WO2019187574A1

WO2019187574A1 - Biosensor

Info

Publication number: WO2019187574A1
Application number: PCT/JP2019/002472
Authority: WO
Inventors: 直林野; 圭吾羽田
Original assignee: Ｐｈｃホールディングス株式会社
Priority date: 2018-03-26
Filing date: 2019-01-25
Publication date: 2019-10-03
Also published as: JP2021089141A

Abstract

Provided is a biosensor used in an electrochemical measurement method, wherein the biosensor is capable of performing measurement with high precision by reducing blank current. This biosensor analyzes a component in a liquid to be tested using a protein and a mediator, wherein the biosensor has one or more space parts formed across a spacer between an insulating substrate and a cover, at least one surface of the space parts has an electroconductive part and a non-electroconductive part, a first reagent part including the protein and a second reagent part including the mediator are individually disposed in different locations on the inner surface, and the first reagent part and/or the second reagent part is disposed in the non-electroconductive part.

Description

Biosensor

The present invention relates to a biosensor, and more particularly to a biosensor using an electrochemical measurement method.

Biosensors are used to measure a substance to be detected in a biological sample in various fields such as the medical field and clinical laboratory field. As an example of a biosensor, a biosensor using an electrochemical measurement method is known (for example, Patent Document 1). This biosensor forms a working electrode, a counter electrode, and a reference electrode on an insulating substrate, and is in contact with these electrodes and includes an enzyme reaction layer (also referred to as a reagent unit) including an enzyme and an electron acceptor (hereinafter referred to as a mediator). ) Is formed. According to such a biosensor, in principle, various substances can be measured by selecting an enzyme that uses the substance to be measured as a substrate. For example, a glucose sensor that measures glucose concentration in a sample solution by selecting glucose oxidase as an enzyme has been put into practical use.

On the other hand, in recent years, glycated proteins such as glycated hemoglobin and glycated albumin have been widely measured as an index for diagnosis of diabetes. For example, hemoglobin A1c (hereinafter abbreviated as HbA1c) is one of glycated hemoglobin, and the HbA1c value is a test value indicating the proportion of hemoglobin bound to sugar in hemoglobin in erythrocytes. Since the HbA1c value reflects the average blood glucose level in the past 1 to 2 months, it is less affected by the diet before the test and is important as an index for managing diabetes. The HbA1c value is measured by an HPLC method or an immunization method using an optical spectroscopic method.

Japanese Patent Laid-Open No. 3-202864

When measuring the HbA1c value, the test sample is diluted by, for example, about 100 times by hemolysis (hereinafter, in this specification, a sample solution to be measured is subjected to a pretreatment such as hemolysis. Called liquid). Therefore, the HbA1c concentration in the test solution is significantly lower than the glucose concentration, and is usually a low concentration on the order of μM. When trying to measure such a low concentration of HbA1c by an electrochemical measurement method, there is a problem that the influence of the blank current increases, the S / N ratio decreases, and the measurement becomes difficult. Therefore, a method for measuring the HbA1c value using an electrochemical measurement method has not been put into practical use. If the blank current can be reduced, it can be expected that the HbA1c value can be measured with high accuracy by using the electrochemical measurement method. As a substance to be detected, proteins other than HbA1c, such as hemoglobin, glycated albumin, glucose, cholesterol, lactic acid, ketone bodies (3-hydroxybutyric acid), antibodies, etc. can be measured with high accuracy if the blank current can be reduced. It becomes possible.

Therefore, an object of the present invention is to provide a biosensor used in an electrochemical measurement method, which can measure with high accuracy by reducing blank current.

In order to solve the above problems, a biosensor of the present invention is a biosensor that analyzes a component in a test solution using a protein and a mediator, and a spacer is interposed between the insulating substrate and the cover. And having at least one formed space portion, and having at least one inner surface of the space portion having a conductive portion and a non-conductive portion, and including a first reagent portion containing the protein and a second mediator. Reagent parts are separately disposed at different locations on the inner surface, and at least one of the first reagent part and the second reagent part is disposed in the non-conductive part.

According to the present invention, a biosensor capable of highly accurate measurement can be provided by reducing the blank current.

2 is a perspective view showing an example of a structure of biosensor A according to Embodiment 1. FIG. It is a disassembled perspective view of the biosensor A of FIG. It is a top view of the insulating base material which comprises the biosensor A of FIG. It is a longitudinal cross-sectional view along the longitudinal direction of the biosensor A of FIG. 6 is an exploded perspective view showing an example of a structure of a biosensor B according to Embodiment 2. FIG. It is a longitudinal cross-sectional view along the longitudinal direction of the biosensor B of FIG. FIG. 6 is a plan view of an insulating base material constituting a biosensor C according to a third embodiment. It is a longitudinal cross-sectional view along the longitudinal direction of the biosensor C which concerns on Embodiment 3. FIG. It is a graph which shows the blank electric current value in a reference example. It is a graph which shows the blank electric current value in a reference example. It is a graph which shows the blank electric current value in a reference example. It is an example of the graph which shows the relationship between the detection electric current value in this invention, and the density | concentration of a to-be-detected substance.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

The biosensor of the present invention is a biosensor that analyzes a component in a test solution using a protein and a mediator, and is one or more formed through a spacer between an insulating substrate and a cover. The inner surface of the at least one space portion has a conductive portion and a non-conductive portion, and the first reagent portion containing the protein and the second reagent portion containing the mediator are different in the inner surface. It is arrange | positioned separately at a place, At least one of the said 1st reagent part and the said 2nd reagent part is arrange | positioned at the said nonelectroconductive part, It is characterized by the above-mentioned.

Embodiment 1
FIG. 1 is a perspective view showing an example of the structure of the biosensor A according to the present embodiment, and FIG. 2 is an exploded perspective view. In the biosensor A, an insulating base material 1 having a conductive portion 4 and a cover 2 are laminated via a spacer 3. FIG. 1 shows an example having a long rectangular piece shape in which the X direction is the longitudinal direction and the Y direction is the width direction.

A conductive portion 4 is formed on one main surface of the insulating substrate 1 having a pair of opposing main surfaces. The conductive portion 4 is a conductive portion, and includes a first electrode pair 41 and a second electrode pair 42, a terminal portion 43 formed at one end of the insulating base material 1, a first electrode pair 41, and a second electrode pair 42. And a lead part 44 for connecting the terminal part 43 to each other. FIG. 3 is a plan view of the insulating substrate 1, and the first electrode pair 41 is disposed in the longitudinal direction and sandwiches the pair of working

electrodes

41 a and 41 a that are electrically connected to each other and the working electrodes 41 a in plan view. Two pairs of

counter electrodes

41b and 41b facing each working electrode 41a through a minute gap (described later) are provided. The second electrode pair 42 acts via a working electrode 42a disposed in the longitudinal direction so as to be separated from the first electrode pair 41, and a minute gap (described later) so as to sandwich the working electrode 42a in plan view. It has a pair of

counter electrodes

42b and 42b facing the pole 42a. The pair of working

electrodes

41a and 41a is connected to the terminal 43a through the lead 44a, the two pairs of

counter electrodes

41b and 41b are connected to the terminal 43d through the lead 44d, and the working electrode 42a is connected to the terminal 43b through the lead 44b. The pair of

counter electrodes

42b and 42b are connected to the terminal 43c via a lead 44c. 2 and FIG. 3, the structure excluding the first reagent part and the second reagent part described later is shown. Further, the two electrode pairs of the first electrode pair 41 and the second electrode pair 42 can be used when measuring two different types of substances to be detected in the test liquid. The case of measuring two kinds of substances to be detected is, for example, the case of measuring the HbA1c value, measuring the concentration of HbA1c with the first electrode pair 41 and measuring the concentration of Hb with the second electrode pair 42. Can do. Moreover, although the example which used two working electrodes for the 1st electrode pair 41 was shown, it can also be set as the structure using one working electrode similarly to the case of the 2nd electrode pair 42. FIG.

The spacer 3 only needs to have at least one opening, but in the present embodiment, the spacer 3 has two

openings

3a and 3b formed to be separated from each other along the longitudinal direction. The spacer 3 is shorter than the insulating base 1 so that the terminal portion 43 is exposed. The cover 2 has an opening 2a at one end in the longitudinal direction, an opening 2c at the other end, and an opening 2b at an intermediate portion between the opening 2a and the opening 2c. Moreover, the cover 2 is also made shorter than the insulating base material 1 so that the terminal part 43 is exposed similarly to the case of the spacer 3. Here, the opening of the spacer 3 and the opening of the cover 2 are such that when the spacer 3 and the cover 2 are stacked, the opening 2 b at the center of the cover 2 is the end on the center side of the opening 3 a of the spacer 3. And the opening 2a at one end of the cover 2 is at least partially overlapped with the side end of the opening 3a of the spacer 3, so that the cover 2 The opening 2c at the other end of the spacer 3 is disposed so as to at least partially overlap the side end of the opening 3b of the spacer 3.

FIG. 4 is a longitudinal sectional view along the longitudinal direction of the biosensor A shown in FIG. The

openings

3 a and 3 b of the spacer 3 form two first space portions 6 and second space portions 7 that are independent from each other when the spacer 3 is sandwiched between the insulating base material 1 and the cover 2. ing. In each space part of the 1st space part 6 and the 2nd space part 7, one main surface of the insulating base material 1 exposed to a space part comprises the bottom face of a space part. Moreover, the cover 2 has a pair of opposing main surfaces, and one main surface exposed to the space portion constitutes the top surface of the space portion. Moreover, the inner surface of the opening part of the spacer 3 comprises the side surface of a space part.

The biosensor of the present invention has a conductive portion and a non-conductive portion on the inner surface of the space portion. Here, as described above, the conductive portion is a conductive portion, and includes an electrode including a working electrode and a counter electrode, a lead portion, and a terminal portion. The conductive portion in the first space portion 6 is the first electrode pair 41 and the lead portion. On the other hand, the non-conductive portion in the first space portion 6 is a portion that is not conductive, specifically, a portion where no electrode or lead is formed on the insulating base material 1, or the opening 3 a of the spacer 3. Among the inner peripheral surface and the pair of opposing main surfaces of the spacer 3, a main surface 5 that is not in contact with the cover 2 (hereinafter also referred to as a back surface) can be exemplified. The conductive part in the second space 7 is the second electrode pair 42 and the lead part. On the other hand, the non-conductive portion of the second space portion 7 is a portion of the insulating base material 1 where no electrodes or leads are formed, the inner peripheral surface of the opening 3b of the spacer 3, and a pair of spacers 3 facing each other. Among the main surfaces, the back surface 5 that is not in contact with the cover 2 can be cited.

In the present invention, the first reagent part containing protein and the second reagent part containing mediator are separately arranged at different locations on the inner surface of at least one space part, and at least one of the first reagent part and the second reagent part is Arranged in the non-conductive portion. In the present embodiment, in the first space portion 6, the first reagent portion 8 is disposed on the surfaces of the first electrode pair 41 and the second electrode pair 42 that are conductive portions, and the second reagent portion 9 is non-conductive. It is arranged on the back surface 5 of the cover 2 which is a part. In the second space portion 7, the second reagent portion 9 is disposed on the surface of the second electrode pair 42.

Note that the opening 2b in the center of the cover 2 can be used as an introduction opening for the test liquid, and the introduction opening communicates with the space. When the test solution is dropped into the opening 2b, the test solution flows into the first space 6 and the second space 7, and in the first space 6, the first reagent unit 8 and the second reagent unit 9 are used. Touch. That is, the space part in the present invention provides a holding area for holding the test liquid and a reaction area for advancing the reaction between the test liquid and the first reagent part and the second reagent part. Here, the reaction region refers to a region occupied by the conductive part and the reagent part arranged in the conductive part. The

openings

2 a and 2 c at both ends of the cover 2 provide air to the inside of the first space portion 6 and the second space portion 7, respectively, and the test liquid is supplied from the opening portion 2 b at the center portion of the cover 2. When it is dropped, it works to release the internal air as the test solution is drawn.

In the present embodiment, the area of one reagent part of the first reagent part and the second reagent part may be larger than the area of the other reagent part. For example, in one space part, one reagent part of the first reagent part and the second reagent part is arranged in the non-conductive part, the other reagent part is arranged in the conductive part, and one of the reagent parts arranged in the non-conductive part The area of the reagent part may be larger than the area of the other reagent part arranged in the conductive part. In FIG. 4, in the first space portion 6, the area of the second reagent portion 9 disposed on the back surface 5 of the cover 2 that is a non-conductive portion is the first space portion disposed on the surface of the first electrode pair 41 that is a conductive portion. An example larger than the area of one reagent part 8 is shown. Here, the area of the reagent part is an area of the reagent part in plan view. For example, when the reagent part is formed by a coating method, it is the area of the coating film in plan view (hereinafter, the area of the coating film in plan view may be referred to as the coating film area). Moreover, in FIG. 4, the two 1st reagent parts 8 exist in the same space part (1st space part 6). When two or more reagent parts exist in the same space part, the area of the reagent part is a total area of two or more reagent parts.

Here, the test liquid flowing in from the introduction opening moves from the upstream to the downstream in the space, and at this time, a part of the reagent in the first reagent part and / or the second reagent part is dissolved in the test liquid. May move out of the reaction zone. However, by making the area of one reagent part larger than the area of the other reagent part, a sufficient amount of reagent can be supplied to the reaction region. In particular, if the area of one reagent part arranged in the non-conductive part is larger than the area of the other reagent part arranged in the conductive part, the reagent contained in one reagent part arranged in the non-conductive part Even if a part flows out of the reaction region together with the test solution, a sufficient amount of reagent can be supplied to the working electrode of the conductive portion. As a result, the response value is increased and the S / N ratio is improved, so that highly accurate measurement is possible. Furthermore, it is preferable to arrange the second reagent part in the non-conductive part and make the area of the second reagent part larger than the area of the first reagent part. By increasing the contact area between the test solution and the second reagent part, a sufficient amount of mediator can be supplied to the reaction region. Specifically, when the area of the reagent part is, for example, in the range of 0.5 mm ² to 200 mm ² , the area of one reagent part is equal to or larger than the area of the other reagent part, preferably 1.05 to 10 times. Times, more preferably 1.5 times to 4 times.

In the present embodiment, the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be larger than the area of the working electrode included in the conductive part. As described above, the test solution flowing in from the introduction opening moves from the upstream to the downstream in the space portion. At this time, a part of the reagent may be dissolved in the test solution and move out of the reaction region. At this time, a part of the reagent contained in the reagent part arranged in the non-conductive part flows out of the reaction region and is not supplied to the working electrode. However, if the area of the reagent part arranged in the non-conductive part is larger than the area of the working electrode of the conductive part, even if a part of the reagent flows out of the reaction region, a sufficient amount of reagent is used as the working electrode. It becomes possible to supply. As a result, the response value increases and the S / N ratio is improved, so that more accurate measurement is possible. Here, the area of the working electrode is the area of the working electrode in plan view. Specifically, for example, when the area of the reagent part is in the range of 0.5 to 200 mm ² , the area of one reagent part is equal to or more than the area of the working electrode, preferably 1.5 to 30 times, More preferably, it is 2 to 20 times.

In the present embodiment, the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is based on the total area of the working electrode area and the counter electrode area of the conductive part. May be larger. As described above, the test solution flowing in from the introduction opening moves from the upstream to the downstream in the space portion. At this time, a part of the reagent may be dissolved in the test solution and move out of the reaction region. At this time, a part of the reagent contained in the reagent part arranged in the non-conductive part flows out of the reaction region and is not supplied to the working electrode. However, if the area of the reagent part arranged in the non-conductive part is larger than the total area of the working electrode area and the counter electrode area, even if a part of the reagent flows out of the reaction region together with the test solution, A sufficient amount of reagent can be supplied to the working electrode and the counter electrode. As a result, a sufficient amount of reagent for the reduction reaction reacts on the counter electrode within a fixed time, and a sufficient amount of reagent for the oxidation reaction reacts on the working electrode, increasing the response value and improving the S / N ratio. Therefore, measurement with high accuracy is possible. Here, the area of the working electrode is the area of the working electrode in plan view, and the area of the counter electrode is the area of the counter electrode in plan view. Specifically, for example, when the area of the reagent part is in the range of 0.5 to 200 mm ² , the area of one reagent part is equal to or larger than the total area of the working electrode area and the counter electrode area, preferably It is 1.05 times to 10 times, more preferably 1.5 times to 4 times.

In the present embodiment, one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is closer to the introduction opening for the test solution than the other reagent part. It may be arranged. As described above, the test solution flowing in from the introduction opening moves from the upstream to the downstream in the space portion. At this time, a part of the reagent may be dissolved in the test solution and move out of the reaction region. At this time, a part of the reagent contained in the reagent part arranged in the non-conductive part flows out of the reaction region and is not supplied to the working electrode. However, one reagent part arranged in the non-conductive part is arranged closer to the introduction opening for the test solution than the other reagent part, that is, upstream of the other reagent part. By disposing, even if a part of the reagent flows out of the reaction region, a sufficient amount of reagent can be supplied to the reaction region. As a result, the response value increases and the S / N ratio is improved, so that more accurate measurement is possible. It should be noted that at least a part of one reagent part may be arranged so as to be closer to the introduction opening for the test solution than the other reagent part.

In the present embodiment, one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be arranged closer to the introduction opening than the working electrode. As described above, the test solution flowing in from the introduction opening moves from the upstream to the downstream in the space portion. At this time, a part of the reagent may be dissolved in the test solution and move out of the reaction region. At this time, a part of the reagent contained in the reagent part arranged in the non-conductive part flows out of the reaction region and is not supplied to the working electrode. However, one of the reagent parts arranged in the non-conductive part is arranged so as to be closer to the introduction opening for the test solution than the working electrode, that is, by arranging it upstream of the working electrode. Even if a part of the reagent flows out of the reaction region, a sufficient amount of the reagent can be supplied to the working electrode. As a result, the response value increases and the S / N ratio is improved, so that more accurate measurement is possible. It should be noted that at least a part of one of the reagent parts may be arranged so as to be closer to the introduction opening for the test solution than the working electrode.

The material used for the insulating substrate is not particularly limited. For example, polyethylene terephthalate, polycarbonate, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, monomer cast nylon, polybutylene terephthalate, methacrylic resin, ABS resin, etc. These resin materials or glass materials can be used. Preferred are polyethylene terephthalate, polycarbonate, and polyimide, and more preferred is polyethylene terephthalate. The size of the insulating substrate is not particularly limited. For example, the total length is 5 to 100 mm, the width is 2 to 50 mm, and the thickness is 0.05 to 2 mm. Preferably, the total length is 7 to 50 mm, the width is 3 to 20 mm, and the thickness is 0.1 to 1 mm. More preferably, the total length is 10 to 30 mm, the width is 3 to 10 mm, and the thickness is 0.1 to 0.6 mm.

The conductive part on the insulating substrate is formed of a conductive layer by sputtering or vapor deposition using, for example, carbon, gold, platinum, palladium or the like, and then formed into a predetermined electrode pattern by laser trimming. It can be formed by processing. Here, by the laser trimming method, the minute gap between the working electrode and the counter electrode, the minute gap between the leads, and the minute gap between the terminals are formed, so that the electrical insulation between the electrodes, between the leads, and between the terminals. Is secured.

The material of the spacer is not particularly limited. For example, the same material as the insulating base material can be used. Further, the size of the spacer is not particularly limited. For example, the total length is 5 to 100 mm, the width is 2 to 50 mm, the thickness is 0.01 to 1 mm, preferably the total length is 7 to 50 mm, the width is 3 to 20 mm, and the thickness is 0.05 to 0.5 mm. Preferably, the total length is 10 to 30 mm, the width is 3 to 10 mm, and the thickness is 0.05 to 0.25 mm.

The cover material is not particularly limited. For example, the same material as the insulating base material can be used. The size of the cover is not particularly limited. For example, the total length is 5 to 100 mm, the width is 3 to 50 mm, the thickness is 0.01 to 0.5 mm, preferably the total length is 10 to 50 mm, the width is 3 to 20 mm, and the thickness is 0.05 to 0.25 mm. More preferably, the total length is 15 to 30 mm, the width is 5 to 10 mm, and the thickness is 0.05 to 0.1 mm. The cover is preferably formed with a plurality of openings used as air holes or test liquid inlets. As the shape of the opening, for example, a circle, an ellipse, a polygon or the like can be used. As for the size of the opening, for example, the maximum diameter is 0.01 to 10 mm, preferably 0.05 to 5 mm, more preferably 0.1 to 2 mm. The opening may be formed by drilling with a laser or a drill, or may be formed by molding using a mold.

A biosensor can be manufactured by laminating an insulating base material, a spacer, and a cover in this order, and bonding and integrating them with an adhesive or heat fusion. An epoxy adhesive, an acrylic adhesive, a polyurethane adhesive, a hot melt adhesive, a UV curable adhesive, or the like can be used as the adhesive.

In the biosensor of the present invention, the first reagent part includes a protein, and the second reagent part includes a mediator. Examples of the protein include enzymes, antibodies, immunoglobulins, bovine serum albumin, human serum albumin and the like. Examples of the enzyme include oxidoreductases such as glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, fructosyl amino acid oxidase, fructosyl peptide oxidase, and 3-hydroxybutyrate dehydrogenase. These oxidoreductases are oxidases or dehydrogenases that act on glucose, lactic acid, cholesterol, bilirubin, glycated amino acids, or glycated peptides, ketone bodies (3-hydroxybutyric acid). The amount of oxidoreductase is, for example, 0.01 to 100 U, preferably 0.05 to 10 U, more preferably 0.1 to 100 U per sensor or per measurement. 5U.

Mediators include, but are not limited to, metal complexes (eg, osmium complexes, ruthenium complexes, iron complexes, etc.), quinone compounds (eg, benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and derivatives thereof). , A phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound. More specifically, potassium ferricyanide, hexaammineruthenium, ferrocene, poly (1-vinylimidazole) -bis (bipyridine) chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfone Acid salt, 9,10-phenanthrenequinone-2-sulfonate, 9,10-phenanthrenequinone-2,7-disulfonate, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonate 1-methoxy-5-methylphenazinium methyl sulfate, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol One or more selected It is. Examples of the salt include, but are not limited to, sodium salt, potassium salt, calcium salt, magnesium salt, lithium salt and the like. The blending amount of the mediator is not particularly limited, and is, for example, 0.1 pmol to 100 μmol, preferably 10 pmol to 10 μmol, more preferably 50 pmol to 1 μmol per measurement or per biosensor. is there.

In the present invention, the first reagent part containing the protein and the second reagent part containing the mediator are separately arranged at different locations on the inner surface of the space part. Therefore, the first reagent part containing the protein and the second reagent part containing the mediator Are prepared separately. If necessary, additives such as a buffer and a hydrophilic polymer can be added to the first reagent part and the second reagent part, respectively. An enzyme stabilizer can also be added to the first reagent part. Each of these substances is dissolved in water, applied to the application part, and dried to form a reagent part. In the present embodiment, the portion to be coated is the first electrode pair that is a conductive portion in the case of the first reagent portion, and the back surface of the cover in the case of the second reagent portion.

Hereinafter, the measurement procedure of the test solution using the biosensor A will be described with reference to FIGS. 1 and 4 in the case of measuring the HbA1c value.
First, a sample collected from a subject is pretreated as necessary to prepare a test solution.

When measuring using the first electrode pair 41 of the biosensor A, the terminal of the terminal portion 43 is connected to a measuring device (not shown).

The test liquid is sucked up with a dropper, and the test liquid is dropped into the opening 2b for introducing the test liquid in the cover 2.

When measuring the HbA1c value, the first electrode pair 41 measures the concentration of HbA1c, and the second electrode pair 42 measures the concentration of Hb. Here, the 1st reagent part 8 of the 1st space part 6 contains fructosyl peptide oxidase, for example, and a 2nd reagent part contains potassium ferricyanide, for example. The fructosyl valyl histidine in the test solution dropped into the test solution introduction opening 2 b of the cover 2 reacts with the fructosyl peptide oxidase of the first reagent unit 8 arranged in the first space 6. The potassium ferricyanide contained in the second reagent unit 9 is reduced to potassium ferrocyanide, but is oxidized to potassium ferricyanide with the application of voltage. Since the oxidation current changes according to the concentration of HbA1c in the test solution, the concentration of HbA1c in the test solution can be measured by measuring the oxidation current.

On the other hand, Hb in the test solution introduced into the second space portion 7 and potassium ferricyanide contained in the second reagent portion 9 undergo a redox reaction. By measuring the current accompanying this reaction with the second electrode pair 42, the concentration of Hb can be measured. Thereby, the HbA1c value can be calculated.

In a conventional biosensor, a coating film obtained by applying a coating liquid containing both an enzyme and a mediator to an electrode and drying it is used as a reagent part. On the other hand, according to the present embodiment, the first reagent part containing the protein and the second reagent part containing the mediator are arranged separately, and at least one of the first reagent part and the second reagent part is made non-conductive. By arranging in the part, the blank current value can be greatly reduced as compared with the conventional biosensor. As a result, the S / N ratio can be increased, so that even a low-concentration target substance can be measured with high accuracy. The reason for this is that conventional biosensors use a coating film that is obtained by applying a coating solution containing both enzyme and mediator on the electrode and drying it as a reagent part. It is conceivable that the amino current having an electron-donating property in the enzyme maintains the state and partially reduces the mediator, thereby increasing the blank current. In addition, the first reagent part and the second reagent part are easily affected by the external environment such as humidity, and cracking may occur on the electrode in contact with the reagent part due to repeated swelling and drying. When the first reagent part and the second reagent part are arranged close to the conductive part, a crack may occur in the conductive part, which may greatly affect the performance, but at least one of the first reagent part and the second reagent part Can be prevented from being generated on the conductive portion.

In the present embodiment, the first reagent unit is arranged on the surface of the first electrode pair and the second reagent unit is arranged on the back surface of the cover. However, the first reagent unit is arranged on the back surface of the cover. The same effect can be obtained even if the second reagent part is disposed on the back surface of the first electrode pair. In the present embodiment, an example has been described in which the second reagent part is provided in the second space part and used for measurement of two types of detected substances. However, by not providing the reagent part in the second space part, Only one electrode pair can be used for measurement of one kind of two kinds of detected substances, and a plurality of electrode pairs can be provided on the insulating substrate in accordance with the number of detected substances.

Embodiment 2
In the first embodiment, an example using two electrode pairs of the first electrode pair and the second electrode pair has been described, but in this embodiment, an example using one electrode pair will be described. In the following description, the description overlapping with the description of Embodiment 1 is omitted.

FIG. 5 is an exploded perspective view showing an example of the structure of the biosensor B according to the present embodiment. In the biosensor B, an insulating base material 21 having a conductive portion 24 and a cover 22 are laminated via a spacer 23. A conductive portion 24 is formed on one main surface of the insulating base material 21 having a pair of opposing main surfaces. The conductive portion 24 is a conductive portion, and includes a first electrode pair 241, a terminal portion 243 formed at one end of the insulating base material 21, and a lead portion 242 that connects the first electrode pair 241 and the terminal portion 43. Have. The first electrode pair 241 has a working electrode 241a and a pair of

counter electrodes

241b and 241b facing the working electrode 241a through a minute gap so as to sandwich the working electrode 241a in plan view. The working electrode 241 is connected to the terminal 243a via the lead 242a, and the pair of

counter electrodes

241b and 241b is connected to the terminal 243b via the lead 242b.

FIG. 6 is a longitudinal sectional view of the biosensor B along the longitudinal direction. The opening 23 a of the spacer 23 forms a first space 26 as one space by sandwiching the spacer 23 between the insulating base material 21 and the cover 22. The conductive portion in the first space portion 26 is the first electrode pair 241 and the lead portion. On the other hand, the non-conductive portion in the first space portion 26 is a portion that is not conductive, specifically, a portion where no electrode or lead is formed on the insulating base material 21, or the opening 23 a of the spacer 23. Among the inner peripheral surface and a pair of opposing main surfaces of the spacer 23, a main surface 25 (hereinafter also referred to as a back surface) that is not in contact with the cover 22 can be given.

In the first space portion 26, the first reagent portion 28 is disposed on the surface of the first electrode pair 241 that is a conductive portion, and the second reagent portion 29 is disposed on the back surface 25 of the cover 22 that is a nonconductive portion. Yes. The opening 22a at the end in the longitudinal direction of the cover 22 can be used as an opening for introducing the test liquid. When the test liquid is dropped into the opening 22a, the test liquid flows into the first space 26. In the first space portion 26, the first reagent portion 28 and the second reagent portion 29 are in contact with each other. The opening 22b at the center of the cover 22 provides air to the inside of the first space 26, and when the test liquid is dropped from the opening 22a at the end of the cover 22, Along with the pull-in, it works to release the air inside.

Biosensor B can be used when measuring one type of substance to be detected. For example, it can be used when measuring glucose. In this case, the 1st reagent part 28 of the 1st space part 26 may contain glucose dehydrogenase, for example, and the 2nd reagent part may contain potassium ferricyanide, for example. The glucose in the test liquid dropped into the test liquid introduction opening 22a of the cover 22 reacts with the glucose dehydrogenase of the first reagent part 28 disposed in the first space part 26, and the second reagent part. The potassium ferricyanide contained in 29 is reduced to potassium ferrocyanide, but is oxidized to potassium ferricyanide with the application of voltage. Since the oxidation current changes according to the glucose concentration in the test solution, the glucose concentration in the test solution can be measured by measuring the oxidation current.

Also in the present embodiment, as in the case of the first embodiment, the following configuration can be adopted. The area of one reagent part of the first reagent part and the second reagent part may be larger than the area of the other reagent part. Moreover, the area of one reagent part of a 1st reagent part and a 2nd reagent part arrange | positioned at a nonelectroconductive part may be larger than the area of the working electrode contained in a conductive part. Further, the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be larger than the total area of the area of the working electrode and the counter electrode of the conductive part. Further, one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be arranged so as to be closer to the introduction opening for the test solution than the other reagent part. . Moreover, one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be arranged so as to be closer to the introduction opening than the working electrode.

The biosensor according to the present embodiment also has the same effect as the biosensor according to Embodiment 1, and can be suitably used when measuring one kind of substance to be detected.

Embodiment 3
In the first embodiment, the example in which the second reagent part is disposed on the back surface of the cover has been described. In the present embodiment, an example in which the second reagent part is disposed in the non-conductive part on the insulating substrate will be described. . In the following description, the description overlapping with the description of Embodiment 1 is omitted.

FIG. 7 is a plan view of the insulating substrate 31 used in the biosensor C according to the present embodiment, and FIG. 8 is a longitudinal sectional view along the longitudinal direction of the biosensor C.

A conductive portion 34 is formed on one main surface of the insulating substrate 31 having a pair of opposing main surfaces. The conductive portion 34 includes a first electrode pair 341 and a second electrode pair 342, a terminal portion 343 formed at one end of the insulating base material 31, a first electrode pair 341 or a second electrode pair 342 and a terminal portion 343. A lead portion 344 to be connected is provided. The first electrode pair 341 has a working electrode 341a and a pair of

counter electrodes

341b and 341b facing the working electrode 341a through a minute gap so as to sandwich the working electrode 341a in plan view. Further, the second electrode pair 342 is disposed in the longitudinal direction apart from the first electrode pair 341, and faces the working electrode 342a through a minute gap so as to sandwich the working electrode 342a in plan view. A pair of

counter electrodes

342b and 342b is provided. The pair of working

electrodes

341a and 341a is connected to the terminal 343a via the lead 344a, and the two pairs of

counter electrodes

341b and 341b are connected to the terminal 343d via the lead 344d. The working electrode 342a is connected to the terminal 343b via a lead 344b, and the pair of

counter electrodes

342b and 342b is connected to the terminal 343c via a lead 344c. Reference numeral 351 denotes a region where a conductive portion is not formed and corresponds to a non-conductive portion. In the case of FIG. 7, a structure excluding a first reagent part and a second reagent part described later is shown.

The

openings

33 a and 33 b of the spacer 33 form two first space portions 36 and second space portions 37 that are independent from each other when the spacer 33 is sandwiched between the insulating base material 31 and the cover 32. ing. The conductive portion in the first space portion 36 is the first electrode pair 341 and the lead portion. On the other hand, the non-conductive portion in the first space portion 36 is an insulating base material surface 352 as a portion where no electrode or lead is formed in the insulating base material 31 and the inner peripheral surface of the opening 33a of the spacer 33. Among the pair of opposing main surfaces of the spacer 33, a main surface 351 that is not in contact with the cover 32 (hereinafter also referred to as a back surface) can be exemplified. The conductive portion in the second space portion 37 is the second electrode pair 42 and the lead portion. On the other hand, the non-conductive portion of the second space portion 37 is a portion of the insulating base 31 where no electrode or lead is formed, an inner peripheral surface of the opening 33b of the spacer 33, and a pair of opposed spacers 33. Among the main surfaces, a back surface 351 that is not in contact with the cover 32 can be cited.

In the present embodiment, in the first space portion 36, the first reagent portion 38 is disposed on the first electrode pair 341 that is a conductive portion, and the second reagent portion 39 is a non-conductive portion. 352. In the second space portion 37, the second reagent portion 39 is disposed on the surface of the second electrode pair 342.

As in the case of the first embodiment, the opening 32b at the center of the cover 32 can be used as an opening for introducing the test liquid. When the test liquid is dropped into the opening 32b, the test liquid is the first. It flows to the space part 36 and the second space part 37, and contacts the first reagent part 38 and the second reagent part 39 in the first space part 36. Further, the

openings

32 a and 32 c at both ends of the cover 32 provide air to the inside of the first space 36 and the second space 37, respectively, and the test liquid is supplied from the opening 32 b at the center of the cover 32. When it is dropped, it works to release the internal air as the test solution is drawn.

In the present embodiment, one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is arranged so as to be closer to the introduction opening for the test solution than the other reagent part. ing. For example, in the example shown in FIG. 8, the second reagent part 39 disposed on the insulating base material surface 352 that is a non-conductive part is more open than the first reagent part 38 as an opening 32 b that is an introduction opening for the test liquid. It is arranged to be close to. The test liquid flowing in from the introduction opening contacts the second reagent part 39 and moves from the upstream to the downstream in the first space 36 while dissolving the reagent contained in the second reagent part 39. However, the second reagent part 39 arranged on the insulating base surface 352 is arranged so as to be closer to the introduction opening than the first reagent part 38, that is, upstream from the first reagent part 38. Therefore, even if a part of the reagent flows out of the reaction region, a sufficient amount of the reagent can be supplied to the working electrode 341a. As a result, the response value increases and the S / N ratio is improved, so that more accurate measurement is possible.

Further, in the present embodiment, one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is arranged so as to be closer to the introduction opening than the working electrode. For example, in the example shown in FIG. 8, the second reagent part 39 disposed on the insulating base material surface 352 that is a non-conductive part is closer to the opening part 32b that is an opening for introducing the test solution than the working electrode 341a. It is arranged to be. The test liquid flowing in from the introduction opening contacts the second reagent part 39 and moves from the upstream to the downstream in the first space 36 while dissolving the reagent contained in the second reagent part 39. However, since the second reagent part 39 disposed on the insulating base material surface 352 is disposed closer to the introduction opening than the working electrode 341a, that is, disposed upstream of the working electrode 341a. Therefore, even if a part of the reagent flows out of the reaction region, a sufficient amount of reagent can be supplied to the working electrode 341a. As a result, the response value increases and the S / N ratio is improved, so that more accurate measurement is possible.

Also in the present embodiment, as in the case of the first embodiment, the following modes can be taken. The area of one reagent part of the first reagent part and the second reagent part may be larger than the area of the other reagent part. Moreover, the area of one reagent part of a 1st reagent part and a 2nd reagent part arrange | positioned at a nonelectroconductive part may be larger than the area of the working electrode contained in a conductive part. Further, the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be larger than the total area of the area of the working electrode and the counter electrode of the conductive part.

According to the present embodiment, in the first space part 36, the first reagent part 38 is disposed on the first electrode pair 341 which is a conductive part, and the second reagent part 39 is provided with an insulating group which is a non-conductive part. By arranging on the material surface 352, the same effect as in the first embodiment can be obtained. Further, since the two electrode pairs of the first electrode pair 341 and the second electrode pair 342 are provided, it can be used when two different kinds of substances to be detected in the test liquid are measured.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Reference Example In a conventional biosensor, a coating film obtained by applying a coating solution containing both a protein such as an enzyme and a mediator to an electrode and drying it is used as a reagent part. In this reference example, as a reagent part, when using a coating film using a coating liquid containing both protein and mediator (hereinafter referred to as a mixed support coating film), a coating film containing protein and a mediator is used. The blank current value was compared for the case (hereinafter referred to as a separate supported coating film).

(experimental method)
In the mixed supported coating film, a predetermined amount of a reagent solution having the following composition is spotted on a palladium sheet prepared by vapor deposition on an insulating substrate constituting the biosensor A in FIG. % And dried for about 3 hours. This coating film was redissolved in the same amount of sodium phosphate buffer (pH 7.0, 50 mM) as the reagent solution, and the redissolved solution was aspirated and collected. On the other hand, the separate supported coating film was prepared using the same method as that for the mixed supported coating film except that a reagent solution containing no mediator was prepared and the reagent solution was spotted on the supporting sheet.
<Reagent solution>
Sodium phosphate buffer (pH 7.0) 10 mM
Mediator 6mM
Protein solution 10 mg / mL or 20 mg / mL
<Mediator>
1-methoxy-5-methylphenazinium methyl sulfate (PMS)
9,10-phenanthrenequinone-2-sulfonic acid sodium salt (PQSA)
<Protein>
Fructosyl peptide oxidase (FPOX)
Glucose dehydrogenase (GDH)
Immunoglobulin G (IgG)
Bovine serum albumin (BSA)

The redissolved solution was spotted on the biosensor A shown in FIG. 1 and subjected to electrochemical measurement. In the electrochemical measurement, the biosensor A was connected to a potentiostat, a voltage of 0.2 V was applied between the working electrode and the counter electrode for 30 seconds, and the current value when 10 seconds had elapsed after application was defined as a blank current.

The results are shown in Figs. Table 1 shows numerical values of the blank current. FIG. 9 shows the result of another supported coating film containing only protein, and FIG. 10 shows the result of the mixed supported coating film using 1-methoxy-5-methylphenazinium methyl sulfate (PMS) as a mediator. FIG. 11 shows the results of a mixed supported coating film using sodium 9,10-phenanthrenequinone-2-sulfonate (PQSA) as a mediator. In the case of another supported coating film of protein, the blank current was 25 nA or less. In the case of another supported coating film of mediator, the blank currents of PMS and PQSA were 235 nA and 18 nA, respectively. On the other hand, in the mixed supported coating film, the blank current increased as compared with the separate supported coating film of protein and the separately supported coating film of mediator, and particularly increased when PMS was used as the mediator. For example, in the case of PQSA, it increased from 75 to 189 nA depending on the type and concentration of protein. On the other hand, in the case of PMS, it increased from 513 to 2707 nA depending on the type and concentration of protein. From this result, it can be expected that the blank current can be suppressed by using another supported coating film in the reagent part instead of the mixed supported coating film. In the case of a mixed supported coating film, the reason why the blank current is large is not always clear, but the high concentration state of the enzyme and the mediator is maintained by drying the coating liquid, and the amino group having an electron donating property in the enzyme is the mediator. It is conceivable that the blank current is increased by partially reducing.

Example 1
<Production of biosensor>
1 μl of the following reagent solution 1 is applied to the surface of the first electrode pair on the insulating base material constituting the biosensor A of FIG. 1 and dried at 25 ° C. and 50% humidity for about 3 hours. Formed. On the other hand, 24 μL of the following reagent solution 2 was applied to the back surface of the cover constituting the biosensor A of FIG. 1 and dried at 25 ° C. and 50% humidity for about 3 hours to form a second reagent part. And the biosensor shown in FIG. 1 was produced.
<Reagent liquid 1>
Sodium phosphate buffer (pH 7.0) 25 mM
Glucose dehydrogenase (GDH) 2000 U / mL
Carboxymethylcellulose (CMC) 0.2%
Dodecyl maltoside 0.0065%
<Reagent liquid 2>
9,10-phenanthrenequinone-2-sulfonic acid sodium salt (PQSA) 10 mM

The biosensor A was spotted with 50 μL of a test solution having the following composition and subjected to electrochemical measurement. In the electrochemical measurement, the biosensor A was connected to a potentiostat, a voltage of 0.2 V was applied between the working electrode and the counter electrode for 30 seconds, and the current value when 10 seconds had elapsed after the application was measured.
<Test solution>
Sodium phosphate buffer (pH 7.0) 25 mM

Glucose

0, 20, 50, 100 μM

FIG. 12 shows the relationship between the glucose concentration and the detected current value (Δ mark). A linear relationship was obtained in the range of 0-100 μM.

Comparative Example 1
<Production of biosensor>
4 μL of the following reagent solution 3 is applied to the surface of the first electrode pair on the insulating substrate constituting the biosensor A of FIG. 1 and dried at 25 ° C. and 50% humidity for about 3 hours to form a reagent part. A biosensor was produced by the same method as in Example 1 except that.
<Reagent liquid 3>
Sodium phosphate buffer (pH 7.0) 25 mM
Glucose dehydrogenase (GDH) 2000 U / mL
9,10-phenanthrenequinone-2-sodium sulfonate (PQSA) 30 mM
Carboxymethylcellulose (CMC) 0.2%
Dodecyl maltoside 0.0065%

The prepared biosensor was spotted, and electrochemical measurement was performed in the same manner as in Example 1.

FIG. 12 shows the relationship between the glucose concentration and the detected current value (◯ mark). A linear relationship was obtained in the range of 0-100 μM.

(result)
As shown in FIG. 12, in the case of Example 1, it can be seen that the blank current value is reduced as compared with Comparative Example 1. When the S / N ratio (100 μM current value / blank current value) was calculated, Comparative Example 1 was 2.29, while Example 1 was 5.27. Thereby, according to this invention, it has confirmed that S / N ratio could be improved significantly compared with the past.

According to the present invention, a biosensor capable of highly accurate measurement can be provided.

1, 21, 31 Insulating

base material

2, 22, 32

Cover

2a, 2b,

2c Cover opening

22a,

22b Cover opening

32a,

32b Cover opening

3, 23, 33

Spacer

3a, 3b Spacer opening 23a

Spacer opening Part

33a,

33b Spacer opening

4, 24, 34

Conductive part

41, 241, 341

First electrode pair

41a, 241a,

341a Working electrode

41b, 241b,

341b Counter electrode

42, 342

Second electrode pair

42a,

342a Working electrode

42b, 342b Counter electrode 43,343

Terminal portion

43a, 43b, 43c,

43d terminal

243a, 243b terminal 343a, 343b, 343c,

343d terminal

44, 242 344

Lead portion

44a, 44b, 44c,

44d Lead

242a,

242b Lead

344a, 344b, 344c,

344d Lead

5, 25, 351, 352

Non-conductive portion

6, 26, 36

First space portion

7, 27, 37

Second space Part

8, 28, 38

First reagent part

9, 29, 39 Second reagent part

Claims

A biosensor that analyzes a component in a test liquid using a protein and a mediator,
Having one or more spaces formed through a spacer between the insulating base and the cover;
The inner surface of at least one of the space portions has a conductive portion and a non-conductive portion,
The first reagent part containing the protein and the second reagent part containing the mediator are separately arranged at different locations on the inner surface, and at least one of the first reagent part and the second reagent part is in the non-conductive part A biosensor is placed.
The biosensor according to claim 1, wherein one of the first reagent part and the second reagent part is disposed in the non-conductive part and the other is disposed in the conductive part.
The biosensor according to claim 1 or 2, wherein the first reagent part is disposed in the conductive part and the second reagent part is disposed in the non-conductive part.
The biosensor according to claim 1, wherein the first reagent part and the second reagent part are arranged at different locations of the non-conductive part.
The insulating substrate has a pair of opposing main surfaces, one main surface exposed to the space portion constitutes a bottom surface of the space portion, and the cover has a pair of opposing main surfaces. The one main surface exposed to the space portion constitutes the top surface of the space portion, the spacer has at least one opening portion, and the inner surface of the opening portion constitutes the side surface of the space portion. The biosensor according to any one of claims 1 to 4.
The biosensor according to claim 5, wherein the bottom surface has the conductive portion and the top surface has the non-conductive portion.
The biosensor according to claim 5, wherein the bottom surface has the conductive portion and the non-conductive portion.
The biosensor according to any one of claims 1 to 7, wherein the conductive portion includes at least a working electrode and a counter electrode.
The biosensor according to any one of claims 1 to 8, wherein an area of one reagent part of the first reagent part and the second reagent part is larger than an area of the other reagent part.
One reagent part of the first reagent part and the second reagent part is arranged in the non-conductive part, the other reagent part is arranged in the conductive part, and the one reagent part arranged in the non-conductive part The biosensor according to claim 9, wherein an area of is larger than an area of the other reagent part arranged in the conductive part.
The conductive part includes a working electrode and a counter electrode, and the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is larger than the area of the working electrode. The biosensor according to any one of claims 1 to 8.
The conductive part includes a working electrode and a counter electrode, and the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is the area of the working electrode and the area of the counter electrode. The biosensor according to any one of claims 1 to 8, wherein the biosensor is larger than the total area.
The cover has an introduction opening for a test liquid that communicates with the space, and one of the first reagent part and the second reagent part arranged in the non-conductive part is the other The biosensor according to any one of claims 1 to 12, wherein the biosensor is disposed so as to be closer to the introduction opening than the reagent portion.
The conductive part includes a working electrode and a counter electrode, and the cover has an introduction opening for a test solution communicating with the space part, and the first reagent part and the first reagent part arranged in the non-conductive part 13. The biosensor according to claim 1, wherein one reagent part of the two reagent parts is disposed closer to the introduction opening than the working electrode.