Biosensor

Abstract

To provide a biosensor that can carry out accurate measurement in one aspect.SOLUTION: The present disclosure relates, as one aspect, to a biosensor comprising: a substrate that is formed, on one principal surface, with a conductive part including an electrode pair having a working electrode and a counter electrode; a reagent layer that is placed on the working electrode and the counter electrode; and a channel for circulating a sample from a sample supply port to the reagent layer. The reagent layer covers the entire area in the channel of the working electrode, extends for at least 50 μm from an upstream-side edge and a downstream-side edge of the working electrode, and is formed to cover an area corresponding to at least 70% of the area of the working electrode, in the area in the channel of the counter electrode. The reagent layer has an average height of 4 μm to 12 μm.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present disclosure relates to biosensors and methods for making biosensors. [Background technology]

[0002] A biosensor that measures a target substance in a sample, such as blood glucose in blood, usually has a pair of electrodes and a reagent layer formed thereon. When blood is supplied to the reagent layer of the biosensor, a reaction system is formed between the electrodes, which makes it possible to measure the target substance in the blood (Patent Document 1).

[0003] The reagent layer is usually formed by applying a reagent solution containing an oxidoreductase, an electron transfer substance, etc., onto the electrode and drying it. Methods for applying the reagent solution include, for example, a dipping method, a dispenser method, a screen printing method, an offset printing method, and an inkjet printing method (Patent Documents 1 to 4). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2003 / 025558 [Patent Document 2] JP 2011-99693 A [Patent Document 3] Japanese Patent Application Publication No. 6-3317 [Patent Document 4] Special Publication No. 2017-520773 Summary of the Invention [Problem to be solved by the invention]

[0005] A biosensor usually has at least a substrate, a conductive part having a working electrode and a counter electrode formed on the substrate, and a reagent layer formed on the conductive part. The size of a biosensor is usually very small, with a longitudinal length of several tens of mm and a lateral length of several mm. Since biosensors are used to measure a measurement target in a sample, such as blood glucose in blood, high measurement accuracy is required.

[0006] The measurement of the object to be measured is performed by detecting a reaction between the object to be measured, such as blood glucose contained in a sample such as blood, and a reagent in the reagent layer disposed on the conductive part. The present inventors have found that the position of the reagent layer disposed on the conductive part has a large effect on the measurement accuracy, and have found that the positional relationship between the working electrode and counter electrode constituting the conductive part and the reagent layer is particularly important.

[0007] The present disclosure provides a biosensor capable of performing measurements with high accuracy. [Means for solving the problem]

[0008] In one aspect, the present disclosure relates to a biosensor comprising a substrate having a conductive portion formed on one main surface thereof, the conductive portion including an electrode pair having a working electrode and a counter electrode, a reagent layer placed on the working electrode and the counter electrode, and a flow path for distributing a sample from a sample supply port to the reagent layer, wherein the reagent layer is formed so as to cover the entire area of ​​the working electrode in the flow path, extend at least 50 μm from each of the upstream and downstream edges of the working electrode, and cover an area of ​​the counter electrode in the flow path that corresponds to at least 70% of the area of ​​the working electrode, and the average height of the reagent layer is 4 μm to 12 μm.

[0009] In another aspect, the present disclosure relates to a biosensor including a substrate having a conductive portion formed on one main surface thereof, the conductive portion including an electrode pair having a working electrode and a counter electrode, a reagent layer placed on the working electrode and the counter electrode, and a flow path for distributing a sample from a sample supply port to the reagent layer, wherein the reagent layer covers the entire surface of the working electrode in the flow path and at least a portion of the surface of the counter electrode in the flow path, and is formed to extend from an upstream edge of the working electrode toward the upstream side and from a downstream edge of the working electrode toward the downstream side, each of the extending ranges being at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, a coverage area of ​​the reagent layer on the counter electrode corresponds to at least 70% of the surface area of ​​the working electrode in the flow path, and an average height of the reagent layer is 4 μm to 12 μm.

[0010] In yet another aspect, the present disclosure relates to a manufacturing method for a biosensor having a conductive portion and a reagent layer in a flow path, the manufacturing method including: preparing a substrate on one main surface of which is formed a conductive portion including an electrode pair having a working electrode and a counter electrode; and forming the reagent layer on the working electrode and the counter electrode, the formation of the reagent layer including dot-printing a reagent solution such that the entire area of ​​the working electrode in the flow path, areas at least 50 μm from each of the upstream and downstream edges of the working electrode, and an area of ​​the counter electrode in the flow path that corresponds to at least 70% of the area of ​​the working electrode in the flow path are covered with reagent, and the average height of the reagent layer is 4 μm to 12 μm. Effect of the Invention

[0011] According to the present disclosure, a biosensor capable of performing measurements with high accuracy can be provided. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram showing a schematic configuration of a biosensor according to an embodiment of the present disclosure. [Diagram 2] FIG. 2 is a schematic diagram showing a schematic configuration of a biosensor according to an embodiment of the present disclosure. [Diagram 3] FIG. 3 is a graph showing an example of the results obtained in Example (glucose current measurement 1), showing the relationship between the glucose concentration and the reaction current value. [Figure 4] FIG. 4 is a graph showing an example of the results obtained in Example (glucose current measurement 1), showing the relationship between the glucose concentration and the reaction current value. [Diagram 5] FIG. 5 is a graph showing an example of the results obtained in Example (glucose current measurement 1), showing the reaction current value of each biosensor in a sample (glucose (GLU) concentration 3000 mg / dl×Hct 42%). [Figure 6] FIG. 6 is a graph showing an example of the results obtained in Example (glucose current measurement 2), showing the reaction current value of each biosensor in a sample (GLU concentration 1500 mg / dl×Hct 42%). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The biosensor of the present disclosure is used to measure a measurement target in a sample. In one or more embodiments, the sample may be a biological sample. In one or more embodiments, the biological sample may be blood, urine, or a sample derived therefrom, a cell extract, or a cell culture medium. In one or more embodiments, the measurement target may be glucose, cholesterol, ethanol, sorbitol, fructose, cellobiose, lactic acid, urea, or the like.

[0014] [Biosensors] The present disclosure relates to, as one aspect, a biosensor including a substrate having a conductive part including an electrode pair having a working electrode and a counter electrode formed on one main surface, a reagent layer placed on the working electrode and the counter electrode, and a flow path for passing a sample from a sample supply port to the reagent layer. In one aspect, the reagent layer in the biosensor of the present disclosure has an average height of 4 μm to 12 μm, and is formed so as to cover the entire area of ​​the working electrode in the flow path, extend at least 50 μm from each of the upstream and downstream edges of the working electrode, and cover an area of ​​the counter electrode in the flow path that corresponds to at least 70% of the area of ​​the working electrode. In another embodiment, the reagent layer in the biosensor of the present disclosure has an average height of 4 μm to 12 μm, covers the entire surface of the working electrode in the flow path and at least a portion of the surface of the counter electrode in the flow path, and is formed to extend from the upstream edge of the working electrode toward the upstream side and from the downstream edge of the working electrode toward the downstream side, each of the extension ranges being at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, and the coverage area of ​​the reagent layer on the counter electrode corresponds to at least 70% of the surface area of ​​the working electrode in the flow path.

[0015] In one or more embodiments, the biosensor of the present disclosure is formed with a sample supply port and a flow path communicating with the sample supply port, the sample supply port being provided for supplying a sample to the flow path, and the flow path being provided for distributing the sample supplied from the sample supply port to a reagent layer in the flow path. In the present disclosure, the "upstream side" refers to the upstream side in the supply direction of the sample to the flow path in the biosensor, i.e., the sample supply port side, and the "downstream side" refers to the downstream side in the supply direction of the sample, i.e., the end side of the flow path located opposite the sample supply port. In the present disclosure, the "sample supply direction" refers to the direction in which the sample supplied to the sample supply port of the biosensor flows through the flow path.

[0016] Considering simplicity and efficiency, the formation of the reagent layer of a biosensor is usually performed by applying a reagent solution onto a conductive part by screen printing, etc. However, in the case of such a method, in order to manufacture a biosensor that can obtain sufficient measurement accuracy, it was thought that it was necessary to form the reagent layer so that the entire surfaces of the electrodes (working electrode and counter electrode) in the flow channel were covered with the reagent (for example, Patent Document 1 (Fig. 1) and Patent Document 4 (Fig. 1)). In response to this, the present inventor has found that, for example, by forming a reagent layer with an average height of 4 μm to 12 μm by dot printing, etc., it is possible to achieve highly accurate measurement even when the entire surface of the electrode in the flow path is not covered with the reagent. Furthermore, the present inventor has found that in order to achieve highly accurate measurement, it is important to cover the edge of the working electrode and the range from the edge to a predetermined region with the reagent.

[0017] According to the biosensor of the present disclosure, the average height of the reagent layer is 4 μm to 12 μm, and the reagent layer is formed so as to extend not only over the entire surface within the flow path of the working electrode but also to a predetermined range from the edge of the working electrode, and further so as to cover an area of ​​the surface within the flow path of the counter electrode that corresponds to at least 70% of the area within the flow path of the working electrode, thereby enabling measurement of the analyte with high accuracy.

[0018] In the present disclosure, "the reagent layer covers the entire area of ​​the working electrode in the flow path" means that the entire surface of the working electrode exposed in the flow path is substantially covered with the reagent. In the present disclosure, the entire surface of the working electrode in the flow path is substantially covered with the reagent, in one or more embodiments, means that 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, 99.99% or more, or 100% of the surface of the working electrode in the flow path is covered with the reagent.

[0019] The reagent layer is formed so as to cover the entire region (surface) in the flow path of the working electrode and to extend from the upstream edge and the downstream edge of the working electrode. In this disclosure, "the reagent layer extends from the edge of the working electrode" means that the reagent layer is disposed on the surface of the substrate (or a member disposed on the substrate) beyond the edge of the working electrode (spreading from the edge). Thus, in this disclosure, the edge of the working electrode in the flow path is covered by the reagent layer.

[0020] The reagent layer extends at least 50 μm from each of the upstream and downstream edges of the working electrode. In order to further improve the measurement accuracy, in one or more embodiments, the reagent layer preferably extends 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 125 μm or more, 150 μm or more, 175 μm or more, 200 μm or more, 225 μm or more, 250 μm or more, 275 μm or more, or 300 μm or more. In one or more embodiments, the range over which the reagent layer extends from each of the upstream and downstream edges of the working electrode is 500 μm or less, 450 μm or less, or 400 μm or less. In one or a plurality of embodiments, the reagent layer extends from the upstream edge of the working electrode by 50 μm to 500 μm, 50 μm to 450 μm, 50 μm to 400 μm, 50 μm to 350 μm, 50 μm to 340 μm, 50 μm to 330 μm, 50 μm to 320 μm, or 50 μm to 310 μm. In one or a plurality of embodiments, the reagent layer extends from the downstream edge of the working electrode by 175 μm to 500 μm, 200 μm to 500 μm, 225 μm to 500 μm, 250 μm to 500 μm, 275 μm to 500 μm, 275 μm to 450 μm, 275 μm to 440 μm, 275 μm to 430 μm, or 280 μm to 420 μm. From the same viewpoint, the extension range of the reagent layer from each of the upstream and downstream edges of the working electrode is preferably 100 μm or more and 450 μm or less, or 100 μm or more and 150 μm or less. In one or more embodiments, the reagent layer extends from 50 μm to 500 μm from the edge of the upstream side of the working electrode and extends from 175 μm to 500 μm from the edge of the downstream side of the working electrode. In one or more embodiments, the reagent layer extends from 50 μm to 450 μm from the edge of the upstream side of the working electrode and extends from 200 μm to 500 μm from the edge of the downstream side of the working electrode. In one or more embodiments, the reagent layer extends from 50 μm to 400 μm from the edge of the upstream side of the working electrode and extends from 225 μm to 500 μm from the edge of the downstream side of the working electrode. In one or more embodiments, the reagent layer extends from 50 μm to 350 μm from the edge of the upstream side of the working electrode and extends from 250 μm to 500 μm from the edge of the downstream side of the working electrode. In one or more embodiments, the reagent layer extends from 50 μm to 340 μm from the upstream edge of the working electrode and from 275 μm to 500 μm from the downstream edge of the working electrode. In one or more embodiments, the reagent layer extends from 50 μm to 330 μm from the upstream edge of the working electrode and from 275 μm to 450 μm from the downstream edge of the working electrode. In one or more embodiments, the reagent layer extends from 50 μm to 320 μm from the upstream edge of the working electrode and from 275 μm to 430 μm from the downstream edge of the working electrode. In one or more embodiments, the reagent layer extends from 50 μm to 310 μm from the upstream edge of the working electrode and from 0 μm to 420 μm from the downstream edge of the working electrode. In one or more embodiments, the distance between the upstream edge of the working electrode and the upstream edge of the reagent layer is 50 μm or more and 500 μm or less, 50 μm or more and 450 μm or less, 50 μm or more and 400 μm or less, 50 μm or more and 350 μm or less, 50 μm or more and 340 μm or less, 50 μm or more and 330 μm or less, 50 μm or more and 320 μm or less, or 50 μm or more and 310 μm or less. In one or more embodiments, the distance between the downstream edge of the working electrode and the downstream edge of the reagent layer is 175 μm or more and 500 μm or less, 200 μm or more and 500 μm or less, 225 μm or more and 500 μm or less, 250 μm or more and 500 μm or less, 275 μm or more and 500 μm or less, 275 μm or more and 450 μm or less, 275 μm or more and 440 μm or less, 275 μm or more and 430 μm or less, or 280 μm or more and 420 μm or less.

[0021] The range over which the reagent layer extends from each of the upstream and downstream edges of the working electrode is at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, and in order to further improve measurement accuracy, in one or more embodiments, the range is 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the area over which the reagent layer extends from the upstream edge of the working electrode is in the range of 12.5% ​​to 100%, 12.5% ​​to 90%, 12.5% ​​to 85%, 12.5% ​​to 80%, or 13% to 75% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the range over which the reagent layer extends from the downstream edge of the working electrode is in the range of 45% to 100%, 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, or 75% to 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the reagent layer has an area extending from the upstream edge of the working electrode that is 12.5% ​​to 100% of the surface area of ​​the working electrode in the flow path, and an area extending from the downstream edge of the working electrode that is 45% to 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the reagent layer has an area extending from the upstream edge of the working electrode that is 12.5% ​​to 90% of the surface area of ​​the working electrode in the flow path, and an area extending from the downstream edge of the working electrode that is 50% to 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the reagent layer has an area extending from the upstream edge of the working electrode that is 12.5% ​​to 85% of the surface area of ​​the working electrode in the flow path, and an area extending from the downstream edge of the working electrode that is 55% to 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the reagent layer has an area extending from the upstream edge of the working electrode that is 12.5% ​​to 80% of the surface area of ​​the working electrode in the flow path, and an area extending from the downstream edge of the working electrode that is 60% to 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the reagent layer has an area extending from the upstream edge of the working electrode that is 13% to 75% of the surface area of ​​the working electrode in the flow path, and an area extending from the downstream edge of the working electrode that is 65% to 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the reagent layer has an area extending from the upstream edge of the working electrode that is 13% to 75% of the surface area of ​​the working electrode in the flow path, and an area extending from the downstream edge of the working electrode that is 70% to 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the reagent layer extends from the upstream edge of the working electrode over a range of 13% to 75% of the surface area of ​​the working electrode in the flow path, and extends from the downstream edge of the working electrode over a range of 75% to 100% of the surface area of ​​the working electrode in the flow path.

[0022] In order to further improve the measurement accuracy, in one or more embodiments, the area of ​​the reagent layer extending from the downstream edge of the working electrode is preferably larger than the area of ​​the reagent layer extending from the upstream edge of the working electrode. In one or more embodiments, the area of ​​the reagent layer extending from the downstream edge of the working electrode is 1.3 times or more, 1.4 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, or 4 times or more the area of ​​the reagent layer extending from the upstream edge of the working electrode.

[0023] The reagent layer is formed so as to cover at least a part of the area (surface) in the flow channel of the counter electrode. The reagent layer is formed so as to cover an area (surface) in the flow channel of the counter electrode that corresponds to at least 70% of the area (area) in the flow channel of the working electrode, and in one or more embodiments, from the viewpoint of further improving the measurement accuracy, the area may be 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. In one or more embodiments, the area (area) of the reagent layer covering the counter electrode may be an area equivalent to 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 89% or less, 88% or less, 87% or less, 86% or less, or 85% or less of the area (area) in the flow channel of the working electrode. In one or more embodiments, the region (area) of the reagent layer covering the counter electrode may be an area corresponding to 70% or more and 100% or less, 75% or more and 100% or less, 80% or more and 100% or less, 85% or more and 100% or less, 90% or more and 100% or less, or 95% or more and 100% or less of the region (area) of the working electrode in the flow path. In addition, in one or more embodiments, the area of ​​the reagent layer covering the counter electrode may be at least 70% of the area (area) in the flow path of the counter electrode, and in one or more embodiments, from the viewpoint of further improving the measurement accuracy, it may be 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. In one or more embodiments, the area of ​​the reagent layer covering the counter electrode may be 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 89% or less, 88% or less, 87% or less, 86% or less, or 85% or less of the area (area) in the flow path of the counter electrode. In one or more embodiments, the region (area) of the reagent layer covering the counter electrode may be 70% or more and 100% or less, 75% or more and 100% or less, 80% or more and 100% or less, 85% or more and 100% or less, 90% or more and 100% or less, or 95% or more and 100% or less of the region (area) of the counter electrode in the flow path.

[0024] The average height of the reagent layer is 4 μm to 12 μm. From the viewpoint of further improving the measurement accuracy, the average height of the reagent layer is preferably 5 μm or more, 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, or 9.5 μm or more. In one or more embodiments, the average height of the reagent layer is 5 μm to 12 μm, 6 μm to 12 μm, 7 μm to 12 μm, 8 μm to 12 μm, 9 μm to 12 μm, or 9.5 μm to 12 μm. In one or more embodiments, the average height of the reagent layer is 4 μm to 11 μm, 4 μm to 10 μm, 4 μm to 9 μm, or 4 μm to 8 μm. The average height of the reagent layer can be measured and calculated by the method described in the Examples.

[0025] In one or more embodiments, the reagent layer is preferably formed by dot printing. According to the dot printing, as described below, the droplets of the ejected reagent liquid are dried, and droplets of the reagent liquid are ejected thereon and dried repeatedly, so that the reagent layer having the above average height can be easily formed. In one or more embodiments, the formation of the reagent layer by dot printing can be performed by taking into consideration the method description described in the examples, EP4266037, or US2023 / 338945, etc.

[0026] The size (area) of the reagent layer in the flow path can be appropriately determined depending on the shape of the substrate, the flow path, and the conductive part, etc. In the case of a rectangular substrate with a short side length of 7 mm and a long side length of 30 mm, a flow path with a width (short side length of the substrate) of 1.8 mm and a length (long side length of the substrate) of 4 mm, and a rectangular shape with a width (short side length of the substrate) of 1.8 mm and a length (long side length of the substrate) of 0.8 mm in the flow path of the working electrode, in one or more embodiments, the size of the reagent layer is a width (short side length of the substrate) of 1.5 mm or more, 2 mm or more, 2.5 mm or more, or 3 mm or more, and / or 5 mm or less, 4.5 mm or less, or 4 mm or less, and a length (long side length of the substrate) of 0.5 mm or more, 0.6 mm or more, 0.8 mm or more, or 0.9 mm or more, and / or 2 mm or less, 1.6 mm or less, or 1.4 mm or less. In one or more embodiments, the size of the reagent layer is such that the width (length in the short direction of the substrate) is 1.5 mm or more and 5 mm or less, 2 mm or more and 5 mm or less, or 2.5 mm or more and 4.5 mm or less, and / or the length (length in the long direction of the substrate) is 0.5 mm or more and 2 mm or less, 0.6 mm or more and 2 mm or less, 0.8 mm or more and 1.6 mm or less, or 0.9 mm or more and 1.4 mm or less.

[0027] In one or more embodiments, the substrate may be a sheet-shaped insulating substrate. In one or more embodiments, the substrate may be made of a thermoplastic resin, a thermosetting resin, glass, ceramic, paper, or the like. In one or more embodiments, the substrate may be made of a material such as polyetherimide (PEI), polyethylene terephthalate (PET), polyethylene (PE), polybutylene terephthalate (PBT), polystyrene (PS), polymethacrylate (PMMA), polypropylene (PP), polyimide resin, acrylic resin, epoxy resin, glass epoxy, or the like.

[0028] The conductive part includes at least one electrode pair having a working electrode and a counter electrode, and is formed on one main surface of the substrate. When the conductive part includes a plurality of electrode pairs, for example, the conductive part may include a first electrode pair for measuring a first analyte (e.g., blood glucose) contained in the same sample such as blood, and a second electrode pair for measuring a second analyte (e.g., hematocrit). Furthermore, the first electrode pair and the second electrode pair may share a counter electrode. Here, when the conductive part includes a plurality of electrode pairs, the reagent layer in the biosensor of the present disclosure may be formed only on at least one electrode pair, and may be formed on a plurality of electrode pairs. In addition, the conductive part may further include a detection electrode in one or more embodiments.

[0029] In one or more embodiments, the working electrode and the counter electrode extend in parallel in a direction perpendicular to the sample supply direction in the flow channel. In one or more embodiments, the working electrode is formed upstream of the counter electrode, and preferably is formed upstream of the counter electrode adjacent to the counter electrode.

[0030] In one or more embodiments, a conductive material can be used as the material of the electrode. In one or more embodiments, examples of the conductive material include metal materials and carbon materials. In one or more embodiments, examples of the metal material include gold (Au), platinum (Pt), silver (Ag), ruthenium (Ru), palladium (Pd), and nickel (Ni) alloys. In one or more embodiments, examples of the nickel alloy include nickel-vanadium alloys, nickel-tungsten alloys, and nickel-ruthenium alloys. In one or more embodiments, examples of the carbon material include graphite, carbon nanotubes, graphene, and mesoporous carbon.

[0031] In one or more embodiments, the electrode may be a thin-film electrode formed by depositing a film of the above-mentioned material, such as a metal material, on a substrate. In one or more embodiments, the deposition method may be screen printing, physical vapor deposition (PVD, e.g., sputtering), chemical vapor deposition (CVD), or the like.

[0032] In one or more embodiments, the working electrode and the counter electrode may be insulated from each other. When the electrodes constituting the conductive part are formed of a metal material formed by physical vapor deposition, in one or more embodiments, the electrodes may be insulated from each other by drawing a predetermined electrode pattern with a laser beam (trimming). When the conductive part is formed of a carbon material, in one or more embodiments, the insulating part may be formed by forming the electrodes at a predetermined interval.

[0033] In one or more embodiments, the biosensor of the present disclosure may include a spacer disposed on a substrate and a cover disposed on the spacer. In one or more embodiments, the spacer may include a cutout. By disposing the spacer on the substrate so that the conductive portion and the reagent layer are located inside the cutout, the space formed by the substrate, the cutout, and the cover can be used as a sample supply port and a flow path.

[0034] The shape of the cutout portion of the spacer may be a U-shape, a C-shape, etc. The material of the spacer is not particularly limited, and the same material as that of the base material can be used.

[0035] In one or more embodiments, the cover may have an air hole, and is preferably disposed on the spacer so that the air hole is located above the cutout of the spacer. The material of the cover is not particularly limited, and may be the same as that of the substrate, and is preferably a hydrophilic film having a higher contact angle (hydrophobicity) than the spacer.

[0036] In one or more embodiments of the biosensor of the present disclosure, the reagent layer may be formed on a conductive portion (particularly a pair of electrodes having a working electrode and a counter electrode) located inside (within the cutout) of the flow path formed by the cutout of the spacer, and may also be formed on a conductive portion located outside the flow path. In other words, the reagent layer may be formed outside the cutout (flow path) of the spacer, that is, beyond the end (boundary) of the flow path. Note that the reagent layer formed outside the cutout (flow path) of the spacer does not substantially come into contact with the sample, so the reagent in the reagent layer in that portion does not contribute (influence) the measurement, and therefore the shape, size, height, etc. of the reagent layer formed outside the cutout (flow path) of the spacer are not particularly limited.

[0037] In one or more embodiments, the reagent contained in the reagent layer includes an oxidoreductase and an electron transfer substance.

[0038] [Oxidoreductase] In one or a plurality of embodiments, examples of the oxidoreductase include glucose dehydrogenase (GDH), glucose oxidase (GOD), cholesterol oxidase, quinohem ethanol dehydrogenase (QHEDH (PQQ Ethanol dh)), sorbitol dehydrogenase, D-fructose dehydrogenase, D-glucoside-3-dehydrogenase, cellobiose dehydrogenase, lactate oxidase (LOD), lactate dehydrogenase (LDH), and urate dehydrogenase. In one or more embodiments, the oxidoreductase may have flavin adenine dinucleotide (FAD), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a coenzyme (also referred to as a catalytic subunit or catalytic domain). In one or more embodiments, examples of the oxidoreductase having a coenzyme include FAD-GDH, PQQ-GDH, NAD-GDH and NADP-GDH. In one or more embodiments, the oxidoreductase may be Aspergillus oryzae type FAD-GDH (flavin adenine dinucleotide-dependent glucose dehydrogenase or flavin adenine dinucleotide-binding glucose dehydrogenase). In one or more embodiments, the Aspergillus oryzae type FAD-GDH may be that disclosed in JP 2013-083634 A. The contents of this document are incorporated by reference as part of the present disclosure.

[0039] In one or more embodiments, the amount of the oxidoreductase in the reagent layer is 1000 KU / cm 3 ≦500 KU / cm 3 More preferably, it is 300 KU / cm or less. 3 The lower limit of the amount of the oxidoreductase in the reagent layer is not particularly limited, and in one or more embodiments, it is 1 KU / cm 3In one or more embodiments, the amount of the oxidoreductase in the reagent layer is 200 KU / cm 3 ~300KU / cm 3 and preferably 200 KU / cm 3 ~280KU / cm 3 and more preferably 210 KU / cm 3 ~260KU / cm 3 In the present disclosure, "U" is a unit of enzyme activity, and refers to the amount of enzyme that acts on 1 μmol of substrate per minute under optimal conditions.

[0040] [Electron transfer substances] In one or a plurality of embodiments, examples of the electron transfer substance include a ruthenium compound, 1-Methoxy-PES (1-Methoxy-5-ethylphenazinium ethylsulfate, 1-mPES), 1-Methoxy-PMS (1-Methoxy-5-methylphenazinium methylsulfate, 1-mPMS), a phenylenediamine compound, a quinone compound, a ferricyanide compound, Coenzyme Q0 (2,3-Dimethoxy-5-methyl-p-benzoquinone), AZURE A Chloride (3-amino-7-(dimethylamino)phenothiazin-5-ium chloride), Phenosafranin (3,7-Diamino-5-phenylphenanzinium chloride), 6-Aminoquinoxaline, and Tetrathiafulvalene. In one or more embodiments, the ruthenium compound may be a ruthenium compound that exists in the reaction system as an oxidized ruthenium complex and functions as an electron transfer substance. The type of ligand of the ruthenium complex is not particularly limited. In one or more embodiments, the ruthenium compound may be an oxidized ruthenium complex represented by the following chemical formula: [Ru(NH 3 ) 5 X] n+ In the above formula, X is NH 3, halogen ions, CN, pyridine, nicotinamide, or H 2 O, especially NH 3 In one or more embodiments, the halogen ion is preferably Cl. - , F - , Br - , and I - In the formula: n+ represents the valence of the oxidized ruthenium (III) complex, which is determined by the type of X. In one or more embodiments, the ruthenium complex disclosed in JP 2018-013400 A can be used. The contents of this document are incorporated by reference as part of the present disclosure. In one or more embodiments, the ruthenium compound can be [Ru(NH 3 ) 6 ] 2+ or [Ru(NH 3 ) 6 ] 3+ In one or more embodiments, the ruthenium compound may be, for example, [Ru(NH 3 ) 6 ]Cl 3 etc. In one or a plurality of embodiments, examples of the phenylenediamine compound include N,N-dimethyl-1,4-phenylenediamine and N,N,N',N'-tetramethyl-1,4-phenylenediamine dihydrochloride. In one or more embodiments, examples of the quinone compound include 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone (VK3), 9,10-phenanthrenequinone, 1,2-naphthoquinone, p-xyloquinone, methylbenzoquinone, 2,6-dimethylbenzoquinone, sodium 1,2-naphthoquinone-4-sulfonate, 1,4-anthraquinone, tetramethylbenzoquinone, and thymoquinone. In one or a plurality of embodiments, the ferricyanide compound may be calcium ferricyanide.

[0041] In one or more embodiments, the amount of the electron mediator in the reagent layer is 0.1 mmol / cm 3 or more, preferably 0.5 mmol / cm 3 More preferably, it is 1 mmol / cm 3 The upper limit of the amount of the electron mediator in the reagent layer is not particularly limited, and in one or more embodiments, it is 50 mmol / cm 3 Less than or equal to 10mmol / cm 3 In one or more embodiments, the amount of the electron mediator in the reagent layer is 0.1 mmol / cm 3 More than 50mmol / cm 3 or less, preferably 0.5 mmol / cm 3 More than 50mmol / cm 3 More preferably, it is 1 mmol / cm or less. 3 More than 10mmol / cm 3 The following is the result.

[0042] In one or more embodiments, the reagent layer may contain components other than the oxidoreductase and the electron transfer substance. In one or more embodiments, the other components include a buffer, an amino acid, a surfactant, an antifoaming agent, a binder, etc.

[0043] In one or more embodiments, the buffering agent may be a phosphate buffering agent, an amine-based buffering agent, a buffering agent having a carboxyl group, or the like. In one or more embodiments, the amine buffer includes Tris(tris(hydroxymethyl)aminomethane), ACES(N-(2-Acetamido)-2-aminoethanesulfonic acid), CHES(N-Cyclohexyl-2-aminoethanesulfonic acid), and CAPSO(3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid). acid), TAPS(N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), CAPS(N-Cyclohexyl-3-aminopropanesulfonic acid), Bis-Tris(Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), TAPSO(2-Hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) acid), TES(N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic Examples of the buffer having a carboxyl group include citrate buffer, phosphate citrate buffer, acetic acid-sodium acetate buffer, malic acid-sodium acetate buffer, malonic acid-sodium acetate buffer, and succinic acid-sodium acetate buffer. One type of buffer may be used, or two or more types may be used in combination. In one or more embodiments, the pH of the buffer is 6.8 to 7.5, and preferably 6.9 to 7.0.

[0044] In one or more embodiments, the amino acid may include glycine, serine, lysine, and histidine.

[0045] In one or more embodiments, examples of the surfactant include TritonX-100 ((p-tert-octylphenoxy)polyethoxyethanol), Tween20 (Polyoxyethylene Sorbitan Monolaurate), sodium dodecyl sulfate, perfluorooctanesulfonic acid, sodium stearate, alkylaminocarboxylic acid (or a salt thereof), carboxybetaine, sulfobetaine, phosphobetaine, etc. One type of surfactant may be used, or two or more types may be used in combination.

[0046] In one or more embodiments, examples of the binder include resin binders and layered inorganic compounds. In one or more embodiments, examples of the resin binder include butyral resin binders and polyester resin binders. Examples of the layered inorganic compounds include the layered inorganic compounds described in WO2005 / 043146. In one or more embodiments, examples of the layered inorganic compounds include swelling clay minerals having ion exchange capacity. In one or more embodiments, examples of the layered inorganic compounds include bentonite, smectite, vermiculite, and synthetic fluorine mica. One type of binder may be used, or two or more types may be used in combination.

[0047] In one or more embodiments, the biosensor of the present disclosure can be manufactured by preparing a substrate having a conductive portion formed on one main surface thereof, the conductive portion including at least one electrode pair having a working electrode and a counter electrode, and dot-printing a reagent liquid onto the conductive portion so that a reagent layer is formed at the position described above.

[0048] [Biosensor manufacturing method] In another aspect, the present disclosure relates to a method for manufacturing a biosensor, which is a method for manufacturing a biosensor having a conductive part and a reagent layer in a flow path, and includes preparing a base material having an electrode pair having a working electrode and a counter electrode, on one main surface of which the conductive part is formed, and forming the reagent layer on the working electrode and the counter electrode, and forming the reagent layer includes dot-printing a reagent solution so that the entire area of ​​the working electrode in the flow path, areas at least 50 μm from each of the upstream and downstream edges of the working electrode, and an area of ​​the counter electrode in the flow path that corresponds to at least 70% of the area of ​​the working electrode in the flow path are covered with the reagent, and the average height of the reagent layer is 4 μm to 12 μm. In another aspect of the manufacturing method of the biosensor of the present disclosure, the formation of the reagent layer includes dot printing of the reagent liquid so as to cover the entire surface within the flow path of the working electrode and at least a portion of the surface within the flow path of the counter electrode, the dot printing including dot printing over an area of ​​at least 12.5% ​​of the surface area within the flow path of the working electrode upstream from the upstream edge of the working electrode and downstream from the downstream edge of the working electrode, and dot printing of the reagent liquid over an area of ​​the surface within the flow path of the counter electrode that corresponds to at least 70% of the surface area within the flow path of the working electrode.

[0049] According to the manufacturing method of the present disclosure, the reagent layer is formed by dot-printing the reagent liquid in the above-mentioned predetermined area on the conductive part, so that the biosensor of the present disclosure capable of highly accurate measurement can be manufactured. Thus, in one or more embodiments, the manufacturing method of the present disclosure can also be said to be a method for manufacturing the biosensor of the present disclosure.

[0050] In one or more embodiments, the dot printing includes repeatedly performing an operation of discharging droplets of the reagent liquid onto the conductive portion, drying the discharged droplets, and discharging droplets of the reagent liquid onto the dried droplets. In one or more embodiments, the dot printing can be performed using an inkjet printer or the like. The composition of the reagent liquid is as described above. In one or more embodiments, the amount of droplets discharged can be appropriately determined depending on the interval between positions where droplets are discharged, the size of the droplets to be formed, etc. In one or more embodiments, the amount of droplets discharged is 10 ng to 30 ng, and is 12 ng to 25 ng or 12 ng to 15 ng from the viewpoint of further reducing unevenness in the concentration and amount of the reagent component in the reagent layer and further improving the reproducibility of the measurement accuracy of the obtained biosensor. In one or more embodiments, the amount of droplets discharged may be changed between the first layer and the subsequent layers. In order to further reduce the unevenness in the concentration and amount of the reagent components in the reagent layer and further improve the reproducibility of the measurement accuracy of the obtained biosensor, it is preferable to make the amount of droplets discharged from the second layer and subsequent layers larger than the amount of droplets discharged from the first layer. In one or more embodiments, but not limited to, the amount of droplets discharged from the first layer may be less than 20 ng, and the amount of droplets discharged from the second layer may be 20 ng or more. In one or more embodiments, the amount of droplets discharged from the first layer is 10 ng to 18 ng, and in order to further reduce the unevenness in the concentration and amount of the reagent components in the reagent layer and further improve the reproducibility of the measurement accuracy of the obtained biosensor, it is preferable to make the amount of droplets discharged from the first layer 11 ng or more. From the same point of view, it is preferable to make the amount of droplets discharged from the first layer 17 ng or less, 15 ng or less, 14 ng or less, or 13 ng or less. In one or more embodiments, the ejection amount of droplets from the second layer onwards is 20 ng to 28 ng, and is preferably 21 ng or more from the viewpoint of further reducing unevenness in the concentration and amount of the reagent components in the reagent layer and further improving the reproducibility of the measurement accuracy of the obtained biosensor. From the same viewpoint, it is preferably 26 ng or less, 25 ng or less, or 24 ng or less. In one or more embodiments, the ejection amount of droplets from the second layer onwards may be the same or different. In one or more embodiments, the formation of the reagent layer includes discharging droplets of the Nth layer (where N is a natural number equal to or greater than 1) of reagent liquid at predetermined intervals, and discharging droplets of the N+1th layer of reagent liquid on the droplets of the Nth layer after drying. In one or more embodiments, N is a natural number of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The manufacturing method of the present disclosure includes repeatedly ejecting droplets of reagent liquid up to at least the fourth layer in order to further reduce unevenness in the concentration and amount of reagent components in the reagent layer and further improve the reproducibility of the measurement accuracy of the obtained biosensor. In one or more embodiments, the formation of the reagent layer may include a step of drying the droplets between the ejection of the Nth layer droplets and the ejection of the (N+1)th layer droplets. When the ejection of the Nth layer droplets and the ejection of the (N+1)th layer droplets are performed by different ejection devices, the transport time of the substrate may be the time for drying the droplets. In one or more embodiments, the interval at which droplets are discharged (the distance between the centers of adjacent droplets) is 50 μm to 150 μm, and is preferably 60 μm to 80 μm, and more preferably 65 μm to 75 μm, from the viewpoint of further reducing unevenness in the concentration and amount of the reagent component in the reagent layer and further improving the reproducibility of the measurement accuracy of the obtained biosensor. In one or more embodiments, the "center of the droplet" in the present disclosure refers to the center of a circle circumscribing the outer periphery of a droplet after a droplet dot is formed on a conductive part and the droplet is dried. In one or more embodiments, the intervals at which the droplets are ejected may be equal or may vary, but equal intervals are preferable in order to further reduce unevenness in the concentration and amount of the reagent components in the reagent layer and further improve the reproducibility of the measurement accuracy of the resulting biosensor. In one or more embodiments, the intervals at which droplets are ejected may be different or the same for each layer, and it is preferable that the intervals be the same for all layers in order to further reduce unevenness in the concentration and amount of the reagent components in the reagent layer and further improve the reproducibility of the measurement accuracy of the resulting biosensor.

[0051] In one or more embodiments, the manufacturing method of the present disclosure may include preparing a spacer having a cutout portion, and preparing a cover having an air hole formed therein, and may further include placing the spacer on a substrate such that the formed reagent layer is located inside the cutout portion of the spacer, and placing the cover on the spacer such that the air hole of the cover is located above the cutout portion of the spacer.

[0052] The electrodes, substrate, position of the reagent layer, and reagent in the manufacturing method of the present disclosure are similar to those of the biosensor of the present disclosure.

[0053] [Measurement method] In another aspect, the present disclosure relates to a method for electrochemically measuring a measurement target in a sample using a biosensor of the present disclosure, the method including contacting a sample with a reagent layer of the biosensor, applying a voltage between electrodes of the biosensor, and measuring an electrical signal generated by the application of the voltage.

[0054] In the present disclosure, "electrochemically measuring" refers to measuring by applying an electrochemical measurement method, and in one or more embodiments, examples of the method include amperometry, potentiometry, coulometry, etc. In one or more embodiments, an example of the amperometry is a method of measuring a current value generated when a reduced electron transfer substance is oxidized by application of a voltage.

[0055] The voltage to be applied is not particularly limited, but in one or more embodiments, it is 10 mV or more or 50 mV or more, and preferably 100 mV or more. In one or more embodiments, the applied voltage is 1000 mV or less or 700 mV or less, and preferably 600 mV or less, 500 mV or less, 400 mV or less, or 300 mV or less.

[0056] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Note that the present disclosure is not limited to these embodiments.

[0057] 1 and 2 are schematic diagrams for explaining the configuration of the biosensor of the present disclosure. In Fig. 1 and 2, the upper side is the upstream side (sample supply port side) and the lower side is the downstream side (insertion side into a measuring device).

[0058] As shown in FIGS. 1 and 2, the biosensor 1 includes a substrate 2, a conductive portion (described later), a reagent layer 50, a spacer 3, and a cover (not shown). The conductive portion is formed on one main surface of the substrate 2, and the reagent layer 50 is formed on one electrode pair 10 in the conductive portion. The spacer 3 has a rectangular cutout portion 3a, and is disposed on the substrate 2 such that the conductive portion and a part of the reagent layer 50 are located inside the cutout portion 3a. The cover has an air hole (not shown), and is disposed on the spacer 3 such that the air hole is located above the cutout portion 3a. A space formed by the substrate 2, the cutout portion 3a of the spacer 3, and the cover becomes a flow path 1d, and an opening portion becomes a sample supply port 1c.

[0059] As the material of the substrate, polyethylene terephthalate (PET) or the like can be preferably used. In this embodiment, the size of the substrate 2 is 7 mm in the short side direction (width), 30 mm in the long side direction (length), and 250 μm in thickness. The flow channel 1d is 2 mm in width and 4 mm in length. The reagent layer 50 is 2700 μm in width (short side direction of the biosensor) and 1000 μm in length (long side direction of the biosensor).

[0060] The conductive part is composed of a first electrode pair 10, a second electrode pair 20, and a blood detection electrode 30. The first electrode pair 10 is composed of a first working electrode 11 and a first counter electrode 12, and the second electrode pair 20 is composed of a second working electrode 21 and a second counter electrode 22. In the flow path 1d, the second working electrode 21, the second counter electrode 22, the first working electrode 11, the first counter electrode 12, and the blood detection electrode 30 are arranged in this order from the upstream side so as to be exposed in a rectangular shape parallel to the short side direction of the base material 2. Between the second working electrode 21 and the second counter electrode 22, and between the second counter electrode 22 and the first working electrode 11, gaps (non-conductive regions) 40b, 40a are provided, respectively. The first counter electrode 12 contacts the first working electrode 11 on the upstream side and contacts the blood detection electrode 30 on the downstream side (the surface opposite to the first working electrode 11), but is insulated between them. In this embodiment, the conductive portions (electrodes) are formed by sputtering a nickel-vanadium alloy, and the electrodes can be insulated from each other by drawing (trimming) a predetermined electrode pattern with a laser beam. The gaps (non-conductive regions) 40b and 40a are regions that are insulated by drawing a rectangular pattern with a laser beam.

[0061] The electrodes (first working electrode 11, first counter electrode 12, second working electrode 21, and second counter electrode 22) constituting the first electrode pair 10 and the second electrode pair 20 and the width of gap 40a (the longitudinal length of substrate 2) are all 400 μm, and the width of gap 40b (the longitudinal length of substrate 2) is approximately 200 μm.

[0062] Each electrode is connected to a lead portion, and the lead portion 21a of the second working electrode 21, the lead portion 22a of the second counter electrode 22, the lead portion 11a of the first working electrode 11, the lead portion 12a of the first counter electrode 12, and the lead portion 30a of the blood detection electrode 30 each extend parallel to the longitudinal direction of the substrate 2 from one end to the other end of the substrate 2. Each electrode is covered with a spacer 3 in a region 1a from the upstream end to the vicinity of the downstream end of the substrate 2, but the downstream end portion is not covered and is exposed, and this portion becomes a connector region 1b that is inserted into an insertion port of a measuring device. In the connector region 1b, the lead portion 21a of the second working electrode 21, the lead portion 22a of the second counter electrode 22, the lead portion 11a of the first working electrode 11, the lead portion 12a of the first counter electrode 12, and the lead portion 30a of the blood detection electrode 30 each become exposed contact points.

[0063] The reagent layer 50 is formed by ejecting droplets of the reagent liquid using an inkjet printer. The size of the reagent layer 50 in this embodiment (reagent layer forming area) is 2700 μm in the short direction of the substrate 2 and 1000 μm in the long direction.

[0064] The reagent layer 50 is formed on the conductive portion exposed in the flow path 1d, as shown in Fig. 2 (not shown in Fig. 1). Specifically, the reagent is formed so that substantially the entire surface of the first working electrode 11, a region (area) corresponding to at least 70% of the area of ​​the first working electrode 11 exposed in the flow path 1d on the surface of the first counter electrode 12 from the edge in contact with the first working electrode 11 toward the downstream side, and a region from the edge on the upstream side (opposite the first counter electrode 12) of the first working electrode 11 toward the upstream side to at least 50 µm, that is, a region (area) corresponding to at least 12.5% ​​of the area of ​​the first working electrode 11 exposed in the flow path 1d on the surface of the non-conductive region (gap 40a) toward the upstream side are covered with the reagent. The reagent layer 50 may be formed on a conductive portion that may be exposed in the flow channel 1d (cutout portion 3a) after the spacer 3 is placed, but may also be formed on a conductive portion that is located (or may be located) outside the flow channel 1d (cutout portion 3a) as shown in Fig. 2. This makes it possible to place the reagent layer in a predetermined region of the working electrode and counter electrode in the flow channel even if a manufacturing error (e.g., a misalignment in the formation location of the cutout portion, a misalignment in the position of the spacer, etc.) occurs during the manufacturing process of the biosensor.

[0065] The reagent layer 50 may or may not be formed on the second working electrode 21 and the second counter electrode 22 that constitute the second electrode pair. In addition, a reagent layer having a composition different from that of the reagent layer 50 may or may not be formed on the second working electrode 21 and the second counter electrode 22 that constitute the second electrode pair.

[0066] The formation of the reagent layer using an inkjet printer can be performed by setting the interval for discharging droplets (the center-to-center distance of dots (discharged droplets of reagent liquid)) at 70 μm, discharging droplets of reagent liquid evenly at a total of 546 locations, 39 locations in the short direction and 14 locations in the long direction of the substrate 2, in the reagent layer formation region, to form a dot group (for example, 546 reagent dots (39 × 14)) containing multiple reagent dots (discharged droplets of reagent liquid), and repeating the formation of this dot group. The formation of the dot group can be performed preferably three times (three layers) or more, and more preferably five times (five layers) or more, in order to easily form a reagent layer with an average height of 4 μm or more, to make the height of the formed reagent layer more uniform, and to further improve the measurement accuracy. In order to make the height of the formed reagent layer more uniform and to improve measurement accuracy, it is preferable that the position where the droplets of reagent liquid are ejected into the odd-numbered dot groups (odd-numbered layers) be different from the position where the droplets of reagent liquid are ejected into the even-numbered dot groups (even-numbered layers). The intervals at which droplets of reagent liquid are ejected in forming the reagent layer 50 and the number (positions) at which droplets of reagent liquid are ejected (number of ejected droplets / dot group) are not limited to the above-mentioned forms, and can be appropriately determined depending on the size (area) of the reagent layer to be formed, the amount of droplets of reagent liquid to be ejected, the composition of the reagent liquid to be ejected, etc.

[0067] In order to make the height of the reagent layer formed more uniform and to further improve the measurement accuracy, a time may be provided for drying the droplets of the reagent liquid ejected between the formation of one dot group and the formation of the next dot group. In one or more embodiments, the drying time may be the time required to transport the substrate in order to eject droplets of the reagent liquid from the nozzles of the inkjet head to predetermined positions, or a time for the drying process may be provided separately from the transport process. When the reagent layer is formed using multiple inkjet printers, the time required for transport between the inkjet printers may be used for the drying process.

[0068] A method for forming a reagent layer using an inkjet printer will be described below by taking as an example a case where the formation of a group of dots is repeated five times (five layers).

[0069] First, a reagent solution is prepared. The composition of the reagent solution can be appropriately determined depending on the type of reagent layer to be formed and the type of biosensor to be produced.

[0070] In one or more embodiments, the amount of oxidoreductase in the reagent solution is 1 KU / mL to 200 KU / mL. In one or more embodiments, the amount of oxidoreductase in the reagent solution is 5 KU / mL or more, 10 KU / mL or more, 15 KU / mL or more, 20 KU / mL or more, 25 KU / mL or more, or 30 KU / mL or more. In one or more embodiments, the amount of oxidoreductase in the reagent solution is 100 KU / mL or less, 90 KU / mL or less, 80 KU / mL or less, 70 KU / mL or less, 60 KU / mL or less, or 55 KU / mL or less. In one or more embodiments, the amount of the electron mediator in the reagent solution is 1 mM to 1000 mM. In one or more embodiments, the amount of the electron mediator in the reagent solution is 2 mM or more, 5 mM or more, 10 mM or more, 50 mM or more, 100 mM or more, 200 mM or more, or 500 mM or more. In one or more embodiments, the amount of the electron mediator in the reagent solution is 900 mM or less, 800 mM or less, or 700 mM or less. In one or more embodiments, the amount of the buffering agent in the reagent solution is 10 mM to 1000 mM. In one or more embodiments, the amount of the buffering agent in the reagent solution is 20 mM or more, 50 mM or more, 100 mM or more, 150 mM or more, or 200 mM or more. In one or more embodiments, the amount of the buffering agent in the reagent solution is 900 mM or less, 800 mM or less, 700 mM or less, 600 mM or less, or 500 mM or less. In one or more embodiments, the amount of the amino acid in the reagent solution is 0.1% by mass to 20% by mass or 0.5% by mass to 10% by mass. In one or more embodiments, the amount of the surfactant in the reagent solution is 0.1% by mass to 20% by mass or 0.5% by mass to 10% by mass.

[0071] The prepared reagent solution is used to form the first row of dots (first dot group). The reagent liquid for the first dot group is ejected at an amount that prevents droplets of the reagent liquid ejected from the nozzle from merging (combining). The amount of the reagent liquid ejected can be, for example, 10 ng to 18 ng (e.g., 12 ng). With this amount, droplets of the reagent liquid ejected onto the conductive portion (substrate) can be prevented from merging into a single droplet, and each droplet is disposed on the conductive portion (substrate) as a single independent droplet. The size of the droplets (long diameter after drying) is 50 μm to 70 μm. After the droplets constituting the first dot group are ejected, it is preferable to dry the reagent of the first dot group before ejecting the reagent liquid for the second dot group (second dot group). By ejecting the next droplet of reagent liquid on top of the first dot group after drying the reagent of the first dot group, the newly ejected droplet of reagent liquid dissolves part of the reagent of the first dot group, and the droplet is dried together with the dissolved reagent, so that it is possible to further reduce reagent unevenness and further improve measurement accuracy. Drying can be performed for 20 seconds or more at room temperature (humidity 50% or less).

[0072] Next, a second row of dots (second dot group) is formed on the dried first dot group. The reagent liquid for the second dot group is ejected at a position different from the position where the reagent liquid for the first dot group was ejected, in order to further reduce the unevenness of the reagent and further improve the measurement accuracy. For example, it is preferable to eject droplets of the reagent liquid at the intersection connecting the centers of the adjacent reagent dots in the first dot group. The ejection of the reagent liquid for the second dot group is preferably performed so that the droplets of the reagent liquid ejected from the nozzles overlap at least a part of the droplets of the adjacent reagent liquid, in order to further reduce the unevenness of the reagent and further improve the measurement accuracy. The ejection amount of the droplets may be, for example, 20 to 28 ng (e.g., 23 ng). With this amount, the droplets of the reagent liquid ejected from the nozzles can be dried in a state where they overlap with the droplets of the adjacent reagent liquid. After the droplets constituting the second dot group are ejected, it is preferable to dry the reagent of the second dot group before ejecting the reagent liquid for the third dot group. By ejecting the next droplet of reagent liquid on top of the second dot group after drying the reagent of the second dot group, the newly ejected droplet of reagent liquid dissolves a part of the reagent of the first dot group and the second dot group, and the droplet is dried together with the dissolved reagent, so that it is possible to further reduce reagent unevenness and further improve measurement accuracy. Drying can be performed in the same manner as for the first dot group.

[0073] Next, a third row of dots (third dot group) is formed on the dried second dot group. The ejection of the reagent liquid for the third dot group is performed at approximately the same position as the position where the reagent liquid was ejected for the first dot group, in order to further reduce the unevenness of the reagent and further improve the measurement accuracy. The ejection amount and drying of the droplets can be performed in the same manner as for the second dot group.

[0074] Next, a fourth row of dots (fourth dot group) is formed on the dried third dot group. The ejection of the reagent liquid for the fourth dot group is performed at approximately the same position as the position where the reagent liquid for the second dot group was ejected, in order to further reduce the unevenness of the reagent and further improve the measurement accuracy. The ejection amount of the droplets and drying can be performed in the same manner as for the second dot group.

[0075] Finally, a fifth row of dots (fifth dot group) is formed on the dried fourth dot group. The ejection of the reagent liquid for the fifth dot group is performed at approximately the same position as the position where the reagent liquid for the first dot group or the third dot group is ejected, in order to further reduce the unevenness of the reagent and further improve the measurement accuracy. The ejection amount of the droplets and drying can be performed in the same manner as for the third dot group.

[0076] By forming these dot groups, in one or a plurality of embodiments, a reagent layer having an average height of 4 μm to 12 μm, 5 μm to 12 μm, 6 μm to 12 μm, 7 μm to 12 μm, 8 μm to 12 μm, 9 μm to 12 μm, or 9.5 μm to 12 μm can be formed.

[0077] Next, a spacer 3 having a cutout portion 3a is placed on the base material 2 on which the reagent layer 50 has been formed. The spacer 3 is placed on the base material 2 so that a part of the conductive portion (first working electrode 11, first counter electrode 12, second working electrode 21, second counter electrode 22, and blood detection electrode 30) on which the reagent layer 50 is arranged is positioned (exposed) inside the cutout portion 3a.

[0078] Finally, a cover (not shown) with air holes is placed on top of the spacer 3.

[0079] When PMMA is used as the material for the spacer 3, the adhesiveness of PMMA allows the base material 2 and the cover to adhere to each other. Alternatively, the spacer 3 and the cover may be laminated in advance, and then laminated on the base material 2 and adhered to each other.

[0080] As an example of a method for electrochemically measuring a measurement target in a sample using biosensor 1, a case in which the sample is whole blood and the measurement target is glucose will be described.

[0081] First, a whole blood sample is brought into contact with the sample supply port 1c of the biosensor 1. When the whole blood sample is deposited on the sample supply port 1c of the biosensor 1, the sample flows downstream in the flow channel 1d by capillary force through the second working electrode 21, the second counter electrode 22, the first working electrode 11, the first counter electrode 12, and the blood detection electrode 30 in that order. When the whole blood sample reaches the reagent layer 50, the reagent components (such as oxidoreductase and electron transfer substance) contained in the reagent layer 50 are dissolved by the whole blood sample.

[0082] When a positive potential is applied to the electrode, electrons are exchanged between the electron mediator present in the reagent layer 50 and the first working electrode 11 located below the reagent layer 50, causing an oxidation current to flow. Based on this, the glucose concentration can be measured. In one or more embodiments, the voltage to be applied is 10 mV to 1000 mV, and preferably 100 mV to 600 mV.

[0083] The above embodiment has been described using the biosensor configuration and measurement method as an example when the sample is blood (e.g., whole blood), but the present disclosure is not limited to this and can similarly measure various biological samples other than blood, such as urine.

[0084] The present disclosure may relate to one or more of the following non-limiting embodiments. [1] A substrate having a conductive portion including an electrode pair having a working electrode and a counter electrode formed on one principal surface thereof; A reagent layer placed on the working electrode and the counter electrode; A flow path for passing a sample from a sample supply port to the reagent layer, The reagent layer comprises: covering the entire area of ​​the working electrode within the flow channel and extending at least 50 μm from each of the upstream and downstream edges of the working electrode; the counter electrode is formed so as to cover an area within the flow path of the counter electrode that corresponds to at least 70% of the area of ​​the working electrode; The biosensor, wherein the average height of the reagent layer is 4 μm to 12 μm. [2] A substrate having a conductive portion including an electrode pair having a working electrode and a counter electrode formed on one main surface thereof; A reagent layer placed on the working electrode and the counter electrode; A flow path for passing a sample from a sample supply port to the reagent layer, the reagent layer is formed so as to cover the entire surface of the working electrode in the flow path and at least a portion of the surface of the counter electrode in the flow path, and to extend from an upstream edge of the working electrode toward the upstream side and from a downstream edge of the working electrode toward the downstream side, each of the extending ranges being at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, and a coverage area of ​​the reagent layer on the counter electrode corresponds to at least 70% of the surface area of ​​the working electrode in the flow path, The biosensor, wherein the average height of the reagent layer is 4 μm to 12 μm. [3] The biosensor according to [1] or [2], wherein an area of ​​the reagent layer extending from the downstream edge of the working electrode is larger than an area of ​​the reagent layer extending from the upstream edge of the working electrode. [4] The working electrode and the counter electrode extend in a direction perpendicular to a sample supply direction in the flow channel; The biosensor according to any one of [1] to [3], wherein the working electrode is formed adjacent to the counter electrode on the upstream side of the counter electrode. [5] The biosensor according to any one of [1] to [4], wherein the reagent layer extends within a range of 100 μm or more and less than 150 μm from the edge of the working electrode. [6] The biosensor according to any one of [1] to [5], wherein the reagent layer is formed by dot printing. [7] A method for manufacturing a biosensor having a conductive portion and a reagent layer in a flow path, comprising: preparing a substrate having a conductive portion formed on one of its principal surfaces, the conductive portion including an electrode pair having a working electrode and a counter electrode; forming the reagent layer on the working electrode and the counter electrode; The formation of the reagent layer includes an entire region of the working electrode within the flow channel; A region of at least 50 μm from each of the upstream and downstream edges of the working electrode; A region of the counter electrode within the flow path that corresponds to at least 70% of a region of the working electrode within the flow path is covered with a reagent; and The average height of the reagent layer is 4 μm to 12 μm. A manufacturing method comprising dot printing of a reagent liquid. [8] A manufacturing method according to [7] for producing a biosensor according to any one of [1] to [6].

[0085] The present disclosure will be described in more detail below with reference to examples, but these are merely illustrative and the present disclosure is not limited to these examples. EXAMPLES

[0086] [Preparation of biosensor 1] (1) As shown in FIG. 1, a substrate 2 (PET, thickness 250 μm, length 30 mm×width 7 mm) having a conductive portion formed on one main surface was prepared. The conductive section is composed of a first electrode pair 10, a second electrode pair 20, and a blood detection electrode 30. The first electrode pair 10 is composed of a first working electrode 11 and a first counter electrode 12, and the second electrode pair 20 is composed of a second working electrode 21 and a second counter electrode 22. The second working electrode 21, the second counter electrode 22, the first working electrode 11, and the first counter electrode 12 are arranged in this order at one end of the longitudinal direction of the substrate 2, parallel to the lateral direction of the substrate 2. Gaps (non-conductive regions) 40b, 40a are provided between the second working electrode 21 and the second counter electrode 22, and between the second counter electrode 22 and the first working electrode 11, respectively. The first working electrode 11 and the first counter electrode 12 are in contact with each other, and the first counter electrode 12 is also in contact with the blood detection electrode 30 on the surface opposite to the first working electrode 11. Each electrode is connected to a lead portion, and the lead portion 21a of the second working electrode 21, the lead portion 22a of the second counter electrode 22, the lead portion 11a of the first working electrode 11, the lead portion 12a of the first counter electrode 12, and the lead portion 30a of the blood detection electrode 30 each extend parallel to the longitudinal direction of the substrate 2 from one end to the other end of the substrate 2. The electrodes (first working electrode 11, first counter electrode 12, second working electrode 21, and second counter electrode 22) constituting the first electrode pair 10 and the second electrode pair 20 and the width of gap 40a (the longitudinal length of substrate 2) are all 400 μm, and the width of gap 40b (the longitudinal length of substrate 2) is approximately 200 μm. (2) Next, a reagent solution was prepared to have the following composition: [Composition of reagent solution] FAD-dependent Glucose Dehydrogenase (product name: Glucose Dehydrogenase "Amano 8", MW: 180,000, manufactured by Amano Enzyme Co., Ltd.): 41 KU / mL Ru complex (Ru(NH 3 ) 6 Cl 3 , manufactured by LT Metal Co. Ltd): 626 mM 1-Methoxy PES (1-methoxy-5-ethylphenazinium ethyl sulfate, Dojindo Laboratories): 824 μg / mL 0.4M phosphate buffer (pH 7.0): 0.29mM Glycine: 2% by mass CHAPS: 2% by mass Antifoaming agent: 0.04% by weight (3) Next, a reagent layer was formed on the base material 2 on which the conductive portion was formed. The reagent layer was formed by dot printing the reagent liquid with an inkjet device under the following conditions of room temperature and humidity of 50% or less. The reagent layer formation area (the range where the reagent is dot printed) was rectangular, with a length of 2700 μm in the short direction of the substrate 2 and a length of 1000 μm in the long direction. The reagent layer was formed in nine patterns (type 1 to type 9) in which the reagent layer 50 covered the entire surface of the first working electrode 11 and at least a part of the first counter electrode 12, and the position where the reagent layer was formed was shifted by 50 μm in the long direction of the substrate 2 as shown in Table 1 below. <Procedure> 1. The above reagent solution was filled into the reservoir of an inkjet device, and droplets of the reagent solution were ejected onto a substrate on which a conductive portion was formed under the following conditions, and the droplets were dried for 30 seconds to form a first layer of reagent (first dot group). The diameter (after drying) of each dot of the printed reagent was 50 to 70 μm. [Discharge conditions (first dot group)] Discharge volume per droplet: 12ng (10pL) Distance between droplets (center-to-center distance of ejected droplets): 70 μm Number of discharges: 546 locations (39 locations in the short direction of the substrate 2 × 14 locations in the long direction of the substrate 2) 2. Next, droplets of reagent liquid were ejected onto the first dot group under the following conditions, and dried for 30 seconds to arrange the second layer of reagent (second dot group). In the second layer, the droplets were ejected so that the position where the droplets of reagent liquid were ejected (the center of the droplet) was approximately the center of a square whose vertices were the centers of the four adjacent reagents in the first dot group. The diameter (after drying) of each dot-printed reagent was 90 to 100 μm. [Discharge conditions (second dot group)] Discharge volume per droplet: 23ng (18pL) Distance between droplets (center-to-center distance of ejected droplets): 70 μm Number of discharges: 546 locations (39 locations in the short direction of the substrate 2 × 14 locations in the long direction of the substrate 2) 3. Next, a third layer of reagent (third dot group) was disposed on top of the second dot group by discharging and drying droplets of reagent liquid. The droplets of the third dot group were discharged in the same manner as the second dot group, except that the droplets of the reagent liquid were discharged at approximately the same positions as the first dot group. 4. Next, droplets of a reagent liquid were ejected onto the third dot group in the same manner as for the second dot group, and then dried, thereby disposing a fourth layer of reagent (fourth dot group). 5. Finally, droplets of reagent liquid were ejected onto the fourth dot group in the same manner as the third dot group, and then dried to form a fifth layer of reagent (fifth dot group). This resulted in a three-dimensionally laminated reagent layer (approximately a rectangular parallelepiped (square prism)). The height of the reagent layer was observed with a laser microscope (product name: VK-X3000, manufactured by KEYENCE). The distribution of the height of the reagent layer was 8 μm to 12 μm, and the average height was approximately 10 μm. The amount of FAD-dependent Glucose Dehydrogenase in the resulting reagent layer was 232 KU / cm. 3 The amount of electron transfer substance is 3.56 mmol / cm 3 It is. (4) On the substrate 2 on which the reagent layer was formed, a spacer 3 having a cutout 3a (length in the longitudinal direction of the substrate 2: 4 mm, length in the lateral direction of the substrate 2: 2 mm) was placed as shown in Fig. 2, and a cover having an air hole (not shown) was placed on top of that. The space formed by the substrate 2, the cutout 3a of the spacer 3, and the cover became the flow path 1d.

[0087] [Measurement of the size of dot-printed reagents] The size of the reagent formed by one droplet ejected from the inkjet printer was measured using a digital microscope (VHX-6000, manufactured by Keyence Corporation).

[0088] [Glucose current measurement 1] Samples with various glucose (GLU) concentrations were prepared as shown below, and the samples were spotted on the biosensor to perform electrochemical measurements. For the electrochemical measurements, a potentiostat (manufactured by Arkray, Inc.) was used to apply a voltage of 0.2 V and measure the current value after 4.0 seconds. The results are shown in Figures 3 to 5. 2 is the coefficient of determination when an approximation curve (linear approximation) is created for each of the graphs of the current values ​​of the biosensors shown in FIGS. <Evaluation conditions> Sample used: Whole blood sample GLU concentration (mg / dl): 0, 45, 330, 800, 1500 and 3000 (all Hct 42%) Number of evaluations: n=10 (for each GLU concentration)

[0089] [Table 1]

[0090] 3 and 4 are graphs showing the relationship between glucose concentration and reaction current value, and FIG. 5 is a graph showing the reaction current value in each biosensor when a sample with a GLU concentration of 3000 mg / dl and Hct of 42% is used. FIG. 3 shows the glucose measurement linearity in biosensors (types 1 to 5) with different regions of the reagent layer covering the counter electrode surface, and FIG. 4 shows the glucose measurement linearity in biosensors (types 1, 6 to 9) with different regions of the reagent layer extending from the working electrode. As shown in FIGS. 3 to 5, in the biosensors in which the reagent layer was formed at positions 4, 5, and 9, the reaction current value decreased in the high glucose concentration range, and sufficient glucose measurement linearity was not obtained. In contrast, in the biosensors in which the reagent layer was formed at positions 1 to 3 and 6 to 8, no decrease in the reaction current value was observed, and high glucose measurement linearity was obtained.

[0091] [Preparation of biosensor 2] As in Biosensor Fabrication 1, a substrate with a conductive part formed was prepared, and seven types of biosensors (types 1 to 3, 5 to 7, and 9) with different positions of the reagent layer were fabricated by arranging the reagents in the first to third layers in the same manner as in <Procedures> 1 to 4. The average height of the resulting reagent layer was approximately 6 μm (height distribution: 4 μm to 8 μm).

[0092] [Glucose current measurement 2] Using the biosensor created in Biosensor Creation 2, measurements were performed in the same manner as in Glucose Current Measurement 1, except that the current value was measured after 4.0 seconds had elapsed. Of the results obtained, the results using a sample with a GLU concentration of 1500 mg / dl × Hct of 42% are shown in Figure 6. As shown in Figure 6, the biosensors in which the reagent layer was formed at positions types 5 and 9 had low reaction current values, whereas the other biosensors (types 1 to 3 and 6 to 7) did not show a decrease in reaction current value, suggesting that high linearity in glucose measurement can be obtained.

[0093] [Preparation of biosensor 3] As in Biosensor Fabrication 1, a substrate with a conductive part formed thereon was prepared, and seven types of biosensors (types 1 to 3, 5 to 7, and 9) with different positions of the reagent layer were fabricated by disposing the first layer of reagent under the ejection conditions of the second dot group in <Procedure> 1. The average height of the resulting reagent layer was approximately 2 μm (height distribution: 1 μm to 5 μm).

[0094] [Glucose current measurement 3] Using the biosensor produced in Biosensor Production 3, samples with the following glucose (GLU) concentrations were used as samples, and measurements were performed in the same manner as Glucose Current Measurement 1, except that the current value was measured after 4.0 seconds had elapsed. <Evaluation conditions> Sample used: Whole blood sample GLU concentration (mg / dl): 0, 45, 330, 800 and 1500 (all Hct 42%) Number of evaluations: n=10 (for each GLU concentration)

[0095] As a result, when the average height of the reagent layer was approximately 2 μm (less than 4 μm), the reagent in the reagent layer was diffused by the sample, and sufficient reagent required for measurement was no longer present on the working electrode, resulting in problems such as a failure to obtain a sufficient measurement current value for a sample with a high glucose concentration (e.g., 800 mg / dl) and, in some cases, an increase in the current value, resulting in unstable measurement and deterioration of measurement performance.

Claims

1. A substrate having a conductive portion formed on one main surface thereof, the conductive portion including an electrode pair having a working electrode and a counter electrode; A reagent layer placed on the working electrode and the counter electrode; A flow path for passing a sample from a sample supply port to the reagent layer, The reagent layer comprises: covering the entire area of ​​the working electrode within the flow channel and extending at least 50 μm from each of the upstream and downstream edges of the working electrode; the counter electrode is formed so as to cover an area within the flow path of the counter electrode that corresponds to at least 70% of an area within the flow path of the working electrode; A biosensor, wherein the average height of the reagent layer is between 4 μm and 12 μm.

2. A substrate having a conductive portion formed on one main surface thereof, the conductive portion including an electrode pair having a working electrode and a counter electrode; A reagent layer placed on the working electrode and the counter electrode; A flow path for passing a sample from a sample supply port to the reagent layer, the reagent layer is formed to cover the entire surface of the working electrode in the flow path and at least a portion of the surface of the counter electrode in the flow path, and to extend from an upstream edge of the working electrode toward the upstream side and from a downstream edge of the working electrode toward the downstream side, respectively, wherein the area extending toward the upstream side and the area extending toward the downstream side are each at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, and a coverage area of ​​the reagent layer on the counter electrode corresponds to at least 70% of the surface area of ​​the working electrode in the flow path, A biosensor, wherein the average height of the reagent layer is between 4 μm and 12 μm.

3. 3. The biosensor according to claim 1, wherein an area of ​​the reagent layer extending from the downstream edge of the working electrode is larger than an area of ​​the reagent layer extending from the upstream edge of the working electrode.

4. the working electrode and the counter electrode extend in a direction perpendicular to a supply direction of the sample in the flow channel, The biosensor according to claim 1 , wherein the working electrode is formed adjacent to the counter electrode on the upstream side of the counter electrode.

5. 3. The biosensor according to claim 1, wherein the reagent layer extends in a range of 100 μm or more and less than 150 μm from an edge of the working electrode.

6. The biosensor according to claim 1 , wherein the reagent layer is formed by dot printing.

7. A method for manufacturing a biosensor having a conductive portion and a reagent layer in a flow path, comprising the steps of: preparing a substrate having a conductive portion formed on one of its principal surfaces, the conductive portion including an electrode pair having a working electrode and a counter electrode; forming the reagent layer on the working electrode and the counter electrode; The formation of the reagent layer includes an entire region of the working electrode within the flow channel; A region of at least 50 μm from each of the upstream and downstream edges of the working electrode; A region of the counter electrode within the flow path that corresponds to at least 70% of a region of the working electrode within the flow path is covered with a reagent; and The average height of the reagent layer is 4 μm to 12 μm. A manufacturing method comprising dot printing of a reagent liquid.

8. A method according to claim 7 for producing the biosensor according to claim 1 or 2.

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