Patent Application
20050247573
The present invention relates to biosensors.
More specifically, the present invention relates to biosensors comprising a structure that can keep the inside of a reaction-detecting section airtight until use; packagings thereof; methods for using the same; and devices thereof.
The present invention also relates to biosensors for simultaneously measuring multiple items, methods for using the same, and devices thereof, as well as methods for determining test compounds using the biosensors for simultaneously measuring multiple items.
To date, modes for packaging disposable sensors by using a container include systems where a number of biosensors are retained in a bottle container, and systems where each individual biosensor is retained in its own container and the opening of the container is thermally compressed using a film (Unexamined Published Japanese Patent Application No. (JP-A) 2000-314711). Dry conditions are maintained in such systems by placing a desiccant or such inside the containers.
However, during use, the former systems are opened and closed an increasing number of times, and thus the moisture-absorbing ability of the desiccant is reduced by the humidity of opening. Such packaging modes are unsuitable for use in humid weather conditions, such as those in Japan. Further, to properly preserve the biosensors, ultraviolet rays and oxygen must be blocked, and these modes of packaging do not do this.
In the latter systems, each biosensor is retained in its own container, thus using a lot of packaging materials. Accordingly, from the viewpoint of effective use of limited resources, and of waste management, such modes of packaging cannot be said to be environmentally friendly.
In addition, packaging methods are used, where biosensors with desiccants are sandwiched between two layers of film, coated with an ultraviolet absorber or a material that does not transmit ultraviolet, and these are adhered by thermal compression from outside the films (JP-A 2003-72861).
However, since this method applies heat at the time of packaging, heat during processing and the influence of thermal oxidation originating from this heat may alter the body of the biosensors, degrade chemical materials developed in reagent layers, and alter biomaterials. Further, it is necessary to consider the influence of heat-derived vapor pressure to maintain a given humidity. Because this packaging mode does not include deoxidants, air oxidation has an effect during the preservation period, since packaging takes place in air. Furthermore, such packages cannot be easily opened since their adhered portions, formed by thermal compression, are firmly stuck with the two films. People with disabilities, the elderly, children, or such may find such packaging difficult to open.
Conventional disposable biosensors have a tertiary structure for maintaining quantitativeness, and known mechanisms for automatically supplying sample solution to a sensor, using capillary action or the like (JP-A Hei 1-291153). A sensor with such a structure is assembled by laminating a spacer and cover on an electrically insulating substrate. An electrode pattern is formed on the substrate, and air holes are opened on the cover, to discharge the air necessary for capillary action. The substrate, spacer, and cover form a sample-inlet port with air holes on one side and a sample-feeding path for providing a given amount of sample solution to the detecting section by capillary action.
So far, systems in which at least two adjacent biosensors share one sample-feeding path have been used for biosensors able to simultaneously measure multiple items (
In conventional systems, bottle container systems are first problematic in that they cannot maintain a dry state inside, due to repeated opening and closing. In addition, since systems that package biosensors in containers use bulky containers compared to the size of the biosensor, they are problematic in that the packaging step is complex and requires lots of materials. Furthermore, although systems which package single biosensors by thermal compression of the biosensor between two films can shield ultraviolet rays and maintain a dry state through use of a desiccant, they cannot eliminate the influence of heat and oxidation, and opening the package can be problematic.
Thus, a first objective of the present invention is to achieve mild manufacturing processes suited to the use of biomaterials, such as processes that do not involve heating, even when a reaction process is required after deploying a reagent layer to an inner reaction-detecting section, or when simple packaging is needed; and to provide biosensors that can reliably shield the inner reaction-detecting section from the outside world until use.
Conventional biosensors for simultaneously measuring multiple items are problematic in that sample solutions supplied to the sample-feeding path are affected by at least one biosensor reagent.
Methods have been adopted in which sample solutions are introduced through holes that communicate with the sample-feeding path to the side of the biosensor on which the substrate or cover lies. In such cases, however, sample solution adheres to the edges of the holes used as sample-inlet ports when that sample solution was supplied to the biosensor feeding path, resulting in the use of sample solution in an amount greater than the inner volume of the sample-feeding path.
Thus, a second objective of the present invention is to provide biosensors that allow simultaneous measurement of multiple items using a reduced amount of sample solution, in which the sample solution supplied to a sample-feeding path to eliminate the influence of at least one other biosensor reagent; and to provide methods for measuring test compounds using these biosensors that simultaneously measure multiple items.
A first invention described herein was made in view of the first objective. By adopting a biosensor structure wherein a sample-inlet port and air-discharge port are exposed as a cross section of the sample-feeding path, achieved by cutting part of the biosensor structure when ready for use, the present inventors discovered biosensors which include a sealed reaction-detecting section comprising at least one set of electrodes and a sealed sample-feeding path, wherein the biosensors enable the reaction-detecting section to be completely sealed prior to use, have excellent sealing ability, and can preserve the inner environment in a preferred state through the use of a desiccant or the like as needed. The present inventors thus completed the present invention.
As this type of biosensor can seal the reaction-detecting section and such by connecting the substrate and cover via a spacer, heating is not involved in the biosensor manufacturing and packaging steps after forming a reaction layer in the reaction-detecting section.
A second invention described herein has been made in view of the second objective. The present inventors discovered that by using biosensors with specific structures to simultaneously measure multiple items, the biosensors could perform simultaneous multi-item measurement even with very little sample solution, and even when a little sample solution is supplied to the sample-feeding path, the influence of at least one biosensor reagent is eliminated. The present inventors thus completed the present invention.
Specifically, the present invention comprises the following:
[1] A biosensor comprising:
[2] The biosensor of [1], wherein the cutting plane line is formed by notches or cuts, and the notches or cuts are laid out to face the same positions on the substrate and cover.
[3] The biosensor of [1] or [2], wherein the substrate and cover each comprise a multilayer structure of at least two or more layers, and the cutting plane line is formed to leave at least an innermost layer of the multilayer structure.
[4] The biosensor of any of [1] to [3], wherein a reagent layer is provided at a region where the sample-feeding path crosses the electrode.
[5] The biosensor of any of [1] to [4], wherein a part of the region sandwiched between the substrate and cover comprises a desiccant and/or deoxidant.
[6] The biosensor of [5], wherein the desiccant and/or deoxidant is comprised in a sealed cap portion.
[7] The biosensor of any of [1] to [6], wherein a part of the region sandwiched between the substrate and cover comprises a humidity indicator and/or oxygen-detecting agent.
[8] The biosensor of [7], wherein a part or all of the substrate and/or cover is of a material transparent to visible rays, and thus the humidity indicator and/or oxygen-detecting agent is visible.
[9] The biosensor of any of [1] to [7], wherein the substrate and/or cover are made of a material that does not transmit ultraviolet.
[10] The biosensor of any of [1] to [9], wherein a top surface of the substrate and/or cover is coated with an ultraviolet absorber or a material that does not transmit ultraviolet.
[11] The biosensor of any of [1] to [10], wherein the substrate or cover comprise a compound with a photocatalytic effect, or where a top surface of the substrate and/or cover is coated with a layer comprising a compound with a photocatalytic effect.
[12] The biosensor of any of [1] to [11], wherein the spacer layer comprises a fluorescent or luminescent agent close to an exposed sample-inlet port and air-discharge port.
[13] The biosensor of any of [1] to [12], wherein the electrodes form an array.
[14] The biosensor of [13], wherein at least one sample-inlet port is exposed when the sealed cap portion is cut along the cutting plane line, and the reaction-detecting section comprising at least one set of electrodes is located ahead of the sample-feeding path connected to the sample-inlet port.
[15] The biosensor of [14], wherein the at least one sample-inlet port is connected to at least two sample-feeding paths branched from the sample-inlet port, and the reaction-detecting section comprising at least one set of electrodes is located ahead of the sample-feeding path.
[16] The biosensor of [8], wherein the substrate and/or cover comprising a material transparent to visible rays is coated with a protective film.
[17] The biosensor of any of [1] to [16], wherein the external terminal is coated with a protective film.
[18] The biosensor of any of [1] to [16], wherein the external terminal is covered with the cover, and the cover has a fold-line foldable in such a way as to expose the external terminal.
[19] A biosensor package retaining a plurality of a biosensor of any of [1] to [18].
[20] A biosensor aggregation sheet comprising a plurality of any of the biosensors of [1] to [18], regularly laid out at predetermined intervals, wherein a cut-away perforation is provided at a substrate of an adjoining biosensor.
[21] A method for using a biosensor of any of [1] to [18], wherein the method comprises the step of cutting off a sealed cap to form a sample-inlet port and an air-discharge port.
[22] A biosensor device comprising:
[23] The biosensor device of [22], wherein the measuring method in the measuring section is any one of potential step chronoamperometry, coulometry, and cyclic voltammetry.
[24] The biosensor device of [22] or [23], wherein the biosensor comprises a wireless means for transmitting measurement data to the measuring section, and the wireless means is a non-contact IC card or Bluetooth.
[25] A biosensor for simultaneously measuring multiple items, comprising:
[26] The biosensor of [25], wherein the sample-feeding path is provided such that the sample-inlet port opens at the cut surface, and an air-discharge port is provided at the surface of the substrate or cover, or at a side surface of the biosensor unit-comprising substrate which differs from the cut surface.
[27] The biosensor of [25], wherein the sample-feeding path is sealed;
[28] The biosensor of [27], equipped with an auxiliary device on a surface of the substrate or cover, such that the substrate or cover are bent along the second cutting plane line in response to bending of the substrate or cover along the first cutting plane line.
[29] The biosensor of [25], wherein the sample-feeding path is provided such that both the sample-inlet port and air-discharge port open to a cut surface of each of the biosensor unit-comprising substrates, and the sample-feeding path is set up in a sealed state, prior to cutting.
[30] The biosensor of any of [25] to [29], wherein the sample-feeding path is laid out such that a sample-inlet port forms for every biosensor unit.
[31] The biosensor of any of [25] to [30], wherein at least one of the substrate or cover comprises a multilayer structure comprising at least two layers, and the cutting plane line is formed at any one of the layers of the multilayer structure, excluding the innermost layer.
[32] The biosensor of any of [25] to [31], wherein the electrodes form an array.
[33] A method for using the biosensor of any one of [25] to [32], wherein said method comprises the steps of:
[34] The method of [33], wherein the bending in step (1) is carried out such that the cut surface is exposed and one substrate is cut while the other substrate is left connected, and wherein step (3) is carried out with the biosensor bent.
[35] The method of [33] or [34], wherein step (3) comprises contacting the sample-inlet ports of two or more biosensor unit-comprising substrates with the solution at one time.
[36] The method of [33], wherein the biosensor unit-comprising substrate comprises two or more biosensor units, and step (2) comprises contacting one sample-inlet port of the biosensor unit-comprising substrates with a solution at the same time.
[37] A method for measuring a measuring target using the biosensor of any one of [25] to [32], wherein the method comprises the steps of:
[38] A biosensor device comprising:
[39] The biosensor device of [38], wherein the connector section comprises a structure for:
[40] The biosensor device of [38], wherein the measuring method in the measuring section is potential step chronoamperometry, coulometry, or cyclic voltammetry.
[41] A connector for use in biosensors, for fixing the biosensor of any one of [25] to [32] to capture electrical signals, wherein the connector comprises:
First Invention: Biosensors
The biosensors of the present invention comprise:
That is, in the biosensors of the present invention, cutting the sealed cap portion along the cutting plane line before use exposes two cross sections of the sample-feeding path in the sensor portion, since the cut surface crosses the sample-feeding path. Accordingly, one of the two exposed cross sections serves as a sample-inlet port, while the other cross section serves as an air-discharge port. The cutting method is not particularly limited, and can be snapping, breaking, or tearing along the cutting plane line.
The sensor portion is the body of the biosensor, comprising a substrate; a cover connected to the substrate via a spacer layer; a reaction-detecting section formed on the substrate at a region sandwiched between the substrate and cover and comprising at least one set of electrodes; an external terminal to be connected to the reaction-detecting section; a sample-feeding path defined between the substrate and cover by the spacer layer; a sample-inlet port; and an air-discharge port. The sealed cap portion is a portion that does not include electrodes and can be disposed of by cutting.
The directions of the openings of the sample-inlet port and air-discharge port of the biosensors are not particularly limited, as long as they are on the same cross section when the biosensor is used. The sample-inlet port and air-discharge port may be formed anywhere inside the cross section which appears at the time of use, at a position where the sample solution can be supplied to the sample-feeding path.
The phrase “on the same cross section” herein means that the entirety of both the sample-inlet port and air-discharge port appear on the same cross section due to biosensor deformation at time of use. The shape of the edges that constitute the cross section of the sensor portion depends on the shape of the corresponding sealed cap portion, and may be linear or curved. With regard to the shape of the cut surface, if the side near the sample-inlet port is curved, the risk of human injury can be reduced in particular uses such as measuring blood glucose, where blood is extracted from the body.
Furthermore, the shape of the sealed cap portion is not particularly limited, and is preferably rectangular, trapezoidal, triangular, or the like.
The sample-feeding path forms a pattern with the spacer layer. Examples of the spacer layer can include an adhesive layer, as well as a spacer layer with an adhesive layer in which adhesive is applied to both sides of the spacer. Therefore, the spacer layer adheres the substrate and cover together, and defines the sample-feeding path.
The electrodes are at least one set of electrodes comprising a positive and negative electrode facing each other. Such electrodes may consist of two electrodes, a positive one and a negative one, or may comprise two or more electrodes.
In the present invention, the area around the sample-inlet port and the top surface of the sample-feeding path can be coated with a surfactant or lipid. This surfactant or lipid coating enables smooth supply of the sample solution.
Before cutting the sealed cap portion, the biosensors of the present invention keep the inside of the sample-feeding path airtight, including the reaction-detecting section. Thus, the internal state of the biosensors can be maintained for long periods after manufacture, and the internal environment of the biosensors, specifically the gas composition (deoxygenated state), atmospheric pressure, humidity (humid conditions) and so on, can be controlled to a certain preferred environment.
Accordingly, even a sample solution that cannot be smoothly supplied via the dry sample-feeding path of the biosensor can be smoothly supplied into the biosensor by uniformly applying a surfactant or the like to the inner wall or the like of the sample-feeding path, maintaining a given humidity. When the sample solution is blood or such, heparin, prolixin-S, or a metal salt of ethylenediaminetetraacetic acid or citric acid may be coated as an anticoagulant agent alone, or together with a surfactant.
Furthermore, it is preferable that the sample-feeding path between at least the sample-inlet port and reaction-detecting section is a straight line or gentle curve. A sample-feeding path of this shape enables smooth supply of the sample solution. It is therefore preferable that corners, particularly areas with acute angles, are absent from the above-described region of the sample-feeding path.
Substrates
The above-described substrates are not particularly limited as long as they provide electrical insulation. For example, any plastic, biodegradable material, or paper can be preferably used.
Plastics include hard polyvinyl chloride, polystyrene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polyester, polyether nitrile, polycarbonate, polyamide imide, phenolic resin, epoxy resin, acrylic resin, and ABS resin. Preferable plastics for use in sheet form are polycarbonate, hard polyvinyl chloride, polyethylene terephthalate, acrylic resin, ABS resin, or the like.
A preferable biodegradable material is polylactic acid.
The substrates may be made of a material that does not transmit ultraviolet light.
The substrate depth is not particularly limited, and for example is preferably in the range of about 10 to 1,000 μm, and more preferably in the range of about 100 to 500 μm.
Covers
The above-described covers may be made of materials similar to those of the substrates described above. Cover depth is not particularly limited, and is preferably, for example, in the range of about 10 to 1000 μm, and more preferably in the range of about 100 to 500 μm.
Spacer Layers
The spacer layers adhere substrates to covers, and define sample-feeding paths.
The spacers may be made of materials similar to the substrates described above, and in such cases, adhesive is applied to the top surface of the spacers to obtain an adhesive layer for connecting the substrate and cover. Alternatively, the spacer itself may be an adhesive layer formed by an adhesive. The adhesive is not particularly limited, as long as it does not react with or is not soluble in the substrate and cover. For example, an acrylic resin can be used as the adhesive.
The spacer itself may be formed by an adhesive and resist. In such cases, as for the adhesive, the resist is not particularly limited, as long as it does not react with or is not soluble in the substrate and cover. Examples of the resist include ultraviolet-curing vinyl-acrylic resin, urethane acrylate resin, and polyester acrylate resin. The resist is mainly used to clarify the electrode pattern, for example, to define the electrode area, and to insulate the sample-feeding path where the reagent layer is not present. Accordingly, the resist layer may or may not form the same pattern as the adhesive layer. Where it does not, the resist layer is preferably formed on the electrode substrate for insulation.
As the above-described acrylic resin, heat-curing and photo-curing types, more specifically, ultraviolet-curing and visible-light-curing types of acrylic resins may be used. The top surface of the spacer may be coated with an ultraviolet-absorber or a material that does not transmit ultraviolet.
Spacer depth is not particularly limited, and is preferably in the range of 5 to 500 μm, and more preferably in the range of about 10 to about 100 μm.
The spacer layer can be formed by screen-printing methods. Reagents such as enzymes, mediators, or surfactants may be comprised in the spacer layer.
Electrode Systems
The above-described electrode systems comprise sets of electrodes consisting of positive and negative electrodes facing each other, as well as lead lines. Such electrode systems may consist of two electrodes, a positive electrode and negative electrode, or may comprise two or more electrodes.
The electrodes can be made of any of carbon, silver, silver/silver chloride, platinum, gold, nickel, copper, palladium, titanium, iridium, lead, tin oxide, and platinum black. As carbon, specifically, carbon nanotubes, carbon microcoils, carbon nanophones, fullerens, dendrimers, and derivatives thereof can be used.
The electrode depth is not limited, as long as it does not interfere with spacer contact. For screen-printing, electrode depth is normally in the range of about 1 to 100 μm, more preferably in the range of about 3 to 20 μm. For vapor deposition, sputtering, film adhesion, and plating, depth is normally in the range of about 200 to 2,000 Angstroms, more preferably in the range of about 500 to 1,000 Angstroms. When electrode depth is within such ranges, the electrode edges formed on the substrate are not serrated, resulting in electrodes with high accuracy. Furthermore, separation and disconnection of the electrodes can also be prevented.
Such electrodes can be formed on the substrate or cover by any methods of screen-printing, vapor deposition, sputtering, film adhesion, or plating.
The cutting plane lines are preferably formed by notches or cuts, and the notches or cuts are laid out at identical substrate or cover positions so they face each other.
Notches or cuts formed on the cutting plane line make cutting easier. Furthermore, if the cutting plane lines are laid out to face the same positions, cutting is easy.
Herein, the term “cuts” refers to cuts on the substrate or cover constituting the biosensor, made from outside to a depth that does not reach the interior. Therefore, the cuts do not penetrate the substrate or cover before cutting.
In the present specification, the phrase “A and/or B” means at least one of A or B.
The substrate or cover may comprise a multilayer structure of at least two or more layers, and the cutting plane line is formed to leave at least an innermost layer of the multilayer structure.
When the substrate or cover comprises a multilayer structure of at least two or more layers, the cutting plane line, as well as notches, perforations, or the like, can be formed to leave at least the innermost layer of the multilayer structure. Furthermore, it is preferable that the notches or perforations are laid out at identical substrate or cover positions so as to face one another. By using a multilayer structure, the cutting plane lines are formed to leave at least an innermost layer, and the resulting biosensors thus are free from damage such as flawed inner layer portions. Therefore, these biosensors are advantageous in that they can endure forces such as sudden bending during the manufacturing process or when in storage state.
Reagent Layers
Reagent layers are preferably provided at a region where the sample-feeding path crosses the electrodes.
In the biosensors of the present invention, reagents react with samples through capillary action used to feed the samples from a sample-inlet port through a sample-feeding path. Samples are then contacted with a reagent layer on electrodes forming a reaction-detecting section. The reaction is monitored as electrical changes on the electrodes. One or a number of such reagent layers can be present where the sample-feeding path passes on the electrodes.
The reagent layers are preferably on the top surface of one or both of the positive and negative electrodes.
Because the biosensors of the present invention are extremely well sealed prior to use, they can maintain a given humidity in the reagent layer. Thus, even when oxygen is present inside the biosensor, degradation or denaturation due to air oxidation can be suppressed in reagents protected by humidity.
According to the present invention, the top surface of a reagent layer can be coated with a compound such as a surfactant or lipid, to enable smooth supply of the sample solution. A surfactant or lipid coating on the top surface of the reagent layer can further suppress degradation due to air oxidation. When the sample solution is blood or such, heparin, prolixin-S, or a metal salt of ethylenediaminetetraacetic acid or citric acid may be used as anticoagulant agents for coating.
The reagent layers can contain, as necessary, any enzyme, antibody, ribosome, nucleic acid, primer, peptide nucleic acid, nucleic acid probe, microorganism, organelle, receptor, cellular tissue, molecular recognizing factor such as crown ether, mediator, intercalating agent, coenzyme, antibody labeling agent, substrate, inorganic salt, surfactant, lipid, sugar such as trehalose, humectant such as glycerin, and stabilizer such as cysteine, or a combination thereof, depending on the test subject.
When the sample solution is blood, the reagent layer may comprise anticoagulant agents. Heparin, prolixin-S, metal salts of ethylenediaminetetraacetic acid or citric acid may be used as anticoagulant agents.
The enzymes include oxidases and dehydrogenases, such as glucose oxidase, fructosylamine oxidase, lactate oxidase, urate oxidase, cholesterol oxidase, alcohol oxidase, glutamate oxidase, pyruvate oxidase, pyruvate kinase, acetate kinase, peroxidase, glucose dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase; cholesterol esterase, inorganic pyrophosphatase, acidic 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, nicotinamide adenine dinucleotide (NADH) oxydase, diaphorase, and osmium peroxidase complex; nucleic acid ligases such as DNA polymerase, RNA polymerase, DNA ligase, and DNase; and restriction enzymes. These enzymes can be used alone or in combination.
Instead of enzymes alone, the reagent layers may also contain enzymes in combination with mediators. The mediators are selected from pigments such as potassium ferricyanide, ferrocene, benzoquinone, osmium peroxidase complex, 1-methoxy-5-methylphenazinium methyl sulfonate (1-M-PMS), 2,6-dichloroindophenol (DCIP), 9-dimethylaminobenzo-α-phenazoxonium chloride, methylene blue, indigo trisulfonic acid, phenosafranin, thionin, new methylene blue, 2,6-dichlorophenol, indophenol, azure B, N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride, resorufin, safranin, sodium anthraquinone β-sulfonate, and indigo carmine; biological oxidation-reduction materials such as riboflavin, L-ascorbic acid, flavin adenine dinucleotide, flavin mononucleotide, nicotine adenine dinucleotide, lumichrome, ubiquinone, hydroquinone, 2,6-dichlorobenzoquinone, 2-methylbenzoquinone, 2,5-dihydroxybenzoquinone, 2-hydroxy-1,4-naphthoquinone, glutathione, peroxydase, cytochrome C, and ferredoxin, or derivatives thereof; and Fe-EDTA, Mn-EDTA, Zn-EDTA, methosulfate, 2,3,5,6-tetramethyl-p-phenylenediamine, and the like.
The reagent layers may comprise inorganic salts such as sodium chloride or potassium chloride, in combination with quinhydrone.
Preferable concentrations of the above-described mediators are approximately 40 nM or more.
Of the above-described compounds, potassium ferricyanide, ferrocene, benzoquinone, osmium peroxidase complex, DCIP, 1-M-PMS, and 9-dimethylaminobenzo-α-phenazoxonium chloride are preferable.
A combination of primers, DNA polymerases, and deoxyribonucleotide triphosphates can be contained in reagent layers. Furthermore, the reagent layers can comprise a combination of inorganic salts, such as sodium chloride or potassium chloride, and quinhydrone, as well as primers, DNA polymerases, and deoxyribonucleotide triphosphates.
When the biosensors are used as DNA chips, fixed nucleic acid probes can be used as reagent layers. In such cases, the electrodes are preferably placed in an array.
The reagent layers are formed near each electrode set, or on a partial or entire electrode surface, to constitute, together with the electrodes, the reaction-detecting section. Such reagent layers can be formed by a dispenser method, where a dispenser or the like is used to add drops which are then dried; by a screen-printing method in which viscosity is adjusted; and so on. Specifically, dispenser methods are preferable. The reagent layers can be fixed to the top surface of the electrode or substrate using an adsorption method involving a drying step or a covalent binding method.
A convex partition section can be provided between reagent layers. The reagent layers can be placed not only at one region but also at two or more regions, where two or more different kinds of reagent layers may be provided.
The reagent layers may be mixed with an adhesive used as a spacer material, or the like.
Such reagent layers can be placed not only at one location, but also at two or more locations. In such cases, two or more different kinds of reagent layers may be provided. When reagent layers are provided at two or more places, a convex partition section can be provided between them. The convex partition section can be formed by screen-printing and can be made of carbon, resist, or water-absorbing materials.
Part of the region sandwiched between the substrate and cover can comprise a desiccant and/or deoxidant. The desiccant and/or deoxidant are preferably comprised in a sealed cap portion.
Thus, since the biosensors of the present invention are sealed with a reaction-detecting section included inside, such desiccants and/or deoxidants provided in the biosensors can maintain an internal dry or oxygen-free state over a long period of time.
By using such desiccants and/or deoxidants, the internal atmosphere of the biosensor can be dry or deoxygenated, even when the biosensors are manufactured and sealed in an atmosphere containing humidity or oxygen during the biosensor assembling step to include the reaction-detecting section.
The above-described desiccants and/or deoxidants are preferably present inside the sealed cap portion, which becomes unnecessary after cutting. This avoids direct contact with the sample solution. The sealed cap portion and sensor portion are connected inside via the spacer or sample-feeding path. Thus, even when the desiccant and/or deoxidant are present in the sealed cap portion, the inner space of the spacer, present between the substrate and cover in the structure of the biosensors in the preserved state before use, can be dried or deoxygenated. In particular, the desiccant and/or deoxidant can keep the inner space of the biosensors in a dry and/or deoxygenated state via a sample-feeding path, where the desiccant and/or deoxidant are arranged in the sealed cap portion to cross the sample-feeding path.
Furthermore, since the biosensor sealed cap portion with desiccant and/or deoxidant inside is cut and removed when the biosensor is used, the sample solution does not contact the desiccant and/or deoxidant when in use.
Examples of the desiccant include porous structures such as silica gel, active alumina, potassium chloride, molecular sieves, and hygroscopic polymer.
Examples of the deoxidant include powder consisting of metal halide and metal such as iron, and organic compounds such as hydrosulfite, active magnesium (see, e.g., JP-A No. 2001-37457), ascorbic acid (see, e.g., JP-A No. Hei 05-7772), catechol compounds (see, for example, JP-A No. Hei 09-75724), and polyvalent alcohols (see, for example, JP-A No. 2003-144113). These deoxidants may be supported by well-known carriers (see, for example, JP-A No. 2001-37457). Commercially available deoxidants include, for example, AGELESS® (produced by Mitsubishi Gas Chemical Company, Inc.) and VITALON® (produced by Toagosei Chemical Co., Ltd.).
In addition, the biosensors of the present invention can comprise a humidity indicator and/or oxygen-detecting agent in a part of the region sandwiched between the substrate and cover. In order to confirm a dry and/or deoxygenated state in the biosensor prior to use, a humidity indicator in combination with a desiccant; and/or oxygen-detecting agent in combination with a deoxidant, can be used.
The humidity indicator is not particularly limited as long as it can be used in the packaging of the present invention.
Commercially available oxygen-detecting agents include, for example, AGELESS EYE® (produced by Mitsubishi Gas Chemical Company, Ltd.) and VITALON®-oxygen-detecting agent (produced by Toagosei Chemical Co., Ltd.).
A part or all of the substrate or cover is preferably a material transparent to visible light, so the humidity indicator and/or oxygen-detecting agent is visible.
A part or all of the substrate or cover is also preferably of a material that can shield ultraviolet rays. In such cases, all of the substrate or cover may be an ultraviolet-shielding material, or the top surface of the substrate or cover may be covered with an ultraviolet-shielding film. Examples of this film include films that comprise an organic compound such as benzotriazole, or a fluorescent agent that converts ultraviolet rays to visible light.
When using a substrate or cover not transparent to visible light, the above-described humidity indicator and/or oxygen-detecting agent can be arranged at the spacer portion of the cut surface that newly appears when the sealed cap portion is cut. In such cases, the humidity indicator and/or oxygen-detecting agent may be contained in the spacer layer or constituted as a part of the spacer layer. This arrangement can allow the state inside a biosensor, indicated by the humidity indicator and/or oxygen-detecting agent, to be confirmed at the cut surface or inside near the cut surface after cutting and immediately before use.
The substrate or cover is preferably made of a material that does not transmit ultraviolet. Alternatively, the top surfaces of the substrate or cover may be coated with an ultraviolet-absorber or a material that does not transmit ultraviolet.
Transmission of ultraviolet rays can be suppressed or blocked when the substrate or cover are made of a material that does not transmit ultraviolet, or are coated with an ultraviolet-absorber or a material that does not transmit ultraviolet, as described above.
The ultraviolet-absorbers are not particularly limited and include, for example, metals such as aluminum, metal halides such as silver chloride, fluorescent agents, and organic compounds such as benzotriazole.
Materials that do not transmit ultraviolet are not particularly limited and include, for example, vapor-deposited film, consisting of metals such as aluminum or of metal halides such as silver chloride, and organic compound films of benzotriazole or the like.
The substrate or cover may comprise a compound with photocatalytic effect, or a top surface of the substrate or cover may be coated with a layer comprising a compound with photocatalytic effect.
Herein, the term “photocatalyst” means a compound excited by light absorption into an active state, exerting a strong oxidation-reduction effect on organic compounds in contact with the top surface of the photocatalyst. A “photocatalytic effect” is such an oxidation-reduction effect.
The above-described light includes ultraviolet rays and/or visible light. An input of ultraviolet rays and/or visible light causes a photocatalytic effect at the top surface of the biosensor. This effect results in self-purification, such as sterilization, decomposition of viruses with capsids and envelopes comprising proteins, and degradation of stains adhering to the top surface. This allows preservation under constantly sanitary conditions. Accordingly, this type of biosensor is particularly effective when used in fields such as the medical field, where biosensors are directly contacted with biological samples, and in fields where food or the like is handled.
Compounds with photocatalytic effect include metal oxides. Metal oxides that can be used specifically include, without limitation, at least one selected from the group consisting of titanium oxide, titanium dioxide, zinc oxide, titanium oxide strontium, tungsten trioxide, ferric oxide, bismuth trioxide, and tin oxide.
Spacer layers may comprise a fluorescent or luminescent agent close to an exposed sample-inlet port, or close to a sample-inlet port and an air-discharge port. It is particularly preferable that the fluorescent or luminescent agent is comprised near the sample-inlet port.
The fluorescent or luminescent agent can form a mark to improve visibility, and thus can prevent mishandling in supplying samples. When the fluorescent or luminescent agent is used at the cut surface portion, it may be comprised in the spacer material or constituted as part of the spacer. When the fluorescent or luminescent agent is used as a mark near the sample-inlet port of the substrate or cover, the mark can be formed by printing or such. The luminescent agent may be one well known in the art, with a light emission reaction that starts upon contact with oxygen in the air.
The electrodes may form an array. Preferable biosensors forming the array are those in which at least one sample-inlet port is exposed when the sealed cap portion is cut along the cutting plane line, and in which the reaction-detecting section comprising at least one set of electrodes is located ahead of the sample-feeding path connected to the sample-inlet port. At least one sample-inlet port may be connected to at least two sample-feeding paths branched from the sample-inlet port, and the reaction-detecting section comprising at least one set of electrodes may be located ahead of the sample-feeding path.
Herein, the term “array” means an arrangement in an arrayed condition.
When at least two sample-feeding paths are branched from one sample-inlet port, a surfactant may be coated inside the sample-feeding path so that the sample solution can reach all of the arrayed reagent layers. Alternatively, when the sample solution is blood or such, heparin, prolixin-S, or a metal salt of ethylenediaminetetraacetic acid or citric acid may be coated as an anticoagulant agent.
The substrate or cover comprising a material transparent to visible rays may be coated with a protective film.
In such cases, the visibility of the oxygen-detecting agent or the humidity indicator located at the spacer layer of the sealed cap can be ensured. The protective films can serve to prevent the influence of visible light and ultraviolet rays on the reagent layer of the biosensors.
The external terminal may be coated with a protective film. The protective film is used to cover the electrode terminals, connecting portions of the measuring unit, or the like, which are exposed on the same surface of the biosensor, as necessary until use. Protective films may have a single or multi layer structure.
Such a protective film may comprise portions with a detachable adhesive layer and a non-adhesive portion. The non-adhesive portion can be used as a grip portion for separating the protective film. The portion with the detachable adhesive layer is generally called the “peel seal” or “weak seal portion”, and can be easily separated with a certain degree of pulling force.
Preferable protective films characteristically block at least one of humidity, ultraviolet rays, or oxygen.
The materials for the protective films are preferably, for example, plastic films such as polyvinylidene chloride, polyethylene, polyester, nylon, ethylene-vinyl alcohol copolymer, and fluorine resin. Such plastics are flexible and excellent at shielding humidity.
Protective films comprising ultraviolet-absorber or a material that does not transmit ultraviolet are preferably used for blocking ultraviolet rays. Protective films comprising a desiccant, deoxidant, or the like are preferably used for blocking oxygen.
An external terminal may be covered with a cover, and the cover may have a fold-line that can be folded to expose the external terminal. In such cases, packaging of the main body and simple packaging with the protective film are unnecessary, since the terminals are stored in the cover before use, i.e., in the main body of the biosensors of the present invention.
Examples of the fold-line include perforations, notches, cuts, and recesses. The fold-back or folding process can be made easier by providing two parallel lines of perforations or the like on the fold-back or folding portion. To facilitate fold-pack or folding, the adhesive layer used to form the spacer is preferably absent from the fold-back or folding portion.
The substrate's detachable adhesive can be provided in at least one location on the inner surface of the fold-back or folding portion of the cover. With a force naturally applied to the fold-back or folding portion of the cover, the detachable adhesive can adhere the cover at a level that does not expose the terminal portion of the biosensor in the preserved state. This can stably maintain the shape of the biosensor in its preserved state.
In such cases, the terminals contact with the detachable adhesive and are separate from the substrate, so the function of the terminals as conducting materials should not be influenced. Accordingly, the detachable adhesive is preferably arranged so as not to contact the terminals.
Furthermore, the two parallel lines of perforations or the like provided at the fold-back or folding portion of the cover enable two cover pieces to be overlapped and folded to face each other. In such cases, the detachable adhesive is preferably placed on the inner side of one of the two cover pieces that cover the end portions of the terminals. This enables the shape of the biosensor in the preserved state to be stably maintained. In addition, the detachable adhesive can also be used to adhesively fix the two cover pieces when they are folded to face each other.
The biosensor packages of the present invention retain a number of the above-mentioned biosensors. More specifically, a number of simply packaged biosensors of the present invention can be packaged together using a bottle container system, a box container system, or the like.
Furthermore, when a number of biosensors of the present invention are aligned in a container using a box container system or the like, and are removed from the container in order, it is possible to print the serial number of each biosensor or the number of the remaining biosensors in the container on the main body or the protective film.
Multiple biosensors can be regularly laid out at predetermined intervals, and cut-away perforations may be provided at the substrates of adjoining biosensors.
This structure can enable efficient manufacture of multiple biosensors at one time. In addition, each of the adjoining biosensors can be connected to a measuring section to provide a measuring device that can simultaneously measure multiple samples. Furthermore, as a number of biosensors are regularly laid out at predetermined intervals in the above-described structure, individual biosensors can be connected to the measuring unit by sequentially shifting the biosensors using rotation or the like. Measuring devices in this form can serially and automatically measure multiple samples. By forming perforations in the adjoining substrates, the retaining space can be smaller, and folding between adjoining biosensors, separation of individual electrodes, and such can be achieved.
Methods for using the biosensors of the present invention comprise the step of cutting a sealed cap to form a sample-inlet port and air-discharge port. As the sample-inlet port and air-discharge port are included in the sample-feeding path inside the biosensors in the manufacturing step, the interior of the biosensor is kept airtight when shipped. Since the part of the biosensor which does not contain the electrodes is separate during use, the sample-inlet port and air-discharge port are formed and exposed for the first time as the cut surface of the sample-feeding path, and the biosensor is ready for use.
The biosensor devices of the present invention comprise:
Further, the biosensors comprise a wireless means for transmitting measurement data to the measuring section, preferably a non-contact IC card or Bluetooth.
Second Invention: Biosensors for Simultaneously Measuring Multiple Items
The biosensors for simultaneously measuring multiple items of the present invention comprise:
Specifically, the biosensors for simultaneously measuring multiple items of the present invention comprise at least two biosensor unit-comprising substrates, in which a cutting plane line is provided to divide each biosensor unit-comprising substrate. Each biosensor unit-comprising substrate comprises at least one biosensor unit. A biosensor unit comprises one reaction-detecting section and one sample-feeding path. A reaction-detecting section comprises one electrode system and one reagent layer, where the electrode system is connected to an external connection terminal by lead lines. A reagent layer is preferably laid out where the sample-feeding path and the electrode system cross.
In the biosensors for simultaneously measuring multiple items of the present invention, one or more biosensor units are present on a single biosensor unit-comprising substrate, where each biosensor unit comprises one reagent layer on one sample-feeding path. Accordingly, as the sample solution supplied to the sample-feeding path reaches only one reagent layer, and is isolated from the other reagent layers, it is not affected by components diffused from other reagent layers.
The biosensors for simultaneously measuring multiple items of the present invention comprise a number of biosensor unit-comprising substrates, where cutting plane lines are provided to divide individual biosensor unit-comprising substrates. Accordingly, substrates or covers can be cut along cutting plane lines to divide each biosensor unit-comprising substrate.
When a substrate or cover is cut along a cutting plane line, the opening of the sample-feeding path is opened on the cut surface of each biosensor unit-comprising substrate formed by the cutting. This cut opening is used as the sample-inlet port. If the biosensors are structured such that the sample-feeding path crosses the cutting plane line twice, two cut openings appear, providing both a sample-inlet port and an air-discharge port.
Since the cut opening is only opened by cutting at the time of use, it can be reliably maintained. This is particularly useful when the cut opening is small. If the sample-inlet port and air-discharge port are both opened by cutting, the sample-feeding path can be sealed until use, and the activity of the reagent layer can be maintained. This can eliminate the need for packaging the biosensors, and thus significantly reduce manufacturing costs. When using the biosensors for simultaneously measuring multiple items of the present invention, the opened sample-inlet port can contact the sample solution to supply solution to the sample-feeding path from the sample-inlet port by capillary action. This procedure can considerably reduce the amount of sample solution used. Accordingly, even small amounts of test compound in the sample solution can be detected with high sensitivity.
A cutting plane line is provided on at least one of the substrate or cover. The cutting plane line is preferably formed from notches or cuts. When the cutting plane line is provided on both the substrate and cover, the notches or cuts are preferably laid out at identical substrate or cover positions so as to face each other.
Notches or cuts on the cutting plane line makes cutting easier. In addition, if the cutting plane lines are laid out in the same position, so as to face each other, substrates or covers which bend outside can be easily cut, and substrates or covers which bend inside can also be easily cut or bent.
Herein, the term “cut” means a cut formed in the substrate or cover constituting the biosensor, made from outside, but not deep enough to reach the interior. Therefore, the cuts do not thoroughly penetrate the substrate and cover before cutting. The cutting method is not particularly limited, and comprises a step of snapping, breaking, or tearing along the cutting plane line.
The sample-feeding path in the present invention is patterned by the spacer layer and provided on the substrate. The depth (height) of the sample-feeding path depends on the depth of the spacer layer, and is preferably in the range of about 5 to 500 μm, and more preferably in the range of about 10 to 100 μm. Capillary action occurs easily in sample-feeding paths with a depth in such a range.
The sample-feeding path preferably connects at least the sample-inlet port and reaction-detecting section, by a straight line or gentle curve. These shapes enable smooth movement of the sample solution. Therefore, the sample-feeding path preferably has no corners between these regions, and particularly no areas with acute angles.
In the present invention, the area around the sample-inlet port and the top surface of the sample-feeding path can be coated with a surfactant or lipid. Coating with a surfactant or lipid can ensure smooth movement of the sample solution.
Herein below, the present specification describes substrates, covers, spacers, electrode systems, reagent layers, etc.
Substrates, Covers, Spacer Layers, Electrode Systems
Substrates, covers, spacers, and electrode systems similar to those described in the aforementioned first invention can be employed in the biosensors for simultaneously measuring multiple items of this invention.
Reagent Layers
While one or a number of reagent layers can be present on the electrodes where the sample-feeding path passes in the aforementioned first invention, in the biosensors for simultaneously measuring multiple items of the second invention, one reagent layer can be present on the electrode system where the sample-feeding path passes. Except for this difference, reagent layers the same as those discussed for the aforementioned first invention can be used.
In the biosensors for simultaneously measuring multiple items, as mentioned above, one reagent layer is placed at one location on the sample-feeding path in each biosensor unit. According to this invention, after cutting the biosensor for simultaneous measurement of multiple items, each biosensor unit-comprising substrate will comprise at least one biosensor unit. When a number of different reagent layers are used, a biosensor unit-comprising substrate can comprise a number of biosensor units.
Since one reagent layer is provided on one sample-feeding path in the biosensors for simultaneously measuring multiple items, sample solutions are not mixed, even when one biosensor unit-comprising substrate comprises two or more biosensor units. When one biosensor unit-comprising substrate comprises two or more biosensor units, however, a convex partition section can be provided between the reagent layers of each biosensor unit to more surely prevent mixing of sample solutions. The convex partition section can be formed by screen-printing, and can be made of carbon, resist, or water-absorbing material.
In the present invention, the sample-feeding path is preferably provided such that the sample-inlet port opens at the cut surface, and an air-discharge port is provided at the surface of the substrate or cover, or at a side surface of the biosensor unit-comprising substrate which differs from the cut surface. In such cases, while the angle at which the cut surface and sample-feeding path cross each other is not limited, it is preferably 0 degrees or more to less than 180 degrees, more preferably 0 degrees or more to 120 degrees or less, and still more preferably 0 degrees or more to less than 100 degrees.
Herein, the sample-feeding path in the biosensors for simultaneously measuring multiple items of the second invention are also preferably sealed to provide the merits of the sealing system described in the aforementioned first invention.
In another preferred embodiment of the present invention, for example, the sample-feeding path is sealed in the biosensors for simultaneously measuring multiple items;
Herein, auxiliary devices may be provided on a top surface of the substrate or cover, such that the substrate or cover are bent along a second cutting plane line in response to bending of the substrate or cover along a first cutting plane line.
Materials for the auxiliary device are not particularly limited, and may stretch or not. Preferably, auxiliary devices are of sufficient strength to cut the cut portion of the substrate or cover to open the air-discharge port along the cutting plane line. Furthermore, auxiliary devices are preferably fixed to a part or all of the portion serving as the opening of the air-discharge port.
Such an auxiliary device may be, for example, strap-shaped stretchable plastic. As shown in
Connectors
Specific connectors are needed to capture electrochemical signals from the biosensors for simultaneously measuring multiple items of the present invention.
Preferable connectors of the present invention fix the biosensors for simultaneously measuring two items so they capture electrical signals. Preferable connectors comprise:
The above-described connector has a structure that alters the structure of a sensor formed flat, to supply a sample solution to the sample-inlet port of the newly opened sensor, and to maintain the biosensor's shape until measurement is complete. Therefore, the shape of the connectors is not particularly limited, as long as it satisfies the above-mentioned conditions and reliably captures electrical signals from the sensor. Furthermore, the connectors may be incorporated in a measuring unit, or used in connection with an electrochemical measuring unit.
For example, preferable connectors comprise a folding portion and a folder that maintains the folded sensor shape. In such cases, the folding portion and folder may be identical or independent. Furthermore, the folding portion is preferably structured such that the biosensor structure can be changed from a flat shape to a non-flat shape, and more preferably, to a V shape. In such cases, the folding portion is preferably designed such that the angle at which the cut surface and sample-feeding path cross is, without limitation, 0 degrees or more to less than 180 degrees, more preferably 0 degrees or more to 120 degrees or less, much more preferably 0 degrees or more to less than 100 degrees. The above-described conditions can be applied even when the folding portion functions as the folder. The folding portion may be, without limitation, a type which changes the shape of the biosensors by sliding and connecting the sensor to the connector, or a type which changes the shape by sandwiching the sensor with the upper and lower angled folding portions.
In another preferable embodiment of the present invention, the sample-feeding path may be provided such that both the sample-inlet port and air-discharge port are open at the cut surface of each of the biosensor unit-comprising substrates, and the sealed sample-feeding path may be laid out before cutting. That is, since the biosensors for simultaneously measuring multiple items of the present invention keep the sample-feeding path airtight before cutting, including the reaction-detecting section, their internal state can be maintained over a long period of time, and the internal environment of the biosensors, specifically, the vapor composition (deoxygenated state), atmospheric pressure, and humidity (humid conditions), can be controlled to a certain preferred environment.
Some sample solutions cannot be smoothly supplied by a dry sample-feeding path of the biosensors for simultaneously measuring multiple items but even these sample solutions can be smoothly supplied to the biosensors by uniformly applying a surfactant or the like to the inner wall or the like of the sample-feeding path, maintaining a given humidity. When the sample solution is blood or such, heparin, prolixin-S, or a metal salt of ethylenediaminetetraacetic acid or citric acid may be coated as an anticoagulant agent, alone or together with a surfactant.
The sample-feeding path of the present invention is preferably laid out such that one sample-inlet port is formed per biosensor unit. Such a sample-feeding path layout better eliminates the influence of components diffused from the other reagent layers.
In the present invention, at least one of the substrate or cover may comprise a multilayer structure comprising at least two layers, and the cutting plane line may be formed at any layer of the multilayer structure, excluding the innermost layer.
When the substrate or cover has a multilayer structure of at least two layers, the cutting plane lines are formed to leave at least the innermost layer of the multilayer structure. Furthermore, the cutting plane lines are preferably laid out at identical positions of the substrate or cover so as to face each other. By forming the cutting plane line to leave at least the innermost layer, the biosensors with a multilayer structure for simultaneously measuring multiple items can be formed without sustaining any damage, such as flaws in the inner layer portion. The above-described biosensors are advantageous in that they can endure forces such as sudden bending applied in the manufacturing process or in storage state.
In the present invention, a part of the region sandwiched between the substrate and cover can comprise a desiccant and/or deoxidant. As the biosensors for simultaneously measuring multiple items of the present invention are sealed with the reaction-detecting section included therein, such a desiccant and/or deoxidant, when provided in the biosensor, can maintain an internal dry state or oxygen-free state over a long period of time. Even when the biosensors are manufactured and sealed such that the reaction-detecting section of the biosensors is included in an environment containing humidity or oxygen at the time of assembling the biosensor, the interior of the biosensor can be set in a dry or deoxygenated state. The desiccant and/or deoxidant preferably avoids direct contact with the sample solution. It is also preferable that the desiccant and/or deoxidant are arranged so as not to cross the sample-feeding path. The same desiccants and deoxidants as described for the aforementioned first invention can be used.
The biosensors for simultaneous measurement of multiple items of the present invention can comprise a humidity indicator and/or oxygen-detecting agent in a part of the region sandwiched between the substrate and cover. To confirm a dry and/or deoxygenated state in the biosensor for simultaneous measurement of multiple items, a desiccant in combination with a humidity indicator, and/or a deoxidant in combination with an oxygen-detecting agent, can be used. Humidity indicators are not particularly limited as long as they can be used in the packaging of the present invention. The same oxygen-detecting agents as those described for the aforementioned first invention can be used.
In such cases, it is preferable that a part or all of the substrate and/or cover are of a material transparent to visible rays, rendering the humidity indicator and/or oxygen-detecting agent visible. It is also preferable that a part or all of the substrate and/or cover are of a material that can shield ultraviolet rays. In such cases, the entire substrate and/or cover may be an ultraviolet shielding material, or the top surface of the substrate and/or cover may be coated with a film of an ultraviolet-shielding material. Examples of the film include films comprising an organic compound such as benzotriazole or the like, and films comprising a fluorescent agent which converts ultraviolet rays to visible light.
When using a substrate or cover that is not transparent to visible light, the humidity indicator and/or oxygen-detecting agent can be arranged to be present at the spacer portion of the cut surface that newly appears when the sealed cap portion is cut. In such cases, the humidity indicator or oxygen-detecting agent may be contained in the spacer layer, or constituted as part of the spacer layer. This arrangement enables confirmation of the state of the interior, as indicated by the humidity indicator and/or oxygen-detecting agent, at the cut surface or inside near the cut surface after cutting and immediately before use.
The substrate or cover is preferably made of a material that does not transmit ultraviolet light. The top surface of the substrate or cover may be coated with an ultraviolet absorber or a material that does not transmit ultraviolet. When the substrate or cover are made of an ultraviolet non-transmitting material, or are coated with an ultraviolet-absorber or a material that does not transmit ultraviolet, transmission of ultraviolet rays can be inhibited or blocked. The ultraviolet-absorbers and materials that do not transmit ultraviolet that can be used are the same as those described for the aforementioned first invention.
The substrate or cover may contain a compound with a photocatalytic effect, or the top surface of the substrate or cover may be coated with a layer containing a compound with a photocatalytic effect. Compounds with a photocatalytic effect that may be used are the same as those described for the aforementioned first invention.
A spacer layer close to an exposed sample-inlet port, or close to a sample-inlet port and an air-discharge port, may comprise a fluorescent or luminescent agent. It is particularly preferable that the fluorescent or luminescent agent is close to the sample-inlet port.
The fluorescent or luminescent agent can form a mark to improve visibility, and thus can prevent mishandling when supplying the sample. When a fluorescent or luminescent agent is used at the cut surface portion, it may be comprised in the spacer member or constituted as part of the spacer. When the fluorescent or luminescent agent is used to form a mark near the sample-inlet port of the substrate or cover, the mark can be formed by printing or such. Luminescent agents may be those well known in the art, where a light emission reaction starts upon contact with oxygen in the air.
The electrodes in the biosensors for simultaneously measuring multiple items of the present invention may form an array. When a biosensor forming an array is cut along the cutting plane line, at least one sample-inlet port is opened, and a reaction-detecting section comprising one electrode system and one reagent layer is located ahead of one sample-feeding path connected to the sample-inlet port. The one sample-inlet port may be connected to one sample-feeding path, or connected to at least two sample-feeding paths branched from the sample-inlet port. The reaction-detecting section comprising one electrode system may be located ahead of the sample-feeding path. In particular, one sample-inlet port is preferably connected to one sample-feeding path. This more surely prevents mixing of sample solutions in the reagent layers. When at least two sample-feeding paths are branched from one sample-inlet port, a surfactant may be coated inside the sample-feeding path so that the sample solution can reach all of the arrayed reagent layers. When the sample solution is blood, heparin, prolixin-S, or a metal salt of ethylenediaminetetraacetic acid or citric acid may be coated as an anticoagulant agent.
The biosensors for simultaneously measuring multiple items of the present invention comprise at least two biosensor unit-comprising substrates. When the biosensors comprise at least three or more biosensor unit-comprising substrates, each of the biosensor unit-comprising substrates is preferably regularly laid out on the serial substrates at predetermined intervals while separated by cutting plane lines. This structure enables efficient manufacture of biosensors for simultaneously measuring multiple items at a time. Each of the biosensor units in the adjacent biosensor unit-comprising substrates can be connected to a measuring section to simultaneously measure multiple samples.
The biosensors for simultaneously measuring multiple items of the present invention can be manufactured, for example, by pre-patterning the electrode systems on the substrate and the spacers on the top surface of the substrate or cover, then laying out the reagent layer, and adhering the substrate and cover using an adhesive. Specifically, for example, the cutting plane line is pre-formed on the outside surface of the substrates, and then the electrode pattern is formed inside the substrates by screen-printing or the like. Meanwhile, similarly, the cutting plane line is pre-formed on the outside surface of the cover as necessary, and then the pattern of the adhesive layer is formed as the spacer inside the cover.
A reagent layer can be formed on the sample-feeding path of the substrates by using a dispenser method to drop an enzyme-comprising reagent solution. The space where a regulating agent or indicator is placed can be simultaneously formed on the substrate or cover as part of the spacer pattern formed inside the cover. The biosensors for simultaneously measuring multiple items can be constructed by adhering the covers and the substrates, formed as described above.
<Method for Using a Biosensor for Simultaneous Measurement of Multiple Items/Method for Measuring Test Compounds>
The present invention provides methods for using the biosensors for simultaneously measuring multiple items, wherein said methods comprise the steps of:
The present invention also provides methods for measuring a measuring target using the biosensors of the present invention, wherein said methods comprise the steps of:
At the above-described step (1), both the substrate and cover can be cut to expose the cut surface, and then step (3) can be performed. Alternatively, one of the substrate or cover can be cut and bent, and the other can be left connected to expose the cut surface, and then step (3) can be performed while the biosensor for simultaneous measurement of multiple items is bent. The latter mode is particularly preferable.
After the above-described cutting step, the cut surface of each biosensor unit-comprising substrate of the biosensors for simultaneously measuring multiple items of the present invention, or the cut surfaces of a number of biosensor unit-comprising substrates at one time, can be contacted with the solution, as in step (3). When the cut surfaces of multiple biosensor unit-comprising substrates are simultaneously contacted with a solution, the biosensor unit-comprising substrates preferably comprise one biosensor unit, or two or more biosensor units. In such cases, to expose the cut surface it is preferable to cut and bend one of the substrate or cover, with the other left connected. When the cut surface of each biosensor unit-comprising substrate is contacted with a solution, the biosensor unit-comprising substrate preferably comprises a number of biosensor units. It is preferable that the substrates or covers are cut to expose the cut surfaces, and that each biosensor unit-comprising substrate is then used. In this way, simultaneous measurement of biosensor units comprising a number of reagent layers can be provided.
The measuring targets are not limited, as long as they can be measured by the biosensors. For example, measuring targets can be the amounts of compounds such as enzymes and DNAs, the quantity of ions, the quantity of oxygen, the pH of the solution, and properties such as conductivity.
<Biosensor Devices>
The biosensor devices of the present invention comprise:
It is preferable that the connector section alters the shape of the biosensor unit-comprising substrate for opening the sample-inlet port; fixes the biosensor unit-comprising substrate with the shape; and then captures electrical signals at the electrodes of the biosensor.
Preferable measuring methods in the measuring section are potential step chronoamperometry, coulometry, or cyclic voltammetry.
<Applications>
The first and second biosensors for simultaneously measuring multiple items of the present invention can be used to measure the following measuring targets, by changing reagent layer types.
In enzyme sensors, for example, the type of enzyme used as a molecular identifier is changed according to the sample's measurement target. For example, glucose oxydase or glucose dehydrogenase is used when the measuring target is blood glucose (glucose) or urine sugar; a mixture of fructosyl amine oxidase and protease is used when the measuring target is glycosylated hemoglobin; lactate oxydase is used when the measuring target is lactic acid; a mixture of cholesterol esterase and cholesterol oxydase is used when the measuring target is the total cholesterol or the like; urate oxydase is used when the measuring target is uric acid; alcohol oxydase is used when the measuring target is ethanol; glutamate oxydase is used when the measuring target is glutamate; pyruvate oxidase is used when the measuring target is pyruvic acid or phosphoric acid; a combination of maltose phosphorylase, alkaline or acid phosphatase, and/or mutarotase, and glucose oxydase is used when the measuring target is maltose or phosphoric acid; and a combination of sucrose phosphorylase, alkaline or acid phosphatase, mutarotase, and glucose oxydase is used when the measuring target is sucrose or phosphoric acid.
In the above-described enzyme sensors, electron carriers (mediators) are used together with enzymes. As the mediators, potassium ferricyanide, ferrocene, ferrocene derivatives, nicotinamide derivatives, flavin derivatives, benzoquinone, quinone derivatives, and the like can be used.
In pH sensors, a reagent layer of a quinhydrone and inorganic salt such as sodium chloride or potassium chloride is provided on the substrate comprising a silver/silver chloride electrode and another electrode. In such cases, a change in potential between electrodes is measured.
In single nucleotide polymorphism (SNP) sensors (A. Ahmadian et al., Biotechniques, 32, 748, 2002), a mixture of primers, DNA polymerases, and deoxyribonucleotide triphosphates is further used as a reagent on the pH sensors to measure a change in pH when the subject DNAs complement to the primers in the sample.
In immunosensors, the antigen-antibody reactions are used. For example, when serum albumin is measured, anti-albumin is uses as a molecular identifier. The immunosensors measure the potential between the electrodes, which changes depending on the formation of an antigen-antibody complex.
In microorganism sensors, microorganisms and soil microbes of the genus Acetobacter, Actinomaaura, Agrobacterium, Alcaligenes, Aphanomyces, Armillaria, Aspergillus, Bacillus, Burkholderia, Candida, Cephalosporium, Ceratocystis, Cladosporium, Clavibacter, Corticium, Corynebacterium, Cylindrocarpon, Cylindrocladium, Enterobacter, Erwinia, Flavobacterium, Fusarium, Gaeumannomyces, Ganoderma, Gibberella, Gliocladium, Gluconobacter, Glycomyces, Helicobasidium, Actobacillus, Leptosphaeria, Micobacterium, Micrococcus, Monosporascus, Mucor, Nocardia, Olpidium, Pasteuria, Penicillium, Phoma, Plasmodiophora, Phytophthora, Polymyxa, Proteus, Pseudomonas, Pyrenochaeta, Pythium, Ralstonia, Rhizobium, Rhizoctonia, Rhizopus, Rhodococcus, Rosellinia, Saccharomonospora, Sclerotina, Scietotium, Serratia, Sphingomonas, Spongospora, Streptococcus, Streptomyces, Streptoverticilium, Synchytrium, Talaromyces, Thanatephorus, Thielaviopsis, Torula, Trichoderma, Typhula, Verticillium, Zymomonas, and Xanthomonas are used as the molecular identifier, such as Pseudomonas fluorescence (measuring target is glucose or BOD (biochemical oxygen demand), soil), Trichosporon cutaneum, Pseudomonas putida (measuring target is BOD), and Trichosporon brassicae (measuring target is ethanol).
As these microorganisms aerobically respire (i.e., are aerobic bacteria), or produce metabolites in an oxygen-free environment, the amount of aerobic respiration or metabolites are to be monitored electrically.
Organella sensors use cell organella as the molecular identifiers. By using mitochondrial electron transport particles, for example, NADH can be measured. This is based on the following principle: NADH is oxidized by mitochondrial electron transport particles, at which time oxygen is consumed; and thus NADH and NADPH can be measured using oxygen as an index.
In receptor sensors, for example, receptors such as cell membranes are used as molecular identifiers. Hormones, neuro transmitters, or the like become target samples. The measuring principle is that a change in reception is converted to an electrical potential and measured via electrodes.
A tissue sensor uses the tissues of plants or animals as molecular identifiers. As plant or animal tissues, for example, frog skin, sliced animal liver, or cucumber or banana peel can be used. For example, the measuring principle with sodium sensors using frog skin tissues is as follows: the frog skin tissue selectively passes sodium ions, changing the potential of the skin tissues; and thus measuring the change in potential will provide the amount of sodium ions.
Another application of the first and second biosensors as described above is a DNA chip. In DNA chips, multiple kinds of single-stranded nucleic acid probes, which are complementary to multiple types of target genes to be detected, are fixed onto an array of electrodes, and one nucleic acid probe is fixed to one electrode. To confirm the presence or absence of multiple target genes, sample genes denatured to single-stranded are hybridized with the nucleic acid probes, and then double strand identifiers, which are electrochemically active and specifically bind to double-stranded nucleotides, are added. After washing, the substrate is folded in buffering solution. A voltage is sequentially applied to each electrode using the arrayed electrodes as the working electrode, and an upper large electrode as the opposite electrode. Double-stranded intercalating agent is oxidized when a double strand is formed, causing an oxidation current to flow. Current caused by the intercalating agent does not flow in electrodes when a double strand does not form. The type of nucleic acid probes can be identified by the position of the electrode which generates the current. Thus, the presence or absence of target genes and their properties can be determined. Intercalators (intercalating agents) such as acridine orange, and metaro intercalators (intercalating agents) such as tris-phenanthroline cobalt complex, can be used as double strand identifiers.
<Manufacturing Methods>
The first and second biosensors of the present invention can be manufactured, for example, by pre-patterning an electrode system on the substrate, and the spacer on the top surface of the substrate or cover; then laying out the reagent layer; and finally adhering the substrate or cover using an adhesive. More specifically, the first biosensors, for example, can be manufactured as follows: A cutting plane line is formed on the outside surface of a substrate, and then an electrode pattern is formed inside by screen printing or the like. Meanwhile, similarly, a cutting plane line is formed on the outside surface of a cover, and a pattern of an adhesive layer is formed inside as a spacer. A portion on the cover where the adhesive layer is not present is used as the space in a sample-feeding path and a sealed cap portion where a regulating agent or indicator is placed.
By using a dispenser method to drop an enzyme-comprising reagent solution, a reagent layer can be formed on the sample-feeding path of a substrate on at least one portion of the sample-inlet port and air-discharge port, which are formed by separating the sealed cap. The space where the regulating agent or indicator is placed can be formed on the cover at the same time, as a part of the adhesive layer pattern formed inside the cover. Biosensors with a boundary with the sealed cap can be constructed by adhering the cover and substrate formed in this manner.
After development of the reagent layer, the biosensor assembling steps do not use packing systems accompanied by heat, such as thermal compression or the like, and can be performed by merely adhering the substrate and cover via the spacer. A sample-feeding path to be the sample-inlet port and air-discharge port are included inside the biosensors manufactured as described, and thus the interior of the biosensors can be kept highly airtight. The second biosensors can be manufactured by using the same methods.
The biosensors of the present invention can be easily manufactured without using thermal compression or the like, and can increase yield, allowing the influence of oxidation of reagent layers to be eliminated, and providing excellent preservation stability over a long period of time. In addition, the essential portions of the biosensors, such as the sample-inlet port, the sample-feeding path, and the reaction-detecting section, are completely sealed after the manufacturing step, ensuring the environment inside the biosensor is extremely airtight. Therefore, a preferred internal environment can be generated by regulating manufacturing steps, and can be maintained over a long period of time. The internal environment can be better maintained over a long period of time by incorporating a regulating agent such as desiccant, to adjust the internal environment as needed. Furthermore, when in use, the incorporated internal-environment regulating agent is separated from the body of the biosensors with a structure of the present invention, and thus contact with sample solutions can be completely eliminated.
Considering the structures, materials, and manufacturing methods of the biosensors of the present invention, the biosensors of the present invention can significantly reduce environmental burdens at the time of manufacture and after use, compared to conventional disposable biosensors.
The biosensors for simultaneously measuring multiple items of the present invention have specific structures and thus by using them, multiple items can be simultaneously measured, even with a small amount of sample solution, and by supplying sample solution to the sample-feeding path the influence of at least one other biosensor reagent can be eliminated.
Japanese Patent Application Nos. 2004-084116 and 2004-127937 are incorporated in the present application.
Any patents, published patent applications, and publications cited herein are incorporated by reference.
Herein below, the present invention will be specifically described using Examples, but it is not to be construed as being limited thereto.
a shows the outside of rectangular substrate 1 of a typical biosensor. On top of substrate 1 is a horizontally made V-shaped notch 7, which becomes a cutting plane line. Notch 7 is provided so that sealed cap portion 10 of the biosensor can be cut along broken line 14, by bending or the like, when using the biosensor.
b shows the inside of substrate 1. Both pattern 4, which includes a pair of electrodes, and reagent layer 6 are formed along the substrate 1 centerline, on the inside upper surface of the substrate. Pattern 4, which includes the electrodes, has two electrode members arranged in parallel from the bottom end until near broken line 14 at the top.
c shows the outside of cover 2. A horizontally formed notch 7, similar to the notch on substrate 1, exists along broken line 14 at the top of cover 2.
e is a structural diagram where the inner surfaces of substrate 1 and cover 2 are overlapped, aligning both top ends. By adhering substrate 1 and cover 2, the portion on cover 2 comprising no spacer becomes a space comprised inside the biosensor, resulting in sample-feeding path 5. By adjusting cover 2 to be shorter than substrate 1, overlapping the two with their top ends aligned causes the bottom of pattern 4, which includes the electrodes, to be exposed, resulting in terminal 8, as shown in
a shows the inner structure of the development of the biosensor in
f shows an A-A′ cross-sectional view of slightly below notch 7 in the sensor portion 9 of the biosensor shown in
The structure of the biosensor of
Furthermore, the biosensors with this structure include the following characteristics: a very small quantity of sample solution can be used; the biosensors have a simple structure and can thus be easily manufactured; and the state of the biosensors at the time of manufacturing can be maintained until use, since the reaction-detecting section is included inside the biosensors, keeping them extremely airtight.
In addition to the above-mentioned characteristics common to the biosensors of the present invention, those for each form of the biosensors described in the present invention will be described hereinafter.
In
b shows the inner surface of cover 2. On the inside upper surface of cover 2, the spacer layer, as well as a portion 5 inside the spacer layer, which comprises no spacer, are provided in a trapezoid shape, with the acute angle down.
The cross-sectional views of this structure shown in
In such biosensors, after sample solution 13 passes through the electrode part intersecting with the sample-feeding path, the feeding of the sample solution can nearly be stopped near the acute angle, since the surface area required for capillary action is partly interrupted in the part bent at the acute angle. Accordingly, these biosensors can measure a smaller amount of sample solution. Alternatively, if the feeding of the sample solution is not stopped, a highly repellent material can be provided as a stopper, by printing or the like at a preferable part where feeding should be stopped, or it can be applied to the wall surface of the sample-feeding path beyond the acute angle.
Since the reagent-feeding path is curved, a sample solution can be smoothly supplied to a position past the electrode part that intersects with the sample-feeding path, and stopped at the bent portion of the sample-feeding path further on. Accordingly, the required amount of sample solution can be reduced.
In this structure, as for
The biosensor shown in
In the biosensor shown in
Desiccant 15 is an example used herein, but other agents can be used in addition to or instead of a desiccant. Therefore, a deoxidant alone or in combination with a desiccant can be used, and furthermore, a humidity indicator or oxygen-detecting agent alone or in combination with the above-described agents can be used.
a to 8f show development diagrams of the above-described biosensors as shown in FIGS. 1 to 6, except for cover 2.
c shows the outer part of cover 2. Horizontal notch 7 is provided along broken line 14 in the top region of cover 2, as for substrate 1.
f shows an exemplary use of the biosensor, and
A variety of DNA sequences can be simultaneously detected in the same sample solution by immobilizing different reagents as probes on each pair of electrodes on arrayed biosensors. These reagents can be, specifically, DNAs comprising different nucleotide sequences. For example, in the case of single nucleotide polymorphism (SNP) sensors (see, for example, A. Ahmadian et al., Biotechniques, 32, 748, 2002), a mixture of primers, DNA polymerases, deoxyribonucleotide triphosphates, and the like can be used as reagents, and electrodes measure change in pH near the electrode, which takes place when test DNAs in a sample are complementary to the primers.
Similarly, by immobilizing a variety of antibodies on each pair of electrodes, the biosensor, as an immune sensor, can simultaneously measure a variety of measuring targets in the same sample solution. For example, anti-albumin is used as a molecular identifier for measuring human serum albumin. Immune sensors measure the potential between electrodes, which changes when antigen-antibody complexes form.
Instead of an above-described structure, the arrayed biosensors may be structured to have at least two sample-inlet ports, where a reaction-detecting section comprising at least one pair of electrodes exists ahead of the sample-feeding path connected with each sample-inlet port, and the sample-inlet ports appear after cutting the sealed cap portion, as for the above biosensor; or structured to have at least two sample-feeding paths branched from at least one sample-inlet port and a reaction-detecting section comprising at least one pair of electrodes exists ahead of the sample feeding path.
c shows an exemplary biosensor structure, where terminal 8 of sensor portion 9 is simply packaged by the protective film, consisting of the top end (protective-film fixing portion 21) fixed to the upper surface of cover 2 by a strong adhesive, and the other part with no detachable adhesive layer.
c is an example of a biosensor where perforations 23 are also provided between the terminal protective cover portion 22; and
a shows the outside of rectangular substrate 101 of a typical biosensor for simultaneously measuring multiple items. On the center portion of substrate 101, a vertical cutting plane line running from top to bottom is provided as V-shaped notch 107. At time of use, notch 107 is used to bend the biosensor for simultaneously measuring multiple items in to a V-shape along the broken line 112.
b shows the inside of substrate 101. Inside substrate 101, pattern 104, including two pairs of electrodes, is arranged in parallel from top to bottom, with the substrate central broken line 112 as a boundary. Reagent layer 106 is formed on parts of the electrode pattern of each pair. To clarify the pattern sections that become reagent layer 106, a resist layer may be provided between an adhesive layer corresponding to spacer 103 in
c shows the outer part of cover 102. On the center portion of cover 102, as for substrate 101, the V-shaped notch 107 runs vertically from top to bottom.
e is a structural diagram where the inner surfaces of substrate 101 and cover 102 are overlapped with their tops aligned, showing the biosensor for simultaneously measuring multiple items 115. By making cover 102 shorter than substrate 101, the bottom of the electrode pattern 104 is exposed when the two are overlapped with their tops aligned. This portion can be used as terminal 108, shown in
f shows an A-A′ cross-sectional view of the sample-feeding path on the upper side of the biosensor for simultaneously measuring multiple items shown in
h shows an exemplary use of the biosensor for simultaneously measuring multiple items of the present invention.
By contacting two adjacent sample-inlet ports 109 in this state with a sample solution 111, the sample solution 111 is independently supplied to the adjacent sample-feeding paths 105 by capillary action. At this time, if sample solution 111 is slightly rounded by surface tension, as shown in the figure, sample solution 111 is effectively supplied to sample-feeding paths 105 since the two biosensor unit-comprising substrates combine to form a V-shape, as shown in
This structure, as shown in
In addition to the above-mentioned common characteristics of the biosensors for simultaneously measuring multiple items of the present invention, characteristics of the biosensors for simultaneously measuring multiple items proposed herein will be described hereinafter.
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. On the center portion of substrate 101, a vertical cutting plane line 112 running from top to bottom is provided as V-shaped notch 107. At time of use, notch 107 is used to bend the biosensor for simultaneously measuring multiple items in to a V-shape along the broken line 112.
b shows the inside of substrate 101. Inside substrate 101, patterns 104 including four pairs of electrodes are symmetrically arranged on substrate 101, with the central broken line 112 of the substrate as a boundary. Also, four reagent layers 106 are formed on a part of each electrode pair pattern. Insulating resist 113 is applied to the inside structure of substrate 101 of
c shows the outer part of cover 102. On the center portion of cover 102, as for substrate 101, vertical cutting plane line 112 runs from top to bottom as V-shaped notch 107.
e shows a structural diagram of a biosensor for simultaneously measuring multiple items 115, where the inside of substrate 1 and cover 102 are overlapped with their tops aligned. By making cover 102 shorter than substrate 101, the bottom of the electrode pattern 104 is exposed when the two are overlapped with their tops aligned. This portion can be used as terminal 108, shown in
f shows an A-A′ cross-sectional view of the sample-feeding path on the top half of a biosensor for simultaneously measuring multiple items shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
Consequently, two biosensor unit-comprising substrates can together form a V-shape, as shown in the figure. At this time, sample-feeding path 105 is divided along the V-shaped notch bordering each of the two biosensor unit-comprising substrates, and the sample-inlet ports 109 of all of the four biosensor units are adjacent and formed in one place.
By contacting four sample-inlet ports 109 in this state with sample solution 111, the sample solution is independently supplied to the sample-feeding paths 105 by capillary action. To smoothly supply sample solution 111 to a sample-feeding path 105, a total of four air-discharge ports 110 are provided on the opposite side of sample-inlet port 109.
Characteristically, the structure in
The biosensor for simultaneously measuring multiple items in
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. On the center portion of substrate 101, a vertical cutting plane line 112, running from top to bottom, is provided as V-shaped notch 107. At time of use, this notch 107 is used to bend the biosensor for simultaneously measuring multiple items in to a V-shape along the broken line 112.
b shows the inside of substrate 101. Inside substrate 101, two pairs of patterns 104, which include electrodes, are symmetrically arranged on substrate 101 at a distance from the top, with the central broken line 112 of the substrate as a boundary. Two reagent layers 106 are formed in an area on each pair of electrode patterns. Although not shown in
c shows the outer part of cover 102. On the center portion of cover 102, as for substrate 101, a vertical cutting plane line 112 running from top to bottom is provided as V-shaped notch 107.
e shows a structural diagram of a biosensor for simultaneously measuring multiple items 115, where the inner surfaces of substrate 101 and cover 102 are overlapped with their tops aligned. By making cover 102 shorter than substrate 101, the bottom of electrode pattern 104 is exposed when the two are overlapped with their tops aligned. This portion can be used as terminal 108, as shown in
f shows an A-A′ cross-sectional view of the sample-feeding path upper portion of the biosensor for simultaneously measuring multiple items 115 shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
At this time, sample-feeding path 105 is divided along the V-shaped notch at the boundary of the two biosensor unit-comprising substrates, to adjacently form sample-inlet port 109 and air-discharge port 110 for each of the two biosensor units in the same position. By contacting two sample-inlet ports 109 in this state with a sample solution 111, the sample solution is independently supplied to the semicircular sample-feeding path 105 by capillary action. To smoothly supply sample solution 111 to sample-feeding path 105, air-discharge ports 110 are provided on the cross sections of the same biosensor unit-comprising substrates that comprise sample-inlet ports 109.
The structure of the biosensor for simultaneously measuring multiple items exemplified in
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. In the center of substrate 101 is a vertical cutting plane line, running from top to bottom as V-shaped notch 107. At time of use, notch 107 is used to bend the biosensor for simultaneously measuring multiple items in to a V-shape along the broken line 112.
b shows the inside of substrate 101. On the inner surface of substrate 101, patterns 104, including two pairs of electrodes, run from top to bottom of the substrate, and two reagent layers 106 are in a section of each electrode pattern, with central broken line 112 of the substrate as a boundary. A resist layer is also provided on part of the two pairs of electrode patterns. As a result, two points of electrode pattern intersection exist per pair of sample-feeding paths, and the lower intersection point of substrate 101 is insulated by resist 113. Thus reagent layer 106 is only provided at the upper intersection point of substrate 101.
c shows the outer part of cover 102. In the center portion of cover 102, as for substrate 101, a vertical cutting plane line 112 runs from top to bottom as V-shaped notch 107.
e shows a structural diagram of the biosensor for simultaneously measuring multiple items 115, where the inner surfaces of substrate 101 and cover 102 are overlapped with their tops aligned. Making cover 102 shorter than substrate 101 exposes the bottom of electrode pattern 104 when the two are overlapped with their tops aligned. This portion can be used as terminal 108, shown in
f shows an A-A′ cross-sectional view of the sample-feeding path on the top side of the biosensor for simultaneously measuring multiple items shown in
g shows a B-B′ cross-sectional view of the electrode pattern in the biosensor for simultaneously measuring multiple items shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
At this time, sample-feeding path 105 is divided along the V-shaped notch at the boundary between the two biosensor unit-comprising substrates, resulting in sample-inlet port 109 and air-discharge port 110 of the two biosensor units being adjacent and in the same place on the cross section.
In the structure in
The biosensor for simultaneously measuring multiple items exemplified in
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. In the center portion of substrate 101, a vertical cutting plane line 112 runs from top to bottom as V-shaped notch 107. When in use, this notch 107 is used to bend the biosensor for simultaneously measuring multiple items in to a V-shape along broken line 112.
b shows the inside of substrate 1. Inside substrate 101, patterns 104 including two pairs of electrodes are symmetrically arranged on substrate 101 at a distance from the top, with the central broken line 112 of the substrate as a boundary. Two reagent layers 106 are formed in an area of each pair of electrode patterns. Although not shown in
c shows the outer part of cover 102. In the center portion of cover 102, as for substrate 101, a vertical cutting plane line 112 runs from top to bottom as a V-shaped notch 107.
e shows a structural diagram of a biosensor for simultaneously measuring multiple items 115, showing the inner surfaces of substrate 101 and cover 102 overlapped with their tops aligned. By making cover 102 shorter than substrate 101, the bottom of electrode pattern 104 is exposed when the two overlap with their tops aligned. This portion can be used as terminal 108, shown in
f shows an A-A′ cross-sectional view of the sample-feeding path on the upper side of the biosensor for simultaneously measuring multiple items shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
By contacting the two sample-inlet ports 109 in this state with sample solution 111, the sample solution is independently supplied to semicircular sample-feeding path 105 by capillary action, and the sample solution stops near sample-feeding path 5 adjacent to the regulating agent layer. To smoothly supply sample solution 111 to sample-feeding path 105, air-discharge port 110 is provided on the same cross section of the biosensor unit-comprising substrate as sample-inlet port 109.
i shows a front view of the two biosensor unit-comprising substrates dividing the substrate in two to form a V-shape.
The biosensor for simultaneously measuring multiple items exemplified in
a-i shows a top view of a biosensor for simultaneously measuring multiple items 115, and measuring unit 116. Terminal 108 is located at the bottom of the biosensor for simultaneously measuring multiple items 115. Supply part 117 of measuring unit 116 is composed of horizontal movement part 118, for sliding the biosensor for simultaneously measuring multiple items 115, horizontal movement guidance part 119, and the upper portion of folding portion 120 to bend the biosensor for simultaneously measuring multiple items 115 along V-shaped notch 107 when moving horizontally to insert the biosensor for simultaneously measuring multiple items into the supply part of the measuring unit.
a-ii shows a biosensor for simultaneously measuring multiple items when connected to the measuring unit. When a biosensor for simultaneously measuring multiple items 115 is connected to measuring unit 116, the biosensor for simultaneously measuring multiple items is transformed into a V-shape in the form shown in
a is a perspective diagram. Electrode patterns 104 are symmetrically arranged in two rows and ten columns, with the V-shaped notches 107 of substrate 101 and cover 102 as a center, and sample-feeding path 105 intersecting each electrode.
c shows an A-A′ cross-sectional view, and
In the arrayed biosensor for simultaneously measuring multiple items of
a is a perspective diagram.
For example, since 20 biosensor units are included in one biosensor unit-comprising substrate in this arrayed biosensor for simultaneously measuring multiple items, a total of 200 sample-inlet ports 109 are formed.
c shows an A-A′ cross-sectional view, and
In the arrayed biosensor for simultaneously measuring multiple items of
In the case of this arrayed biosensor for simultaneously measuring multiple items, wiring 122 orthogonal to a sample-feeding path does not necessarily have to be insulated by resist layer 113, which is different from the arrayed simultaneous multi-item measuring biosensors shown in
a is a perspective diagram.
c shows an A-A′ cross-sectional view, and
In the arrayed biosensor for simultaneously measuring multiple items of
When in actual use, as for Example 22, biosensors with sample solution 111 pre-arranged on a flat substrate so as to have a certain contact angle can be used for measurement. Also, by facing the sample-inlet port 109 upwards, sample solution can be directly supplied using a sample-dispensing apparatus, such as a spotter. In these cases, for the reason mentioned in Example 23, less sample solution is needed for measurement, compared with Example 22.
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. Horizontally formed V-shaped notch 107 is in the center portion of substrate 101.
b shows the inside of substrate 101. Inside substrate 101, patterns 104, including two pairs of electrodes, are arranged to face each other, with the central broken line 112 of the substrate as a boundary. Also, reagent layer 106 is formed in a portion of the electrode pattern of each pair. Although not shown in
c shows the outer part of cover 102. In the center portion of cover 102, a horizontal cutting plane line is located in the form of V-shaped notch 107, as for substrate 101.
e shows a structural diagram of a biosensor for simultaneously measuring multiple items 115, where the inner surfaces of substrate 101 and cover 102 are overlapped while aligned along horizontal central line 112. Making cover 102 shorter than substrate 101 forms terminal 108 at both ends. Two biosensor unit-comprising substrates 128, each including one biosensor unit 127, exist with notch 107 as a boundary.
f shows an A-A′ cross-sectional view of the sample-feeding path on the upper side of the biosensor for simultaneously measuring multiple items, shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
At this time, sample-feeding path 105 is divided along the V-shaped notch at the boundary of the two biosensor unit-comprising substrates, and sample-inlet ports 109 for each biosensor units are formed adjacently in one place. By contacting two adjacent sample-inlet ports 109 in this state with sample solution 111, the sample solution is independently supplied to sample-feeding paths 105 of the adjacent biosensor units by capillary action. To smoothly supply sample solution 111 to sample-feeding path 105, air-discharge port 110 is located on the opposite side of sample-inlet port 109.
The structure of the biosensor for simultaneously measuring multiple items in
The outside structure of
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. Horizontally formed V-shaped notch 107 is located in the center portion of substrate 101.
b shows the inside of substrate 101. Inside substrate 101, patterns 104, including two pairs of electrodes, are arranged to face each other, with central broken line 112 of the substrate as a boundary. Also, reagent layer 106 is formed in a portion of each pair of electrode patterns. Although not shown in
c shows the outer part of cover 102. In the center portion of cover 102, a horizontally formed cutting plane line is located in the form of V-shaped notch 107, as for substrate 101. Furthermore, in contrast to
e shows a structural diagram of a biosensor for simultaneously measuring multiple items 115, where the inner surfaces of substrate 101 and cover 102 are overlapped while aligned alone each horizontal central line 112. By making cover 102 shorter than substrate 101, terminal 108 is formed at both ends. Also, one of the two biosensor units 127 are included in each of the two biosensor unit-comprising substrates 128, with notch 107 as a boundary, and each biosensor unit-comprising substrate 128 is has one air-discharge port 124.
f shows an A-A′ cross-sectional view of the sample-feeding path on the upper side of the biosensor for simultaneously measuring multiple items shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
In contrast to the biosensor for simultaneously measuring multiple items exemplified in
On the outside,
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. The horizontally formed V-shaped notch 107 is located in the center portion of substrate 101.
b shows the inside of substrate 101. Inside substrate 101, four pairs of patterns 104, including electrodes, are arranged to face each other in two sets of two pairs, with the central broken line 112 of the substrate as a boundary. Also, reagent layer 106 is formed in a portion of each pair of electrode patterns. Although not shown in
c shows the outer part of cover 102. In the center portion of cover 102, a horizontally formed cutting plane line is located in the form of V-shaped notch 107, as for substrate 101.
e shows a structural diagram of the biosensor for simultaneously measuring multiple items 115, where the inner surfaces of substrate 101 and cover 102 are overlapped with their horizontal central line 112 aligned. By making cover 102 shorter than substrate 101, a terminal 108 forms at both ends. Also, the four biosensor units 127 exist in groups of two, in two biosensor unit-comprising substrates 128, with notch 107 as a boundary. Furthermore, air-discharge ports 110, derived from each biosensor unit, are provided on different side faces to the cross section of biosensor unit comprising substrate 128.
f shows an A-A′ cross-sectional view of the sample-feeding path on the upper side of the biosensor for simultaneously measuring multiple items shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
At this time, in sample-feeding path 105, the four biosensor units are divided along the V-shaped notch into two lots of two, and sample-inlet ports 109 are formed adjacently in one place for each biosensor unit. By contacting four adjacent sample-inlet ports 109 in this state with sample solution 111, the sample solution is independently supplied to each of the sample-feeding paths 105 of the adjacent biosensor units. To smoothly supply sample solution 111 to sample-feeding path 105, air-discharge port 110 is provided on the side face of the biosensor unit-comprising substrate, located at the back of reagent layer 106 where the electrodes exist.
In contrast to the biosensor for simultaneously measuring multiple items exemplified in
The outer structure of
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. The horizontally formed V-shaped notch 107 is located in the center portion of substrate 101.
b shows the inside of substrate 101. Inside substrate 101, patterns 104, including four pairs of electrodes, are arranged to face each other in two lots of two, with the central broken line 112 of the substrate as a boundary. Also, reagent layer 106 is formed in a portion on each pair of electrode patterns. Although not shown in
c shows the outer part of cover 102. A horizontally formed cutting plane line is located in the form of V-shaped notch 107 in the center portion of cover 102, as for substrate 101. Furthermore, in contrast to
e shows a structural diagram of the biosensor for simultaneously measuring multiple items 115, where the inner surfaces of substrate 101 and cover 102 are overlapped with their horizontal central lines 112 aligned. By making cover 102 shorter than substrate 101, terminals 108 are formed at both ends. Also, four biosensor units 127 are separated into two with notch 107 as a boundary, included in two biosensor unit-comprising substrates 128, where each biosensor unit has one air-discharge port 124.
f shows an A-A′ cross-sectional view of the sample-feeding path on the upper side of the biosensor for simultaneously measuring multiple items shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
Consequently, the two biosensor unit-comprising substrates can be folded as shown in the figure. At this time, the four biosensor units of sample-feeding path 105 are divided along the V-shaped notch into two groups of two, and the sample-inlet ports 109 of the respective biosensor units are formed adjacently in one place. By contacting four adjacent sample-inlet ports 109 in this state with sample solution 111, the sample solution is independently supplied to sample-feeding paths 105 of the adjacent biosensor units by capillary action. To smoothly supply sample solution 111 into sample-feeding path 105, air-discharge port 124 is provided so as to penetrate cover 102 at the back of the reagent layer 106, where the electrodes exist.
In contrast to the biosensor for simultaneously measuring multiple items exemplified in
a, b, and f are identical to
By using cover 102 to cover the biosensor for simultaneously measuring multiple items up to terminal part 108, terminal 108 can be protected until use.
a shows the outside of rectangular substrate 101 of a biosensor for simultaneously measuring multiple items. In the center portion of substrate 101, a vertically formed cutting plane line 112 runs from top to bottom, and upper outward-folding portions (the air-discharge port openings) 131 of substrate 101 are formed so as to form a small rectangle. Also, cutting plane line 112 is provided as a V-shaped notch 107. These notches 107 are to bend the biosensor for simultaneously measuring multiple items in to a V-shape along the center broken line 112 when in use, and further, to bend the upper outward-folding portion 131 along broken line 112, in the direction of the center broken line 112. Furthermore, in the upper outward-folding portion 131, an auxiliary device 129 is provided so that the air-discharge port openings 131 open in conjunction with opening a sample-inlet port. Two auxiliary devices 129 are provided to connect both ends of upper outward-folding portion 131. Auxiliary device 129 is provided with a fixed portion for fixing to the upper outward-folding portion 131.
b shows the inside of substrate 101. Inside substrate 101, patterns 104 including two pairs of electrodes are arranged in parallel from top to bottom, with the center broken line 112 of the substrate as a boundary. Similarly, at both inner tops of substrate 101, the broken line 112 showing the air-discharge port opening 131 is parallel to the outsides of patterns 104, which include two pairs of electrodes. Also, reagent layer 106 is formed in a portion of the electrode pattern of each pair. Although not shown in
c shows the outer portion of cover 102. In the center portion of cover 102, as for substrate 101, the vertically formed V-shaped notches 107 run from top to bottom, and the upper outward-folding portions 131 of substrate 101 form a small rectangle. Cutting plane line 112 is provided as a V-shaped notch 107.
e is a structural diagram of a biosensor for simultaneously measuring multiple items 115, showing the inner surfaces of substrate 101 and cover 102 superimposed on each other with their tops aligned. By making cover 102 shorter than substrate 101, the bottom portion of electrode pattern 104 is exposed when the two are superimposed with their tops aligned. This becomes terminal 108, shown in
f shows an A-A′ cross-sectional view of the sample-feeding path portion on the upper side of the biosensor for simultaneously measuring multiple items shown in
h shows an exemplary use of a biosensor for simultaneously measuring multiple items of the present invention.
By contacting two adjacent sample-inlet ports 109 in this state with sample solution 111, the sample solution 111 is independently supplied to the adjacent sample-feeding paths 105 by capillary action. If sample solution 111 is slightly rounded by surface tension at this time, as shown in the figure, it can be effectively supplied to the sample-feeding path 105, since the two biosensor unit-comprising substrates form a V-shape together, as shown in
In the case of
a shows a biosensor before use for measurement, showing an example where the reagent layer 6 is developed in sample-feeding path 5. In the biosensor shown in
This biosensor of the present invention employs glucose oxidase and potassium ferricyanide as a reagent layer. The measurement principle of this glucose sensor, shown in
A sample is supplied to this biosensor by capillary action from a sample-inlet port to the inside. As indicated by Formula 1 below, in the supplied glucose solution, catalytic action of GOD in the reagent layer converts ferricyanide ions into ferrocyanide ions, and oxidizes glucose:
The generated ferrocyanide ions are oxidized by a carbon electrode according to the electrode reaction of the following Formula 2, and then detected electrochemically:
In detection methods using the glucose sensors of the present invention, the generated ferrocyanide ions are oxidized by an anode electrode, an anode current flows, and the ferrocyanide ions become ferricyanide ions again. The glucose quantity can thus be determined by observing changes in the current value due to the concentration of ferrocyanide ions generated by enzyme reaction.
Next, the production methods and measurement methods of the biosensor are described.
Polyethylene terephthalate (PET) (length 40 mm, width 6 mm, depth 188 μm) was employed as a biosensor substrate and cover. On the biosensor substrate, two carbon electrodes of 1.3 mm in width were formed at an interval of 0.5 mm using a screen printer. Screen printing was also used to form resist and adhesive as a spacer layer. Notches for cutting off biosensor units and sealing cap portions were formed 10 mm from the upper portion of the biosensor substrate, so their depth was half, or more than half the depth of the substrate and cover. Perforations were provided at two positions 5 mm and 10 mm from the lower portion of the biosensor substrate, in a bent portion of the terminal protective cover.
The amount of sample was about 0.28 μl, but the amount of sample actually required, when measured in whole blood (specific gravity: 1.05), was 0.34±0.023 mg (n=10, coefficient of variation CV=6.7%). This difference between sample amounts is assumed to be because the spacer layer was actually deeper than expected, or the sample solution adhered near the sample-inlet port, or the like.
The reagent layer of enzyme and mediator, made with 5.5 units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator) dissolved in distilled water, was formed on both electrodes by application to the electrode surface and vacuum drying.
The results of measuring blood sugar (glucose in the blood) using this glucose sensor will now be described: For blood sugar measurement using this glucose sensor, whole blood samples with a hematocrit value of 40% were prepared, with glucose concentrations set at 0, 100, 300, and 500 mg/dl, and these were used as specimen solutions. An electrochemical measuring unit (ALS/CHI-1202, BAS Inc.) was used for measurement, and potential step chronoamperometry was employed as a measurement method. Twenty seconds after supplying about 0.3 μl blood into the sample-inlet port by capillary action, an electric potential of 900 mV was applied between the two electrodes in the biosensor, and the current value ten seconds after application was used as the measured value.
a is the biosensor before use for measurement, showing an example where reagent layer 106 is developed in the sample-feeding path 105 in each biosensor unit;
b shows an example where the biosensor for simultaneously measuring two items 115 is connected to connector 132, and sample-inlet port 109 of each biosensor unit 127 opens downward in a lower portion near the bottom of the V-shaped biosensor.
In one example of application of this biosensor, glucose oxidase and potassium ferricyanide were employed as reagent layers, as for Example 35. By separately supplying samples from the sample-inlet port of each biosensor unit 127 to the inside by capillary action, these biosensors electrochemically measured the quantity of glucose in whole blood.
Next, the production methods and measurement methods of the biosensors are described:
Production methods used PET (length 35 mm, width 12 mm, depth 188 μm) as a biosensor substrate, and PET (length 30 mm, width 12 mm, depth 188 μm) as a cover. Two pairs of biosensor units 127 were arranged on the biosensor substrate, with notch 107 as a boundary. The notch was formed so that its depth was half that of the substrate and cover or more. Using a screen printer, two carbon electrodes 1.3 mm in width were formed on the biosensor substrate of each biosensor unit 127, at an interval of 0.5 mm. Screen printing was also used to form resist and adhesive as a spacer layer.
In theory, the total amount of sample of the two biosensors was about 0.90 μl, but the sample amount actually required when measuring whole blood (specific gravity: 1.05) was 1.13±0.064 mg (n=10, coefficient of variation CV=5.7%). The difference between these sample volumes is assumed to be because the spacer layer was in fact deeper than expected, or the sample solution adhered near the sample-inlet port, or the like.
A reagent layer of enzyme and mediator, made with nine units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator) dissolved in distilled water, was formed on both electrodes by application to electrode surfaces in each biosensor, and then vacuum drying.
The results of measuring blood sugar (glucose in blood) using this glucose sensor are described. For blood sugar measurement using this glucose sensor, whole blood samples with a hematocrit value of 40% were prepared to have glucose concentrations set at 0, 100, 300, and 500 mg/dl, and these were used as specimen solutions. The responses of the two left and right biosensors were compared. The electrochemical measuring unit (ALS/CHI-1202, BAS Inc.) used in Example 35, which can measure two items simultaneously, was used for measurement. Potential step chronoamperometry was employed as the measurement method. Twenty seconds after supplying about 1.1 μl blood to the sample-inlet port by capillary action, an electric potential of 900 mV was applied between the two electrodes in the biosensor, and the current value ten seconds after application was used as the measured value.
Subsequently, under the same conditions, storage stability tests for the biosensors of
Next, this biosensor was used to simultaneously measure both glucose and lactic acid. The reagent layer of enzyme and mediator in the two left and right biosensors was different. The reagent layer of the biosensors of one side was formed on both electrodes by dissolving 1.65 units of glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator) in distilled water, applying this to the electrode surface of each biosensor, and then vacuum drying. The reagent layer of the biosensors on the other side was formed on both electrodes by dissolving 3.7 units of lactic acid oxidase (LOD) and 0.1 mg potassium ferricyanide (mediator) in distilled water, applying this to the electrode surface of each biosensor, and then vacuum drying.
As sample solutions, 0.1 M, pH7.4 phosphate buffer comprising 100 mg/dl lactic acid, phosphate buffer comprising 100 mg/dl lactic acid and 300 mg/dl glucose, and phosphate buffer comprising 300 mg/dl glucose were used.
The results of simultaneously measuring glucose and lactic acid using these biosensors for simultaneously measuring multiple items is described. For the measurement of glucose and lactic acid using a biosensor for simultaneously measuring two items, four kinds of mixed solutions were used as specimen solutions, with glucose and lactic acid prepared at prescribed concentrations (glucose+lactic acid: 0+0 mg/dl, 100+50 mg/dl, 300+100 mg/dl, and 500+140 mg/dl). Also, 0.1M, pH7.4 phosphate buffer was used to prepare these mixed solutions. The measuring unit used was the electrochemical measuring unit (ALS/CHI-1202, BAS Inc.) used in Example 35, which can be used to simultaneously measure two items. Potential step chronoamperometry was employed as the measurement method. Twenty seconds after supplying about 1.1 μl of sample solution to the sample-inlet port by capillary action, 900 mV of electric potential was applied between the two electrodes in the biosensor, and the current value ten seconds after application was used as the measured value.
Thus, to solve the problems of the open-type biosensor for simultaneously measuring multiple items in Example 36, the following Examples describe the results of study on a sealed-type biosensor for simultaneously measuring multiple items of the present invention for which no wrapping is required.
a shows the biosensor before use for measurement, showing an example where reagent layer 106 is developed in the sample-feeding path 105 in each biosensor unit; where the sample-feeding path is in a hermetically sealed state, shut off from the outside by an upper outward-folding portion 131.
Next, the production methods and measurement methods of the biosensors are described:
Production methods used PET (length 35 mm, width 15 mm, depth 188 μm) as a biosensor substrate, and PET (length 30 mm, width 15 mm, depth 188 μm) as a cover. Two pairs of biosensor units 127 were arranged on the biosensor substrate, with notch 107 as a boundary. The opening 131 of air-discharge port is also provided with the notch 107 on upper of the outer surface of each biosensor unit. The notch was formed so that its depth was half that of the substrate and cover or more. Using a screen printer, two carbon electrodes 1.3 mm in width were formed on the biosensor substrate of each biosensor unit 127, at an interval of 0.8 mm. Screen printing was also used to form resist and adhesive as a spacer layer.
In theory, the total amount of sample of the two biosensors was about 0.90 μl, but the sample amount actually required when measuring whole blood (specific gravity: 1.05) was 1.12±0.077 mg (n=10, coefficient of variation CV=6.9%). The difference between these sample volumes is assumed to be because the spacer layer was in fact deeper than expected, or the sample solution adhered near the sample-inlet port, or the like.
A reagent layer of enzyme and mediator, made with nine units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator) dissolved in distilled water, was formed on both electrodes by application to electrode surfaces in each biosensor, and then vacuum drying.
The results of measuring blood sugar (glucose in blood) using this glucose sensor are described. For blood sugar measurement using this glucose sensor, whole blood samples with a hematocrit value of 40% were prepared to have glucose concentrations set at 0, 100, 300, and 500 mg/dl, and these were used as specimen solutions. The responses of the two left and right biosensors were compared. The electrochemical measuring unit (ALS/CHI-1202, BAS Inc.) used in Example 35, which can measure two items simultaneously, was used for measurement. Potential step chronoamperometry was employed as the measurement method. Twenty seconds after supplying about 1.1 μl blood to the sample-inlet port by capillary action, an electric potential of 900 mV was applied between the two electrodes in the biosensor, and the current value ten seconds after application was used as the measured value.
Subsequently, under the same conditions, storage stability tests were conducted for these biosensors on day 0, week 2, month 1, month 2, and month 3. The biosensors were stored in a (room temperature) laboratory table drawer in a laboratory, for tests under the same conditions as in Example 35. The results are shown in
a shows the biosensor before use for measurement, illustrating an example where reagent layer 106 is developed in sample-feeding path 105 in each biosensor unit; and where the sample-feeding path is in a hermetically sealed state, shut off from the outside.
Next, the production methods and measurement methods of the biosensors are described:
Production methods used PET (length 35 mm, width 12 mm, depth 188 μm) as a biosensor substrate, and PET (length 30 mm, width 12 mm, depth 188 μm) as a cover. Two pairs of biosensor units 127 were arranged on the biosensor substrate, with notch 107 as a boundary. The notch was formed so that its depth was half that of the substrate and cover or more. Using a screen printer, two carbon electrodes 1.3 mm in width were formed on the biosensor substrate of each biosensor unit 127, at an interval of 0.5 mm. Screen printing was also used to form resist and adhesive as a spacer layer.
In theory, the total amount of sample of the two biosensors was about 2.0 μl, but the sample amount actually required when measuring whole blood (specific gravity: 1.05) was 2.71±0.097 mg (n=10, coefficient of variation CV=3.6%), indicating high reproducibility. The difference between these sample volumes is assumed to be because the spacer layer was in fact deeper than expected, or the sample solution adhered near the sample-inlet port, or the like.
A reagent layer of enzyme and mediator, made with 20 units glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator) dissolved in distilled water, was formed on both electrodes by application to electrode surfaces in each biosensor, and then vacuum drying.
The results of measuring blood sugar (glucose in blood) using this glucose sensor are described. For blood sugar measurement using this glucose sensor, whole blood samples with a hematocrit value of 40% were prepared to have glucose concentrations set at 0, 100, 300, and 500 mg/dl, and these were used as specimen solutions. The responses of the two left and right biosensors were compared. The electrochemical measuring unit (ALS/CHI-1202, BAS Inc.) used in Example 35, which can measure two items simultaneously, was used for measurement. Potential step chronoamperometry was employed as the measurement method. Twenty seconds after supplying about 3 μl blood to the sample-inlet port by capillary action, an electric potential of 900 mV was applied between the two electrodes in the biosensor, and the current value ten seconds after application was used as the measured value.
Subsequently, under the same conditions, storage stability tests were conducted for these biosensors on day 0, week 2, month 1, month 2, and month 3. The biosensors were stored in a (room temperature) laboratory table drawer in a laboratory, for tests under the same conditions as in Example 35. The results are shown in
These biosensors thus obtained relatively excellent results regarding the correlation between glucose concentration in blood and output current value. These biosensors, which are sealed-type biosensors, were found able to conduct measurements for at least three months, without the need for wrapping.
Next, this biosensor was used to simultaneously measure both glucose and lactic acid. The reagent layer of enzyme and mediator in the two left and right biosensors was different. The reagent layer of the biosensors of one side was formed on both electrodes by dissolving nine units of glucose oxidase (GOD) and 0.1 mg potassium ferricyanide (mediator) in distilled water, applying this to the electrode surface of each biosensor, and then vacuum drying. The reagent layer of the biosensors on the other side was formed on both electrodes by dissolving 20 units of lactic acid oxidase (LOD) and 0.1 mg potassium ferricyanide (mediator) in distilled water, applying this to the electrode surface of each biosensor, and then vacuum drying.
As sample solutions, 0.1 M, pH7.4 phosphate buffer comprising 100 mg/dl lactic acid, phosphate buffer comprising 100 mg/dl lactic acid and 300 mg/dl glucose, and phosphate buffer comprising 300 mg/dl glucose were used.
The results of simultaneously measuring glucose and lactic acid using these sealed-type biosensors for simultaneously measuring multiple items is described. For the measurement of glucose and lactic acid using a biosensor for simultaneously measuring two items, four kinds of mixed solutions were used as specimen solutions, with glucose and lactic acid prepared at prescribed concentrations (glucose+lactic acid: 0+0 mg/dl, 100+50 mg/dl, 300+100 mg/dl, and 500+140 mg/dl). Also, 0.1M, pH7.4 phosphate buffer was used to prepare these mixed solutions. The measuring unit used was the electrochemical measuring unit (ALS/CHI-1202, BAS Inc.) used in Example 35, which can be used to simultaneously measure two items. Potential step chronoamperometry was employed as the measurement method. Twenty seconds after supplying about 3 μl of sample solution to the sample-inlet port by capillary action, 900 mV of electric potential was applied between the two electrodes in the biosensor, and the current value ten seconds after application was used as the measured value.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-084116 | Mar 2004 | JP | national |
| 2004-127937 | Apr 2004 | JP | national |
| 2005-014376 | Jan 2005 | JP | national |
| 2005-014377 | Jan 2005 | JP | national |
