crosses the sample transport path; The biosensor according to claim 1, wherein the cutting line is present at a position where the air outlet is exposed.   The biosensor according to claim 1, wherein the cutting line is formed by a groove or a cut, and the groove or the cut is arranged so as to face the same position of the substrate and the cover.   3. The bio according to claim 1, wherein the substrate and the cover each have a multilayer structure of at least two layers, and the cutting line is formed leaving at least the innermost layer of the multilayer structure. Sensor.   The biosensor according to any one of claims 1 to 3, wherein a reagent layer is provided in a region where the sample transport path and the electrode intersect.   The biosensor according to any one of claims 1 to 4, wherein a desiccant and / or an oxygen scavenger is included in a part of a region sandwiched between the substrate and the cover.   The biosensor according to claim 5, wherein the desiccant and / or oxygen scavenger is included in the sealing cap part.   The biosensor according to any one of claims 1 to 6, wherein a humidity indicator and / or an oxygen detector is included in a part of a region sandwiched between the substrate and the cover.   The biosensor according to claim 7, wherein 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 can be visible. .   The biosensor according to any one of claims 1 to 7, wherein the substrate and the cover are made of an ultraviolet light impermeable substance.   The biosensor according to any one of claims 1 to 9, wherein surfaces of the substrate and the cover are coated with an ultraviolet absorber or an ultraviolet non-transparent substance.   11. The substrate according to claim 1, wherein the substrate and the cover contain a compound having a photocatalytic action, or the surface of the substrate and the surface of the cover are coated with a film containing a compound having a photocatalytic action. The biosensor according to crab.   The biosensor according to any one of claims 1 to 11, wherein a fluorescent agent or a luminescent agent is contained in the spacer layer in the vicinity of the exposed sample inlet and the air outlet.   The biosensor according to claim 1, wherein the electrodes form an array. The biosensors forming the array are:
When the sealing cap part is cut along the cutting line, at least one sample introduction port is exposed,
The biosensor according to claim 13, wherein a reaction detection unit including at least one pair of electrodes is present at the tip of the sample conveyance path communicating with the sample introduction port.   The at least one sample introduction port communicates with at least two sample conveyance paths branched from the sample introduction port, and a reaction detection unit including at least one pair of electrodes exists at the tip of the sample conveyance path. 14. The biosensor according to 14.   The biosensor according to claim 8, wherein the substrate and / or cover having a material transparent to visible light is covered with a protective film.   The biosensor according to claim 1, wherein the external terminal is covered with a protective film.   The biosensor according to any one of claims 1 to 16, wherein the external terminal is covered with the cover, and the cover has a folding line that can be folded back so that the external terminal is exposed.   A biosensor package comprising a plurality of biosensors according to claim 1.   A biosensor according to claim 1, wherein a plurality of biosensors are regularly arranged at a predetermined interval, and a perforation for separation is provided on a substrate of the connected biosensors. Sensor assembly sheet.   The biosensor according to claim 1, wherein the sealing cap is cut to form a sample introduction port and an air discharge port. The method for using the biosensor according to any one of claims 1 to 18, comprising the following steps;
(1) cutting the biosensor sealing cap to open the sample inlet and the air outlet;
(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 a biosensor. The biosensor according to any one of claims 1 to 18,
A measurement unit for measuring an electrical value in the reaction detection unit of the biosensor;
A display unit for displaying a measurement value in the measurement unit;
A biosensor device comprising a memory unit for storing the measurement value.   24. The biosensor device according to claim 23, wherein any one of a potential step chronoamperometry method, a coulometry method, and a cyclic voltammetry method is used as a measurement method in the measurement unit. 25. The biosensor device according to claim 23, wherein the biosensor further includes a wireless unit that sends measurement data to a measurement unit, and the wireless unit is a non-contact type IC card or Bluetooth (registered trademark) .