Biosensor and method for manufacturing biosensor

Abstract

The invention provides a biosensor capable of performing high-precision measurement and a method for manufacturing the biosensor. A biosensor according to one embodiment of the present invention is provided with: a substrate on which a conductive part including a pair of electrodes having a working electrode and a counter electrode is formed on one main surface; a reagent layer placed on the working electrode and the counter electrode; and a flow path through which a sample flows from a sample supply port to the reagent layer, the reagent layer being formed so as to cover the entire region of the working electrode in the flow path and extending at least 50 [mu] m from each of the upstream and downstream edges of the working electrode. The reagent layer is formed so as to cover a region corresponding to at least 70% of the region of the working electrode in the region of the counter electrode in the flow path, and the average height of the reagent layer is 4 [mu] m to 12 [mu] m.

Description

Technical Field

[0001] The present invention relates to a biosensor and a method for manufacturing the biosensor. Background Art

[0002] A biosensor for measuring an object to be measured in a sample such as blood sugar in blood generally comprises a pair of electrodes and a reagent layer formed on the pair of electrodes. When blood is supplied to the reagent layer of the biosensor, a reaction system is formed between the electrodes, thereby enabling the object to be measured in the blood to be measured (WO2003 / 025558).

[0003] The reagent layer is usually formed by coating a reagent solution containing an oxidoreductase and an electron transfer substance on the electrode and drying the reagent solution. As a coating method of the reagent solution, for example, there are an immersion method, a dispensing method, a screen printing method, an offset printing method, and an inkjet printing method (WO2003 / 025558, Japanese Patent Publication No. 2011-99693, Japanese Patent Publication No. 6-3317, Japanese Patent Table No. 2017-520773). Summary of the invention

[0004] A biosensor generally has at least a substrate, a conductive portion having a working electrode and a counter electrode formed on the substrate, and a reagent layer formed on the conductive portion. The size of a biosensor is generally very small, with a length of tens of mm in the long side direction and a length of several mm in the short side direction. Biosensors are used to measure the measurement object in a sample such as blood sugar in the blood, so high-precision measurement is required.

[0005] The determination of the object to be determined is performed by reacting the object to be determined such as blood sugar contained in a sample such as blood with a reagent in the reagent layer disposed on the conductive part, and detecting the reaction. The inventors have found that the position of the reagent layer disposed on the conductive part has a great influence on the determination accuracy, and in particular, have found that the positional relationship between the working electrode and the counter electrode constituting the conductive part and the reagent layer is very important.

[0006] The present invention provides a biosensor capable of performing high-precision measurements.

[0007] A biosensor as one embodiment of the present invention comprises: a substrate having a conductive portion formed on one main surface thereof including an electrode pair, the electrode pair having a working electrode and a counter electrode; a reagent layer carried on the working electrode and the counter electrode; and a flow path for allowing a sample to flow from a sample supply port to the reagent layer, the reagent layer being formed to cover the entire area of ​​the working electrode in the flow path and extending at least 50 μm from respective edges of the upstream and downstream sides of the working electrode, and being formed to cover an area of ​​the counter electrode in the flow path that is equivalent 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.

[0008] As another embodiment of the present invention, a biosensor comprises: a substrate having a conductive portion formed on one main surface including an electrode pair, wherein the electrode pair has a working electrode and a counter electrode; a reagent layer carried on the working electrode and the counter electrode; and a flow path for allowing the sample to flow from a sample supply port to the reagent layer, wherein 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 extends from an edge on the upstream side of the working electrode to the upstream side, and extends from an edge on the downstream side of the working electrode to the downstream side, and the extension ranges are at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, respectively, and the coverage area of ​​the reagent layer at the counter electrode is equivalent to at least 70% of the surface area of ​​the working electrode in the flow path, and the average height of the reagent layer is 4μm to 12μm.

[0009] In another embodiment of the present invention, a method for manufacturing a biosensor comprises a conductive portion and a reagent layer in a flow path, wherein the method for manufacturing the biosensor comprises the following steps: preparing a substrate having the conductive portion including an electrode pair formed on one main surface, the 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 comprising the following steps: dot-matrix printing a reagent solution so that the entire area of ​​the working electrode in the flow path, an area of ​​at least 50 μm from the respective edges of the upstream and downstream sides of the working electrode, and an area of ​​the counter electrode in the flow path corresponding 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.

[0010] According to the present invention, a biosensor capable of performing high-precision measurements can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic diagram showing a schematic structure of a biosensor according to one embodiment of the present invention.

[0012] Figure 2 It is a schematic diagram showing a schematic structure of a biosensor according to one embodiment of the present invention.

[0013] Figure 3 This is a graph showing an example of the results obtained in Example (Glucose Current Value Measurement 1), and shows the relationship between the glucose concentration and the reaction current value.

[0014] Figure 4 This is a graph showing an example of the results obtained in Example (Glucose Current Value Measurement 1), and shows the relationship between the glucose concentration and the reaction current value.

[0015] Figure 5 This is a graph showing an example of the results obtained in Example (glucose current value measurement 1), and shows the reaction current value of each biosensor in a sample (glucose (GLU) concentration 3000 mg / dl×Hct 42%).

[0016] Figure 6 This is a graph showing an example of the results obtained in Example (glucose current value measurement 2), and shows the reaction current value of each biosensor in a sample (GLU concentration 1500 mg / dl×Hct 42%). DETAILED DESCRIPTION

[0017] The biosensor of the present invention is used for measuring the measurement object in the sample. As the sample, in one or more embodiments, biological samples can be cited. As the biological sample, in one or more embodiments, blood and urine and samples derived therefrom, cell extraction samples and cell culture fluids can be cited. As the measurement object, in one or more embodiments, glucose, cholesterol, ethanol, sorbitol, fructose, cellobiose, lactic acid and urea can be cited.

[0018] [Biosensor]

[0019] As one embodiment, the present invention relates to a biosensor comprising: a substrate having a conductive portion formed on one main surface thereof including an electrode pair, the 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 allowing a sample to flow from a sample supply port to the reagent layer. As one embodiment, the average height of the reagent layer in the biosensor of the present invention is 4 μm to 12 μm, the reagent layer is formed to cover the entire area of ​​the working electrode in the flow path, and extends at least 50 μm from the edges of the upstream and downstream sides of the working electrode, and covers an area of ​​the counter electrode in the flow path that is equivalent to at least 70% of the area of ​​the working electrode. In addition, as another embodiment, the reagent layer in the biosensor of the present invention is formed to have an average height of 4μm to 12μm, covering 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 extending from the edge of the upstream side of the working electrode to the upstream side, and extending from the edge of the downstream side of the working electrode to the downstream side, and the extension range is at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, respectively, and the coverage area of ​​the reagent layer at the counter electrode is equivalent to at least 70% of the surface area of ​​the working electrode in the flow path.

[0020] In one or more embodiments, the biosensor of the present invention is formed with a sample supply port and a flow path connected to the sample supply port. The sample supply port is provided for supplying the sample to the flow path, and the flow path is provided for allowing the sample supplied from the sample supply port to flow to the reagent layer in the flow path. In the present invention, the "upstream side" refers to the upstream side in the supply direction of the sample to the flow path in the biosensor, that is, the sample supply port side, and the "downstream side" refers to the downstream side in the supply direction of the sample, that is, the end side of the flow path located on the opposite side of the sample supply port. In the present invention, the "supply direction of the sample" refers to the direction in which the sample supplied to the sample supply port of the biosensor flows in the flow path.

[0021] In consideration of simplicity and efficiency, the reagent layer of a biosensor is usually formed by applying a reagent solution onto the conductive portion by screen printing or the like. On the other hand, in such a method, in order to manufacture a biosensor with sufficient measurement accuracy, it is considered necessary to form the reagent layer in such a way that the surfaces of the electrodes (working electrode and counter electrode) in the flow path are completely covered with the reagent (e.g., WO2003 / 025558 ( Figure 1 ) and Japanese Patent Publication No. 2017-520773 ( Figure 1 )wait).

[0022] In contrast, the present inventors have found that, for example, by forming a reagent layer with an average height of 4 μm to 12 μm by dot printing or the like, high-precision measurements can be achieved even when the surface of the electrode in the flow path is not completely covered with the reagent. Furthermore, the present inventors have found that, in order to achieve high-precision measurements, it is important to cover the edge of the working electrode and the range from the edge to a predetermined area with the reagent.

[0023] According to the biosensor of the present invention, the average height of the reagent layer is 4μm to 12μm, and the reagent layer is formed to not only cover the entire surface of the working electrode in the flow path, but also extend from the edge of the working electrode to a specified range, and further, is formed to cover an area of ​​the surface of the counter electrode in the flow path that is equivalent to at least 70% of the area of ​​the above-mentioned working electrode in the flow path, thereby enabling high-precision measurement of the analysis object.

[0024] In the present invention, "the reagent layer covers the entire area of ​​the working electrode in the flow path", as long as the surface of the working electrode exposed in the flow path is substantially covered by the reagent. In the present invention, as the surface of the working electrode in the flow path is substantially covered by the reagent, in one or more embodiments, 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 by the reagent.

[0025] The reagent layer is formed to cover the entire area (surface) of the working electrode in the flow path, and extends from the edge of the upstream side and the edge of the downstream side of the working electrode, respectively. In the present invention, "the reagent layer extends from the edge of the working electrode" means that the reagent layer exceeds the edge of the working electrode (extends from the edge) and is arranged on the surface of the substrate (or a component arranged on the substrate). Therefore, in the present invention, the edge of the working electrode in the flow path is covered by the reagent layer.

[0026] The reagent layer extends at least 50 μm from the edges of the upstream and downstream sides of the working electrode. From the viewpoint of further improving the measurement accuracy, in one or more embodiments, the reagent layer preferably extends more than 60 μm, more than 70 μm, more than 80 μm, more than 90 μm, more than 100 μm, more than 125 μm, more than 150 μm, more than 175 μm, more than 200 μm, more than 225 μm, more than 250 μm, more than 275 μm or more than 300 μm. In one or more embodiments, the range in which the reagent layer extends from the edges of the upstream and downstream sides of the working electrode is less than 500 μm, less than 450 μm or less than 400 μm.

[0027] In one or more embodiments, the reagent layer extends from the edge on the upstream side of the working electrode by more than 50 μm and less than 500 μm, more than 50 μm and less than 450 μm, more than 50 μm and less than 400 μm, more than 50 μm and less than 350 μm, more than 50 μm and less than 340 μm, more than 50 μm and less than 330 μm, more than 50 μm and less than 320 μm, or more than 50 μm and less than 310 μm. In one or more embodiments, the reagent layer extends from the edge on the downstream side of the working electrode by more than 175 μm and less than 500 μm, more than 200 μm and less than 500 μm, more than 225 μm and less than 500 μm, more than 250 μm and less than 500 μm, more than 275 μm and less than 500 μm, more than 275 μm and less than 450 μm, more than 275 μm and less than 440 μm, more than 275 μm and less than 430 μm, or more than 280 μm and less than 420 μm.

[0028] From the same viewpoint, the range in which the reagent layer extends from each edge portion on the upstream side and the downstream side of the working electrode is preferably 100 μm to 450 μm, or 100 μm to 150 μm.

[0029] In one or more embodiments, the reagent layer extends from the edge of the upstream side of the working electrode by more than 50 μm and less than 500 μm, and extends from the edge of the downstream side of the working electrode by more than 175 μm and less than 500 μm. In one or more embodiments, the reagent layer extends from the edge of the upstream side of the working electrode by more than 50 μm and less than 450 μm, and extends from the edge of the downstream side of the working electrode by more than 200 μm and less than 500 μm. In one or more embodiments, the reagent layer extends from the edge of the upstream side of the working electrode by more than 50 μm and less than 400 μm, and extends from the edge of the downstream side of the working electrode by more than 225 μm and less than 500 μm. In one or more embodiments, the reagent layer extends from the edge of the upstream side of the working electrode by more than 50 μm and less than 350 μm, and extends from the edge of the downstream side of the working electrode by more than 250 μm and less than 500 μm. In one or more embodiments, the reagent layer extends from the edge of the upstream side of the working electrode by more than 50 μm and less than 340 μm, and extends from the edge of the downstream side of the working electrode by more than 275 μm and less than 500 μm. In one or more embodiments, the reagent layer extends from the edge of the upstream side of the working electrode by more than 50 μm and less than 330 μm, and extends from the edge of the downstream side of the working electrode by more than 275 μm and less than 450 μm. In one or more embodiments, the reagent layer extends from the edge of the upstream side of the working electrode by more than 50 μm and less than 320 μm, and extends from the edge of the downstream side of the working electrode by more than 275 μm and less than 430 μm. In one or more embodiments, the reagent layer extends from the edge of the upstream side of the working electrode by more than 50 μm and less than 310 μm, and extends from the edge of the downstream side of the working electrode by more than 0 μm and less than 420 μm.

[0030] In one or more embodiments, the distance between the edge on the upstream side of the working electrode and the edge on the upstream side of the reagent layer is greater than 50 μm and less than 500 μm, greater than 50 μm and less than 450 μm, greater than 50 μm and less than 400 μm, greater than 50 μm and less than 350 μm, greater than 50 μm and less than 340 μm, greater than 50 μm and less than 330 μm, greater than 50 μm and less than 320 μm, or greater than 50 μm and less than 310 μm. In one or more embodiments, the distance between the downstream edge of the working electrode and the downstream edge of the reagent layer is greater than 175 μm and less than 500 μm, greater than 200 μm and less than 500 μm, greater than 225 μm and less than 500 μm, greater than 250 μm and less than 500 μm, greater than 275 μm and less than 500 μm, greater than 275 μm and less than 450 μm, greater than 275 μm and less than 440 μm, greater than 275 μm and less than 430 μm, or greater than 280 μm and less than 420 μm.

[0031] The range in which the reagent layer extends from the respective edges of the upstream and downstream sides of the working electrode is at least 12.5% ​​of the surface area of ​​the working electrode in the flow path. From the viewpoint of further improving the measurement accuracy, in one or more embodiments, it is more than 13%, more than 14%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 70%, more than 80%, more than 90% or 100% of the surface area of ​​the working electrode in the flow path.

[0032] In one or more embodiments, the range in which the reagent layer extends from the edge on the upstream side of the working electrode is greater than 12.5% ​​and less than 100%, greater than 12.5% ​​and less than 90%, greater than 12.5% ​​and less than 85%, greater than 12.5% ​​and less than 80%, or greater than 13% and less than 75% of the surface area of ​​the working electrode in the flow path.

[0033] In one or more embodiments, the range in which the reagent layer extends from the edge on the downstream side of the working electrode is greater than 45% and less than 100%, greater than 50% and less than 100%, greater than 55% and less than 100%, greater than 60% and less than 100%, greater than 65% and less than 100%, greater than 70% and less than 100%, or greater than 75% and less than 100%, of the surface area of ​​the working electrode in the flow path.

[0034] In one or more embodiments, the range in which the reagent layer extends from the edge of the upstream side of the working electrode is 12.5% ​​to 100% of the surface area of ​​the working electrode in the flow path, and the range in which the reagent layer extends from the edge of the downstream side of the working electrode is greater than 45% and less than 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the range in which the reagent layer extends from the edge of the upstream side of the working electrode is greater than 12.5% ​​and less than 90% of the surface area of ​​the working electrode in the flow path, and the range in which the reagent layer extends from the edge of the downstream side of the working electrode is greater than 50% and less than 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the range in which the reagent layer extends from the edge of the upstream side of the working electrode is greater than 12.5% ​​and less than 85% of the surface area of ​​the working electrode in the flow path, and the range in which the reagent layer extends from the edge of the downstream side of the working electrode is greater than 55% and less than 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the range in which the reagent layer extends from the edge on the upstream side of the working electrode is greater than 12.5% ​​and less than 80% of the surface area of ​​the working electrode in the flow path, and the range in which the reagent layer extends from the edge on the downstream side of the working electrode is greater than 60% and less than 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the range in which the reagent layer extends from the edge on the upstream side of the working electrode is greater than 13% and less than 75% of the surface area of ​​the working electrode in the flow path, and the range in which the reagent layer extends from the edge on the downstream side of the working electrode is greater than 65% and less than 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the range in which the reagent layer extends from the edge on the upstream side of the working electrode is greater than 13% and less than 75% of the surface area of ​​the working electrode in the flow path, and the range in which the reagent layer extends from the edge on the downstream side of the working electrode is greater than 70% and less than 100% of the surface area of ​​the working electrode in the flow path. In one or more embodiments, the range of the reagent layer extending from the edge on the upstream side of the working electrode is greater than 13% and less than 75% of the surface area of ​​the working electrode in the flow path, and the range of the reagent layer extending from the edge on the downstream side of the working electrode is greater than 75% and less than 100% of the surface area of ​​the working electrode in the flow path.

[0035] From the viewpoint of further improving the measurement accuracy, in one or more embodiments, the area of ​​the reagent layer extending from the edge on the downstream side of the working electrode is preferably larger than the area of ​​the reagent layer extending from the edge on the upstream side of the working electrode. In one or more embodiments, the area of ​​the reagent layer extending from the edge on the downstream side 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 of the area of ​​the reagent layer extending from the edge on the upstream side of the working electrode.

[0036] The reagent layer is formed to cover at least a portion of the region (surface) of the counter electrode in the flow path. The reagent layer is formed to cover the region (surface) of the counter electrode in the flow path, and the region (area) of the working electrode in the flow path is at least 70% equivalent. From the perspective of further improving the measurement accuracy, in one or more embodiments, it can be an area equivalent to 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 region (area) of the counter electrode covered by the reagent layer can 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 region (area) of the working electrode in the flow path. In one or more embodiments, the region (area) of the reagent layer covering the counter electrode can be an area equivalent to more than 70% and less than 100%, more than 75% and less than 100%, more than 80% and less than 100%, more than 85% and less than 100%, more than 90% and less than 100%, or more than 95% and less than 100% of the region (area) of the working electrode in the flow path.

[0037] In addition, in one or more embodiments, the area of ​​the reagent layer covering the electrode can be at least 70% of the area (area) of the electrode in the flow path. From the perspective of further improving the measurement accuracy, in one or more embodiments, it can be more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or less than 100%. In one or more embodiments, the area of ​​the reagent layer covering the electrode can be less than 100%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, or less than 85% of the area (area) of the electrode in the flow path. In one or more embodiments, the area of ​​the reagent layer covering the counter electrode can be greater than 70% and less than 100%, greater than 75% and less than 100%, greater than 80% and less than 100%, greater than 85% and less than 100%, greater than 90% and less than 100%, or greater than 95% and less than 100% of the area (area) of the counter electrode in the flow path.

[0038] 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 embodiments.

[0039] In one or more embodiments, the reagent layer is preferably formed by dot matrix printing. According to dot matrix printing, as described later, by repeatedly drying the droplets of the ejected reagent solution, ejecting the droplets of the reagent solution thereon and drying them, a reagent layer with the above-mentioned average height can be easily formed. In one or more embodiments, the formation of the reagent layer based on dot matrix printing can be carried out with reference to the methods described in the embodiments, EP4266037 or US2023 / 338945, etc.

[0040] The size (area) of the reagent layer in the flow path can be appropriately determined according to the shape of the substrate, flow path and the conductive part. In a rectangular substrate with a length of 7mm in the short side direction and a length of 30mm in the long side direction, the flow path is a rectangular shape with a width (length in the short side direction of the substrate) of 1.8mm and a length (length in the long side direction of the substrate) of 4mm, and the working electrode is a rectangular shape with a width (length in the short side direction of the substrate) of 1.8mm and a length (length in the long side direction of the substrate) of 0.8mm in the flow path. Regarding the size of the reagent layer, in one or more embodiments, the width (length in the short side direction of the substrate) is 1.5mm or more, 2mm or more, 2.5mm or more or 3mm or more, and / or 5mm or less, 4.5mm or less or 4mm or less, and the length (length in the long side direction of the substrate) is 0.5mm or more, 0.6mm or more, 0.8mm or more or 0.9mm or more, and / or 2mm or less, 1.6mm or less or 1.4mm or less. Regarding the size of the reagent layer, in one or more embodiments, the width (the length in the short side direction of the substrate) is greater than 1.5 mm and less than 5 mm, greater than 2 mm and less than 5 mm, greater than 2.5 mm and less than 4.5 mm, and / or the length (the length in the long side direction of the substrate) is greater than 0.5 mm and less than 2 mm, greater than 0.6 mm and less than 2 mm, greater than 0.8 mm and less than 1.6 mm, or greater than 0.9 mm and less than 1.4 mm.

[0041] As a substrate, in one or more embodiments, a sheet-like insulating substrate can be cited. As a material of the substrate, in one or more embodiments, thermoplastic resins, thermosetting resins, glass, ceramics, and paper can be cited. As a material, in one or more embodiments, polyetherimide (PEI), polyethylene terephthalate (PET), polyethylene (PE), polybutylene terephthalate (PBT), polystyrene (PS), polymethacrylate (PMMA), polypropylene (PP), polyimide resins, acrylic resins, epoxy resins, and glass epoxy resins can be cited.

[0042] The conductive portion includes at least one electrode pair having a working electrode and a counter electrode, formed on a main surface of the substrate. In the case where the conductive portion includes multiple electrode pairs, for example, it is also possible to have a first electrode pair for measuring a first measurement object (e.g., blood sugar) contained in the same sample such as blood and a second electrode pair for measuring a second measurement object (e.g., hematocrit). Furthermore, the counter electrodes of the first electrode pair and the second electrode pair can also be shared. Here, in the case of having multiple electrode pairs, the reagent layer in the biosensor of the present invention can be formed only on at least one electrode pair, or it can be formed on multiple electrode pairs. In addition, in one or more embodiments, the conductive portion can also have a detection electrode.

[0043] 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 path. In one or more embodiments, the working electrode is formed upstream of the counter electrode, preferably formed adjacent to the counter electrode upstream of the counter electrode.

[0044] As the material of the electrode, in one or more embodiments, a conductive material can be used. As the material with conductivity, in one or more embodiments, metal materials and carbon materials can be cited. As the metal material, in one or more embodiments, gold (Au), platinum (Pt), silver (Ag), ruthenium (Ru), palladium (Pd) and nickel (Ni) alloys can be cited. As a nickel alloy, in one or more embodiments, nickel-vanadium alloys, nickel-tungsten alloys and nickel-ruthenium alloys can be cited. As a carbon material, in one or more embodiments, graphite, carbon nanotubes, graphene and mesoporous carbon can be cited.

[0045] In one or more embodiments, the electrode may include a thin film electrode formed by forming a film of the above-mentioned material such as a metal material on a substrate. As a film forming method, in one or more embodiments, screen printing, physical vapor deposition (PVD, such as sputtering) or chemical vapor deposition (CVD) may be included.

[0046] In one or more embodiments, the working electrode and the counter electrode may be insulated. In the case where the electrode constituting the conductive portion is formed of a metal material formed by physical evaporation, in one or more embodiments, each electrode may be insulated by drawing a prescribed electrode pattern (trimming) using a laser. In the case where the conductive portion is formed using a carbon material, in one or more embodiments, the insulating portion may be formed by forming each electrode at a prescribed interval.

[0047] In one or more embodiments, the biosensor of the present invention may include a spacer disposed on the substrate and a cover disposed on the spacer. In one or more embodiments, the spacer may also include a cutout portion. The spacer is disposed on the substrate in a manner such that the conductive portion and the reagent layer are located inside the cutout portion of the spacer, thereby enabling the space formed by the substrate, the cutout portion and the cover to be used as a sample supply port and a flow path.

[0048] Examples of the shape of the cutout portion of the spacer include a U-shape and a U-shape. The material of the spacer is not particularly limited, and the same material as that of the substrate can be used.

[0049] In one or more embodiments, the cover may have air holes, preferably the air holes are arranged on the spacer so as to be located on the cutout of the spacer. The material of the cover is not particularly limited, and the same material as the substrate can be used, preferably a hydrophilic film with a higher contact angle (hydrophobicity) than the spacer.

[0050] In the biosensor of the present invention, in one or more embodiments, the reagent layer is formed on a conductive portion (particularly a pair of electrodes having a working electrode and a counter electrode), and the conductive portion is located on the inner side (inside the cutout portion) of the flow path formed by the cutout portion of the above-mentioned spacer. The reagent layer can also be formed on a conductive portion located on the outer side of the flow path. In other words, the reagent layer can also exceed the outer side of the cutout portion (flow path) of the spacer, that is, the end (boundary) of the flow path to form a reagent layer. It should be noted that the reagent layer formed on the outer side of the cutout portion (flow path) of the spacer does not substantially contact the sample, so the reagent in the reagent layer of this part will not contribute (affect) the determination, and therefore, the shape, size and height of the reagent layer formed on the outer side of the cutout portion (flow path) of the spacer are not particularly limited.

[0051] In one or more embodiments, examples of the reagent contained in the reagent layer include oxidoreductase and electron transfer substances.

[0052] [Oxidoreductase]

[0053] As oxidoreductases, in one or more embodiments, glucose dehydrogenase (GDH), glucose oxidase (GOD), cholesterol oxidase, quinone heme alcohol 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, etc. can be cited.

[0054] 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). As an oxidoreductase having a coenzyme, in one or more embodiments, FAD-GDH, PQQ-GDH, NAD-GDH and NADP-GDH can be cited.

[0055] As the oxidoreductase, in one or more embodiments, Aspergillus oryzae type FAD-GDH (flavin adenine dinucleotide-dependent glucose dehydrogenase or flavin adenine dinucleotide-binding glucose dehydrogenase) etc. can be cited. As the Aspergillus oryzae type FAD-GDH, in one or more embodiments, the substance disclosed in Japanese Patent Application Laid-Open No. 2013-083634 can be used. The content of this document is cited as a part of the content constituting the present invention.

[0056] In one or more embodiments, the amount of the oxidoreductase in the reagent layer is 1000 KU / cm 3 Below, preferably 500KU / cm 3 Below, more preferably 300KU / cm 3 The lower limit of the amount of the oxidoreductase in the reagent layer is not particularly limited, but in one or more embodiments, it is 1 KU / cm 3 The amount of the oxidoreductase in the reagent layer is 200 KU / cm in one or more embodiments. 3 ~300KU / cm 3 , preferably 200KU / cm 3 ~280KU / cm 3 , more preferably 210KU / cm 3 ~260KU / cm 3 In the present invention, "U" is the unit of enzyme activity, which refers to the amount of enzyme that acts on 1 μmol of substrate in 1 minute under optimal conditions.

[0057] [Electron transfer substances]

[0058] Regarding electron transfer materials, in one or more embodiments, ruthenium compounds, 1-methoxy-PES (1-methoxy-5-ethylphenazine ethyl sulfate, 1-mPES), 1-methoxy-PMS (1-methoxy-5-methylphenazine methyl sulfate, 1-mPMS), phenylenediamine compounds, quinone compounds, ferrocyanide, coenzyme Q0 (2,3-dimethoxy-5-methyl-p-benzoquinone), azure A chloride (3-amino-7-(dimethylamino)phenothiazine-5-ium chloride), phenol safranin (3,7-diamino-5-phenylphenazinium chloride), 6-aminoquinoxaline, tetrathiafulvalene, etc. can be cited.

[0059] As the ruthenium compound, in one or more embodiments, a ruthenium compound that is present in the reaction system in the form of an oxidized ruthenium complex and functions as an electron transfer material can be used. The type of the ligand of the ruthenium complex is not particularly limited. As the ruthenium compound, in one or more embodiments, an oxidized ruthenium complex shown in the following chemical formula can be cited.

[0060] [Ru(NH3)5X] n+

[0061] In the above formula, as X, NH3, halogen ion, CN, pyridine, nicotinamide, or H2O can be cited, among which NH3 or halogen ion is preferred. As halogen ion, in one or more embodiments, Cl - 、F - Br - , and I - In the formula n+ represents the valence of the oxidized ruthenium (III) complex determined by the type of X. As the ruthenium complex, in one or more embodiments, the ruthenium complex disclosed in Japanese Patent Publication No. 2018-013400 can be used. The content of this document is cited as a part of the present invention. As the ruthenium compound, in one or more embodiments, [Ru(NH3)6] 2+ or [Ru(NH3)6] 3+ Ruthenium hexamine, etc. As the ruthenium compound, in one or more embodiments, [Ru(NH3)6]Cl3 and the like can be cited.

[0062] As the phenylenediamine compound, in one or a plurality of embodiments, N,N-dimethyl-1,4-phenylenediamine and N,N,N',N'-tetramethyl-1,4-phenylenediamine dihydrochloride can be mentioned.

[0063] In one or more embodiments, the quinone compound includes 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone (VK3), 9,10-phenanthrenequinone, 1,2-naphthoquinone, p-dimethylquinone, methylbenzoquinone, 2,6-dimethylbenzoquinone, 1,2-naphthoquinone-4-sodium sulfonate, 1,4-anthraquinone, tetramethylbenzoquinone, thymoquinone, and the like.

[0064] In one or more embodiments, examples of the ferrocyanide include calcium ferrocyanide and the like.

[0065] In one or more embodiments, the amount of the electron transfer substance in the reagent layer is 0.1 mmol / cm 3 Above, preferably 0.5 mmol / cm 3 More preferably, 1 mmol / cm 3 The upper limit of the amount of the electron transfer substance in the reagent layer is not particularly limited, but in one or more embodiments, it is 50 mmol / cm 3 Below or 10mmol / cm 3 In one or more embodiments, the amount of the electron transfer substance in the reagent layer is 0.1 mmol / cm 3 Above and 50mmol / cm 3 Below, preferably 0.5 mmol / cm 3 Above and 50mmol / cm 3 Below, more preferably 1 mmol / cm 3 Above and 10mmol / cm 3 the following.

[0066] In one or more embodiments, the reagent layer may contain other components in addition to the oxidoreductase and the electron transfer substance. In one or more embodiments, other components include buffers, amino acids, surfactants, defoaming agents, and binders.

[0067] In one or more embodiments, examples of the buffer include a phosphate buffer, an amine buffer, and a buffer having a carboxyl group. As the amine buffer, in one or more embodiments, tris (tris (hydroxymethyl) aminomethane), ACES (N- (2-acetylamino) -2-aminoethane sulfonic acid), CHES (N-cyclohexyl-2-aminoethane sulfonic acid), CAPSO (3- (cyclohexylamino) -2-hydroxy-1-propane sulfonic acid), TAPS (N-tris (hydroxymethyl) methyl-3-aminopropane sulfonic acid), CAPS (N-cyclohexyl-3-aminopropane sulfonic acid), Bis-Tris (bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane), TAPSO (2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropane sulfonic acid), TES (N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid), Tricine (N- [tris (hydroxymethyl) methyl] glycine), and ADA (N- (2-acetylamino) iminodiacetic acid) and the like can be cited. As the buffer having a carboxyl group, in one or more embodiments, citric acid buffer, phosphocitrate buffer, acetic acid-sodium acetate buffer, malic acid-sodium acetate buffer, malonic acid-sodium acetate buffer, and succinic acid-sodium acetate buffer can be mentioned. The buffer may be one kind or two or more kinds may be used in combination. The pH of the buffer is 6.8 to 7.5 in one or more embodiments, preferably 6.9 to 7.0.

[0068] In one or more embodiments, examples of the amino acid include glycine, serine, lysine, and histidine.

[0069] As the surfactant, in one or more embodiments, Triton X-100 ((p-tert-octylphenoxy) polyethoxyethanol), Tween 20 (polyoxyethylene sorbitan monolaurate), sodium lauryl sulfate, perfluorooctane sulfonic acid, sodium stearate, alkylaminocarboxylic acid (or its salt), carboxybetaine, sulfobetaine, and phosphobetaine, etc. can be mentioned. The surfactant can be one kind or two or more kinds can be used in combination.

[0070] As the binder, in one or more embodiments, resin binders and layered inorganic compounds can be cited. As the resin binder, in one or more embodiments, butyral resin binders and polyester resin binders can be cited. As the layered inorganic compound, the layered inorganic compound described in WO2005 / 043146 can be used. As the layered inorganic compound, in one or more embodiments, swelling clay minerals with ion exchange capacity can be cited. As the layered inorganic compound, in one or more embodiments, bentonite, montmorillonite, vermiculite and synthetic fluorine mica can be cited. The binder can be one kind, or two or more kinds can be used in combination.

[0071] In one or more embodiments, the biosensor of the present invention can be manufactured by the following method: preparing a substrate having a conductive portion formed on a main surface, the conductive portion comprising at least one electrode pair having a working electrode and a counter electrode, and dot-matrix printing a reagent solution on the conductive portion in a manner to form a reagent layer at the above-mentioned position.

[0072] [Method for producing biosensor]

[0073] The present invention relates to a method for manufacturing a biosensor in another embodiment. The method for manufacturing the biosensor is a method for manufacturing a biosensor having a conductive portion and a reagent layer in a flow path, and the method comprises the following steps: preparing a substrate having a conductive portion including an electrode pair formed on one main surface, the 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 comprising: dot-matrix printing a reagent solution so that the entire area of ​​the working electrode in the flow path, an area of ​​at least 50 μm from the respective edges of the upstream and downstream sides of the working electrode, and an area of ​​the counter electrode in the flow path corresponding 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.

[0074] As another method of manufacturing the biosensor of the present invention, formation of the reagent layer includes: dot-matrix printing the reagent solution in a manner that 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, the dot-matrix printing including: dot-matrix printing on a range of at least 12.5% ​​of the surface area of ​​the working electrode in the flow path from the edge of the upstream side of the working electrode to the upstream side, and from the edge of the downstream side of the working electrode to the downstream side, respectively; and dot-matrix printing the reagent solution on a range of the surface of the counter electrode in the flow path that is equivalent to at least 70% of the surface area of ​​the working electrode in the flow path.

[0075] According to the manufacturing method of the present invention, the reagent layer is formed by dot-matrix printing the reagent solution within the above-mentioned prescribed range on the conductive portion, so that the biosensor of the present invention capable of high-precision measurement can be manufactured. Therefore, in one or more embodiments, the manufacturing method of the present invention can also be referred to as a method for manufacturing the biosensor of the present invention.

[0076] In one or more embodiments, dot matrix printing includes the steps of repeatedly performing the following operations: spraying droplets of a reagent solution onto the conductive portion, drying the sprayed droplets, and spraying droplets of a reagent solution onto the dried droplets. Thus, the reagents can be overlapped to form a reagent layer with an average height of 4 μm to 12 μm. In one or more embodiments, dot matrix printing can be performed using an inkjet printer or the like. The composition of the reagent solution is as described above.

[0077] In one or more embodiments, the ejection amount of the droplets can be appropriately determined according to the interval between the positions of the ejected droplets and the size of the formed droplets, etc. In one or more embodiments, the ejection amount of the droplets is 10 ng to 30 ng, and from the viewpoint of further reducing the concentration and amount unevenness of the reagent components in the reagent layer and further improving the reproducibility of the measurement accuracy of the obtained biosensor, it is 12 ng to 25 ng or 12 ng to 15 ng.

[0078] In one or more embodiments, the ejection amount of the droplets may also vary between the first layer and thereafter. From the viewpoint of further reducing 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, it is preferred that the ejection amount of the droplets after the second layer is more than the ejection amount of the droplets of the first layer. In one or more embodiments that are not particularly limited, the ejection amount of the droplets of the first layer may be set to less than 20ng, and the ejection amount of the droplets of the second layer may be set to more than 20ng. In one or more embodiments, the ejection amount of the droplets of the first layer is 10ng to 18ng, and from the viewpoint of further reducing 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, it is preferably more than 11ng. From the same viewpoint, it is preferably less than 17ng, less than 15ng, less than 14ng or less than 13ng. In one or more embodiments, the ejection amount of the droplets after the second layer is 20ng to 28ng, and from the viewpoint of further reducing 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, it is preferably 21ng or more. From the same viewpoint, it is preferably 26ng or less, 25ng or less, or 24ng or less. In one or more embodiments, the ejection amount of the droplets after the second layer can be the same or different.

[0079] In one or more embodiments, the formation of the reagent layer includes the following steps: ejecting droplets of the Nth layer (where N is a natural number greater than 1) of the reagent solution at a predetermined interval; and ejecting droplets of the N+1th layer of the reagent solution onto the dried droplets of the Nth layer. In one or more embodiments, N is a natural number of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater.

[0080] From the viewpoint of further reducing the concentration and amount variations of reagent components in the reagent layer and further improving the reproducibility of the measurement accuracy of the obtained biosensor, the manufacturing method of the present invention includes the step of repeatedly ejecting droplets of the reagent solution at least up to the fourth layer.

[0081] In one or more embodiments, the formation of the reagent layer may include a step for drying the droplets between the ejection of the droplets of the Nth layer and the ejection of the droplets of the N+1th layer. When the ejection of the droplets of the Nth layer and the ejection of the droplets of the N+1th layer are performed by different ejection devices, the transport time of the substrate may also be set as the time for drying the droplets.

[0082] In one or more embodiments, the interval between the ejected droplets (the distance between the centers of adjacent droplets) is 50 μm to 150 μm, preferably 60 μm to 80 μm, and more preferably 65 μm to 75 μm, from the viewpoint of further reducing 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. In one or more embodiments, the "center of the droplet" in the present invention refers to the center of a circle circumscribed with the outer periphery of the droplet after the droplet is dried and forms a droplet point on the conductive portion.

[0083] In one or more embodiments, the intervals between ejected droplets may be equal or variable, but are preferably equal from the viewpoint of further reducing the concentration and amount variations of the reagent components in the reagent layer and further improving the reproducibility of the measurement accuracy of the obtained biosensor.

[0084] In one or more embodiments, the intervals between ejected droplets may be different or the same in each layer. From the perspective of further reducing the concentration and amount unevenness of the reagent components in the reagent layer and further improving the reproducibility of the measurement accuracy of the obtained biosensor, it is preferably the same in all layers.

[0085] In one or more embodiments, the manufacturing method of the present invention may include the following steps: preparing a spacer having a cutout portion; and preparing a cover having air holes, and may also include the following steps: configuring the spacer on the substrate in a manner such that the reagent layer formed is located on the inner side of the cutout portion of the spacer; and configuring the cover on the spacer in a manner such that the air holes of the cover are located on the cutout portion of the spacer.

[0086] The positions of the electrodes, substrate, reagent layer, and reagents in the production method of the present invention are the same as those of the biosensor of the present invention.

[0087] [Measurement method]

[0088] As another embodiment, the present invention relates to a method for performing electrochemical measurement of a measurement object in a sample using the biosensor of the present invention, the method comprising the following steps: bringing the sample into contact with a reagent layer of the biosensor; applying a voltage between electrodes of the biosensor; and measuring an electrical signal generated by applying the voltage.

[0089] In the present invention, "electrochemical measurement" refers to measurement using an electrochemical measurement method, and in one or more embodiments, examples include current measurement, potential difference measurement, coulometry, etc. As the current measurement method, in one or more embodiments, a method of measuring the current value generated when a reduced electron transfer substance is oxidized by applying a voltage can be cited.

[0090] The applied voltage is not particularly limited, but in one or more embodiments, it is 10 mV or more or 50 mV or more, preferably 100 mV or more. In addition, in one or more embodiments, the applied voltage is 1000 mV or less or 700 mV or less, preferably 600 mV or less, 500 mV or less, 400 mV or less, or 300 mV or less.

[0091] Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to these embodiments.

[0092] Figure 1 and 2 1 is a schematic structural diagram for explaining the structure of the biosensor of the present invention. Figure 1 and 2 In the diagram, the upper side is the upstream side (sample supply port side), and the lower side is the downstream side (insertion side into the measuring device).

[0093] like Figure 1 and 2 As shown, 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 3a, and the spacer 3 is arranged on the substrate 2 in such a manner that the conductive portion and a part of the reagent layer 50 are located inside the cutout 3a. The cover has an air hole (not shown), and the cover is arranged on the spacer 3 in such a manner that the air hole is located on the cutout 3a. The space formed by the substrate 2, the cutout 3a of the spacer 3, and the cover becomes a flow path 1d, and the opening becomes a sample supply port 1c.

[0094] As the material of the substrate, polyethylene terephthalate (PET) or the like can be preferably used. In the present 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 width of the flow path 1d is 2 mm, and the length is 4 mm. The width (short side direction of the biosensor) of the reagent layer 50 is 2700 μm, and the length (long side direction of the biosensor) is 1000 μm.

[0095] 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 pair of electrodes 12, and the second electrode pair 20 is composed of a second working electrode 21 and a second pair of electrodes 22. In the flow path 1d, the second working electrode 21, the second pair of electrodes 22, the first working electrode 11, the first pair of electrodes 12 and the blood detection electrode 30 are sequentially arranged from the upstream side in a rectangular exposed manner parallel to the short side direction of the substrate 2. Gaps (non-conductive areas) 40b and 40a are provided between the second working electrode 21 and the second pair of electrodes 22, and between the second pair of electrodes 22 and the first working electrode 11, respectively. The first pair of electrodes 12 is in contact with the first working electrode 11 on the upstream side and is in contact with the blood detection electrode 30 on the downstream side (the surface on the opposite side of the first working electrode 11), but they are insulated from each other. In this embodiment, the conductive part (electrode) is formed by sputtering a nickel-vanadium alloy, and the electrodes can be insulated by drawing a predetermined electrode pattern (trimming) using a laser. The gaps (non-conductive regions) 40 b and 40 a are regions insulated by drawing rectangular patterns with laser.

[0096] The electrodes constituting the first electrode pair 10 and the second electrode pair 20 (the first working electrode 11, the first pair of electrodes 12, the second working electrode 21 and the second pair of electrodes 22) and the width of the gap 40a (the length in the long side direction of the substrate 2) are both 400 μm, and the width of the gap 40b (the length in the long side direction of the substrate 2) is approximately 200 μm.

[0097] 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 pair of electrodes 22, the lead portion 11a of the first working electrode 11, the lead portion 12a of the first pair of electrodes 12, and the lead portion 30a of the blood detection electrode 30 extend from one end to the other end of the substrate 2 in parallel with the long side direction of the substrate 2. The region 1a from the upstream end to the vicinity of the downstream end of each electrode is covered by the spacer 3, but the downstream end portion is not covered and exposed, and this portion becomes a connector region 1b inserted into the insertion port of the measurement device. In the connector region 1b, the lead portion 21a of the second working electrode 21, the lead portion 22a of the second pair of electrodes 22, the lead portion 11a of the first working electrode 11, the lead portion 12a of the first pair of electrodes 12, and the lead portion 30a of the blood detection electrode 30 become exposed contacts.

[0098] The reagent layer 50 is formed by ejecting droplets of a reagent solution using an inkjet printer. The size (reagent layer formation region) of the reagent layer 50 in this embodiment is 2700 μm in the short side direction of the substrate 2 and 1000 μm in the long side direction of the substrate 2.

[0099] like Figure 2 As shown ( Figure 1 ), the reagent layer 50 is formed on the conductive portion exposed in the flow path 1d. Specifically, the reagent is formed so that substantially the entire surface of the first working electrode 11, the area from the edge in contact with the first working electrode 11 toward the downstream side to at least 70% of the surface of the first working electrode 11 on the surface of the first pair of electrodes 12, the area from the edge on the upstream side (the side opposite to the first pair of electrodes 12) of the first working electrode 11 toward the upstream side to at least 50 μm, that is, the area from the edge in contact with the first working electrode 11 toward the upstream side to at least 12.5% ​​of the surface of the first working electrode 11 on the surface of the non-conductive region (gap 40a) is covered with the reagent.

[0100] After the spacer 3 is arranged, the reagent layer 50 may be formed on the conductive portion that can be exposed in the flow path 1d (the cutout portion 3a). Figure 2 As shown, it can also be formed on the conductive portion located (or possibly located) outside the flow path 1d (cutout portion 3a). Thus, even if a manufacturing error occurs during the manufacturing process of the biosensor (for example, a shift in the formation position of the cutout portion and a shift in the configuration of the spacer, etc.), the reagent layer can be arranged in the specified area of ​​the working electrode and the counter electrode in the flow path.

[0101] The reagent layer 50 may be formed on the second working electrode 21 and the second counter electrode 22 constituting the second electrode pair, or may not be formed on the second working electrode 21 and the second counter electrode 22. In addition, a reagent layer having a composition different from that of the reagent layer 50 may be formed on the second working electrode 21 and the second counter electrode 22 constituting the second electrode pair, or may not be formed.

[0102] The formation of the reagent layer 50 using an inkjet printer can be performed by the following method: the interval of the ejected droplets (the distance between the centers of the dots (the ejected droplets of the reagent solution)) is set to 70 μm, and in the reagent layer formation area, the droplets of the reagent solution are uniformly ejected to a total of 546 locations, 39 locations in the short side direction of the substrate 2 × 14 locations in the long side direction of the substrate 2, thereby forming a point group (for example, 546 reagent dots (39 × 14)) containing a plurality of reagent dots (droplets of the ejected reagent solution), and the formation of the point group is repeated. From the viewpoint of being able to easily form a reagent layer with an average height of 4 μm or more, making the height of the formed reagent layer more uniform, and further improving the measurement accuracy, the formation of the point group is preferably repeated 3 times (3 layers) or more, and more preferably repeated 5 times (5 layers) or more. From the perspective of making the height of the formed reagent layer more uniform and further improving the measurement accuracy, it is preferred that the position of the reagent liquid droplets ejected in the odd-numbered point group (odd-numbered layers) is different from the position of the reagent liquid droplets ejected in the even-numbered point group (even-numbered layers).

[0103] In addition, the intervals between the droplets of the sprayed reagent liquid and the number (positions) of the droplets of the sprayed reagent liquid (the number of sprayed droplets / point group) in the formation of the reagent layer 50 are not limited to the above-mentioned method, and can be appropriately determined according to the size (area) of the reagent layer to be formed, the amount of the droplets of the sprayed reagent liquid, and the composition of the sprayed reagent liquid.

[0104] From the viewpoint of making the height of the formed reagent layer more uniform and further improving the measurement accuracy, a time for drying the droplets of the ejected reagent solution can be set between the formation of one dot group and the formation of the next dot group. Regarding the time for drying, in one or more embodiments, the time required for transporting the substrate for ejecting the droplets of the reagent solution from the nozzle of the inkjet head to a specified position can be set as the drying time, or the time for the drying process can be set separately from the transport process. In the case of forming the reagent layer by multiple inkjet printers, the time required for transport between the inkjet printers can also be used for the drying process.

[0105] Hereinafter, a method of forming a reagent layer using an inkjet printer will be described by taking the case where formation of a dot group is repeated five times (five layers) as an example.

[0106] First, a reagent solution is prepared. The composition of the reagent solution can be appropriately determined according to the type of reagent layer to be formed and the type of biosensor to be manufactured.

[0107] In one or more embodiments, the amount of the oxidoreductase in the reagent solution is 1KU / mL to 200KU / mL. In one or more embodiments, the amount of the oxidoreductase in the reagent solution is 5KU / mL or more, 10KU / mL or more, 15KU / mL or more, 20KU / mL or more, 25KU / mL or more, or 30KU / mL or more. In one or more embodiments, the amount of the oxidoreductase in the reagent solution is 100KU / mL or less, 90KU / mL or less, 80KU / mL or less, 70KU / mL or less, 60KU / mL or less, or 55KU / mL or less.

[0108] In one or more embodiments, the amount of the electron transfer substance in the reagent solution is 1mM to 1000mM. In one or more embodiments, the amount of the electron transfer substance in the reagent solution is 2mM or more, 5mM or more, 10mM or more, 50mM or more, 100mM or more, 200mM or more, or 500mM or more. In one or more embodiments, the amount of the electron transfer substance in the reagent solution is 900mM or less, 800mM or less, or 700mM or less. In one or more embodiments, the amount of the buffer in the reagent solution is 10mM to 1000mM. In one or more embodiments, the amount of the buffer in the reagent solution is 20mM or more, 50mM or more, 100mM or more, 150mM or more, or 200mM or more. In one or more embodiments, the amount of the buffer in the reagent solution is 900mM or less, 800mM or less, 700mM or less, 600mM or less, or 500mM or less.

[0109] In one or more embodiments, the amount of the amino acid in the reagent solution is 0.1% to 20% by mass or 0.5% to 10% by mass. In one or more embodiments, the amount of the surfactant in the reagent solution is 0.1% to 20% by mass or 0.5% to 10% by mass.

[0110] Using the prepared reagent solution, a first-stage point group (first point group) is formed.

[0111] The reagent solution for the first point group is ejected in such an amount that the droplets of the reagent solution ejected from the nozzle do not merge (combine) with each other. The ejection amount of the reagent solution can be set to 10ng to 18ng (e.g., 12ng), for example. If it is this amount, it is possible to suppress the droplets of the reagent solution ejected onto the conductive portion (substrate) from merging with each other to form one droplet, and each droplet is arranged on the conductive portion (substrate) as an independent droplet. The size of the droplet (after drying, the major diameter) is 50μm to 70μm.

[0112] Preferably, after ejecting the droplets constituting the first point group, the reagent of the first point group is dried before ejecting the reagent solution for the second stage point group (second point group). After the reagent of the first point group is dried, droplets of the next reagent solution are ejected thereon, whereby the newly ejected droplets of the reagent solution dissolve a portion of the reagent of the first point group, and the droplets are dried together with the dissolved reagent, thereby further reducing the unevenness of the reagent and further improving the measurement accuracy. The drying can be performed at room temperature (humidity below 50%) for more than 20 seconds.

[0113] Next, a second point group (second point group) is formed on the dried first point group.

[0114] From the viewpoint of further reducing reagent unevenness and further improving measurement accuracy, the reagent solution for the second point group is ejected at a position different from the position where the reagent solution was ejected for the first point group. For example, it is preferred to eject droplets of the reagent solution to the intersection of the centers of adjacent reagent points of the first point group.

[0115] From the viewpoint of further reducing reagent unevenness and further improving measurement accuracy, the ejection of the reagent solution for the second point group is preferably performed in a manner that the droplets of the reagent solution ejected from the nozzle overlap at least a portion of the droplets of the adjacent reagent solution. The ejection amount of the droplets can be set to 20 to 28 ng (e.g., 23 ng), for example. With this amount, the droplets of the reagent solution ejected from the nozzle can dry while overlapping with the droplets of the adjacent reagent solution.

[0116] Preferably, after ejecting the droplets constituting the second point group, the reagent of the second point group is dried before ejecting the reagent solution for the third stage point group. After the reagent of the second point group is dried, droplets of the next reagent solution are ejected thereon, whereby the newly ejected droplets of the reagent solution dissolve a portion of the reagent of the first point group and the second point group, and the droplets are dried together with the dissolved reagent, thereby further reducing the unevenness of the reagent and further improving the measurement accuracy. The drying can be performed in the same manner as the first point group.

[0117] Next, a third point group (third point group) is formed on the dried second point group.

[0118] In order to further reduce reagent unevenness and improve measurement accuracy, the reagent solution for the third point group is ejected at substantially the same position as the first point group. The ejection amount and drying of droplets can be performed in the same manner as the second point group.

[0119] Next, a fourth point group (fourth point group) is formed on the dried third point group.

[0120] In order to further reduce reagent unevenness and improve measurement accuracy, the reagent solution for the fourth point group is ejected at substantially the same position as the second point group. The ejection amount and drying of droplets can be performed in the same manner as the second point group.

[0121] Finally, a fifth point group (fifth point group) is formed on the dried fourth point group.

[0122] In order to further reduce reagent unevenness and improve measurement accuracy, the reagent solution for the fifth point group is ejected at substantially the same position as the first or third point group. The ejection amount and drying of droplets can be performed in the same manner as the third point group.

[0123] By forming these dot groups, in one or more embodiments, a reagent layer having an average height of 4 to 12 μm, 5 to 12 μm, 6 to 12 μm, 7 to 12 μm, 8 to 12 μm, 9 to 12 μm, or 9.5 to 12 μm can be formed.

[0124] Next, a spacer 3 having a cutout 3a is disposed on the substrate 2 having the reagent layer 50 formed thereon. The spacer 3 is disposed on the substrate 2 in such a manner that a portion of the conductive portion (the first working electrode 11, the first counter electrode 12, the second working electrode 21, the second counter electrode 22, and the blood detection electrode 30) on which the reagent layer 50 is disposed is located (exposed) inside the cutout 3a.

[0125] Finally, a cover (not shown) having air holes is placed on the spacer 3 .

[0126] When PMMA is used as the material of the spacer 3, the base material 2 and the cover can be bonded together by the adhesive property of PMMA. The spacer 3 and the cover may be laminated in advance and then laminated and bonded to the base material 2.

[0127] As an example of a method of electrochemically measuring a measurement target in a sample using the biosensor 1 , a case where the sample is whole blood and the measurement target is glucose will be described as an example.

[0128] 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 dropped onto the sample supply port 1c of the biosensor 1, the sample flows toward the downstream side in the flow path 1d in the order of 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 due to capillary force. At this time, when the whole blood sample reaches the reagent layer 50, the reagent components (oxidoreductase and electron transfer substances, etc.) contained in the reagent layer 50 are dissolved by the whole blood sample.

[0129] When a positive potential is applied to the electrode, electron transfer occurs between the electron transfer substance in the reagent layer 50 and the first working electrode 11 located below the reagent layer 50, and an oxidation current flows. Based on this, the glucose concentration can be measured. In one or more embodiments, the applied voltage is 10 mV to 1000 mV, preferably 100 mV to 600 mV.

[0130] The above embodiment describes the form and measurement method of the biosensor when the sample is blood (eg, whole blood) as an example, but the present invention is not limited thereto, and various biological samples other than blood, such as urine, can be measured in the same manner.

[0131] The present invention may relate to one or more of the following non-limiting embodiments.

[0132] [1] A biosensor comprising: a substrate having a conductive portion including an electrode pair formed on one main surface, the electrode pair including a working electrode and a counter electrode;

[0133] a reagent layer placed on the working electrode and the counter electrode; and

[0134] a flow path for allowing the sample to flow from the sample supply port to the reagent layer,

[0135] The reagent layer is formed to cover the entire region of the working electrode in the flow path and to extend at least 50 μm from the edges of the upstream and downstream sides of the working electrode, and is formed to

[0136] covering an area of ​​the counter electrode in the flow path that corresponds to at least 70% of the area of ​​the working electrode,

[0137] The average height of the reagent layer is 4 μm to 12 μm.

[0138] [2] A biosensor comprising:

[0139] 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;

[0140] a reagent layer placed on the working electrode and the counter electrode; and

[0141] a flow path for allowing the sample to flow from the sample supply port to the reagent layer,

[0142] 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 extends from the edge of the upstream side of the working electrode to the upstream side, and extends from the edge of the downstream side of the working electrode to the downstream side, and the extension ranges are at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, respectively, and the coverage area of ​​the reagent layer at the counter electrode is equivalent to at least 70% of the surface area of ​​the working electrode in the flow path,

[0143] The average height of the reagent layer is 4 μm to 12 μm.

[0144] [3] The biosensor according to [1] or [2], wherein the area of ​​the reagent layer extending from the downstream edge of the working electrode is larger than the area of ​​the reagent layer extending from the upstream edge of the working electrode.

[0145] [4] The biosensor according to any one of [1] to [3], wherein the working electrode and the counter electrode extend in a direction perpendicular to a supply direction of the sample in the flow path,

[0146] The working electrode is formed adjacent to the counter electrode on the upstream side of the counter electrode.

[0147] [5] The biosensor according to any one of [1] to [4], wherein the reagent layer extends from the edge of the working electrode within a range of 100 μm to less than 150 μm.

[0148] [6] The biosensor according to any one of [1] to [5], wherein the reagent layer is formed by dot matrix printing.

[0149] [7] A method for manufacturing a biosensor, the biosensor comprising a conductive portion and a reagent layer in a flow path, wherein:

[0150] The method for manufacturing the biosensor comprises the following steps:

[0151] preparing a substrate having the conductive portion including an electrode pair formed on one main surface thereof, the electrode pair including a working electrode and a counter electrode; and

[0152] forming the reagent layer on the working electrode and the counter electrode,

[0153] The formation of the reagent layer comprises the following steps:

[0154] The reagent liquid is dot-printed so that the entire area of ​​the working electrode in the flow path, an area of ​​at least 50 μm from the respective edges of the upstream and downstream sides of the working electrode, and an area of ​​the counter electrode in the flow path equivalent 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.

[0155] [8] The production method according to [7], which is used to produce the biosensor according to any one of [1] to [6].

[0156] Hereinafter, the present invention will be described in more detail with reference to Examples, but these are merely illustrative and the present invention is not limited to these Examples.

[0157] Example

[0158] [Production of biosensor 1]

[0159] (1) Figure 1 As shown, a substrate 2 (PET, thickness 250 μm, length 30 mm×width 7 mm) having a conductive portion formed on one main surface was prepared.

[0160] 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 pair of electrodes 12, and the second electrode pair 20 is composed of a second working electrode 21 and a second pair of electrodes 22. At one end of the long side direction of the substrate 2, the second working electrode 21, the second pair of electrodes 22, the first working electrode 11 and the first pair of electrodes 12 are sequentially arranged in parallel with the short side direction of the substrate 2. Gaps (non-conductive areas) 40b and 40a are provided between the second working electrode 21 and the second pair of electrodes 22, and between the second pair of electrodes 22 and the first working electrode 11, respectively. The first working electrode 11 is in contact with the first pair of electrodes 12, and the first pair of electrodes 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, respectively. The lead portion 21a of the second working electrode 21, the lead portion 22a of the second pair of electrodes 22, the lead portion 11a of the first working electrode 11, the lead portion 12a of the first pair of electrodes 12, and the lead portion 30a of the blood detection electrode 30 extend from one end of the substrate 2 to the other end parallel to the long side direction of the substrate 2.

[0161] The electrodes constituting the first electrode pair 10 and the second electrode pair 20 (the first working electrode 11, the first pair of electrodes 12, the second working electrode 21 and the second pair of electrodes 22) and the width of the gap 40a (the length in the long side direction of the substrate 2) are both 400 μm, and the width of the gap 40b (the length in the long side direction of the substrate 2) is approximately 200 μm.

[0162] (2) Next, a reagent solution was prepared to have the following composition.

[0163] [Composition of reagent solution]

[0164] FAD-dependent glucose dehydrogenase (trade name: glucose dehydrogenase "Amano 8", MW: 180,000, manufactured by Amano Enzyme Co., Ltd.): 41 KU / mL

[0165] Ru complex (Ru(NH3)6Cl3, manufactured by LT Metal Co., Ltd.): 626 mM

[0166] 1-Methoxy PES (1-methoxy-5-ethylphenazine ethyl sulfate, manufactured by Dojin Chemical Research Institute Co., Ltd.): 824 μg / mL

[0167] 0.4M phosphate buffer (pH 7.0): 0.29mM

[0168] Glycine: 2 mass%

[0169] CHAPS: 2 mass%

[0170] Defoamer: 0.04 mass%

[0171] (3) Next, a reagent layer is formed on the substrate 2 having the conductive portion formed thereon.

[0172] The reagent layer was formed by dot-printing the reagent solution with an inkjet device at room temperature and a humidity of 50% or less according to the following steps. The reagent layer formation area (the range of the dot-printed reagent) was rectangular, and the length of the short side direction of the substrate 2 was set to 2700 μm, and the length of the long side direction of the substrate 2 was set to 1000 μm. The reagent layer was formed in 9 modes (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 portion of the first counter electrode 12, and the position of the reagent layer was moved by 50 μm in the long side direction of the substrate 2 as shown in Table 1 below.

[0173] <Steps>

[0174] 1. The storage unit of the inkjet device is filled with the above reagent solution, and droplets of the reagent solution are ejected on the substrate having the conductive portion under the following conditions, and the droplets are dried for 30 seconds to thereby configure the first layer of reagent (first dot group). The diameter of each reagent printed by dot matrix (after drying) is 50 to 70 μm.

[0175] [Ejection conditions (first point group)]

[0176] Ejection volume per droplet: 12ng (10pL)

[0177] Interval between droplets (distance between the centers of ejected droplets): 70 μm

[0178] Number of ejections: 546 (39 in the short side direction of substrate 2 × 14 in the long side direction of substrate 2)

[0179] 2. Next, on the first dot group, droplets of reagent solution were ejected under the following conditions, and the droplets were dried for 30 seconds, thereby configuring the second layer of reagent (second dot group). In the second layer, droplets were ejected so that the position of the droplets of reagent solution (the center of the droplets) became the approximate center of a square, and the center of the four adjacent reagents of the first dot group was the vertex. The diameter of each reagent printed by the dot matrix (after drying) was 90 to 100 μm.

[0180] [Ejection conditions (second point group)]

[0181] Ejection volume per droplet: 23ng (18pL)

[0182] Interval between droplets (distance between the centers of ejected droplets): 70 μm

[0183] Number of ejections: 546 (39 in the short side direction of substrate 2 × 14 in the long side direction of substrate 2)

[0184] 3. Next, the third layer of reagent (third dot group) is arranged by spraying droplets of reagent solution on the second dot group and drying them. The spraying of droplets of the third dot group is the same as the second dot group except that the positions of the droplets of reagent solution sprayed are substantially the same as those of the first dot group.

[0185] 4. Next, on the third dot group, droplets of the reagent solution are ejected and dried in the same manner as on the second dot group, thereby disposing a fourth layer of the reagent (fourth dot group).

[0186] 5. Finally, on the fourth point group, droplets of the reagent solution were sprayed and dried in the same manner as the third point group, thereby configuring the fifth layer of reagent (fifth point group). Thus, a three-dimensionally stacked reagent layer (roughly a rectangular parallelepiped (quadrangular prism)) was obtained. The height of the reagent layer was observed using a laser microscope (product name: VK-X3000, manufactured by KEYENCE). The height distribution of the reagent layer was 8 μm to 12 μm, and the average height was about 10 μm. In addition, the amount of FAD-dependent glucose dehydrogenase in the obtained reagent layer was 232 KU / cm 3 The amount of electron transfer material is 3.56mmol / cm 3 .

[0187] (4) Figure 2 As shown, a spacer 3 having a cutout 3a (length of the long side direction of the substrate 2: 4 mm, length of the short side direction of the substrate 2: 2 mm) is arranged on the substrate 2 on which the reagent layer is formed, and a cover (not shown) having air holes is arranged on the spacer 3. The space formed by the substrate 2, the cutout 3a of the spacer 3 and the cover becomes a flow path 1d.

[0188] [Measurement of the size of the reagent printed by dot matrix]

[0189] The size of a reagent formed by one droplet ejected from an inkjet printer was measured using a digital microscope (VHX-6000, manufactured by KEYENCE).

[0190] [Glucose current value measurement 1]

[0191] The following samples of various glucose (GLU) concentrations were prepared and dropped onto the prepared biosensor for electrochemical measurement. The electrochemical measurement was performed using a potentiostat (manufactured by ARKRAY, Inc.), with a voltage of 0.2 V applied and the current value after 4.0 seconds was measured. The results are shown in Figure 3 to Figure 5 The R shown in Table 1 2 It is for Figure 3 and Figure 4The graphs of the current values ​​of the respective biosensors shown are the coefficients of determination when approximate curves (linear approximation) are prepared.

[0192] <Evaluation conditions>

[0193] Sample used: Whole blood sample

[0194] GLU concentration (mg / dl): 0, 45, 330, 800, 1500 and 3000 (all Hct 42%)

[0195] Number of evaluations: n = 10 (for each GLU concentration)

[0196]

[0197] Figure 3 and Figure 4 is a graph showing the relationship between glucose concentration and reaction current value. Figure 5 This is a graph showing the reaction current value in each biosensor when a sample having a GLU concentration of 3000 mg / dl and a Hct of 42% was used. Figure 3 Glucose measurement linearity in biosensors (types 1 to 5) with different areas of the reagent layer covering the counter electrode surface, Figure 4 The linearity of glucose measurement in biosensors (types 1, 6 to 9) with different areas of the reagent layer extending from the working electrode is shown. Figure 3 to Figure 5 As shown, in the biosensors having the reagent layer formed at the positions of type 4, type 5, and type 9, the reaction current value in the high glucose concentration region decreased, and sufficient glucose measurement linearity could not be obtained. In contrast, in the biosensors having the reagent layer formed at the positions of type 1 to type 3 and 6 to 8, no decrease in the reaction current value was observed, and high glucose measurement linearity was obtained.

[0198] [Production of biosensor 2]

[0199] As in the preparation of the biosensor 1, a substrate having a conductive portion formed thereon was prepared, and the first to third layers of reagents were arranged in the same manner as in <Steps> 1 to 4, thereby preparing 7 types of biosensors (types 1 to 3, types 5 to 7, and type 9) having different positions of reagent layers. The average height of the obtained reagent layers was about 6 μm (height distribution: 4 μm to 8 μm).

[0200] [Glucose current value measurement 2]

[0201] The measurement was performed in the same manner as in Glucose Current Value Measurement 1 except that the biosensor prepared in Biosensor Preparation 2 was used to measure the current value after 4.0 seconds. Among the obtained results, the results using the sample with a GLU concentration of 1500 mg / dl×Hct 42% are shown in Figure 6 .

[0202] like Figure 6 As shown, the reaction current values ​​of the biosensors having the reagent layer formed at positions 5 and 9 were low, whereas no decrease in the reaction current values ​​was observed in the other biosensors (types 1 to 3 and types 6 to 7), suggesting that high glucose measurement linearity can be obtained.

[0203] [Production of biosensor 3]

[0204] As in the preparation of the biosensor 1, a substrate having a conductive portion formed thereon was prepared, and the first layer of reagent was arranged under the ejection conditions of the second point group in <Step> 1, thereby preparing 7 types of biosensors (types 1 to 3, types 5 to 7, and type 9) having different positions of the reagent layer. The average height of the obtained reagent layer was about 2 μm (height distribution: 1 μm to 5 μm).

[0205] [Glucose current value measurement 3]

[0206] The biosensor prepared in Preparation of Biosensor 3 was used, and the measurement was performed in the same manner as in Glucose Current Value Measurement 1 except that a sample having the following glucose (GLU) concentration was used as a sample and the current value after 4.0 seconds had passed was measured.

[0207] <Evaluation conditions>

[0208] Sample used: Whole blood sample

[0209] GLU concentration (mg / dl): 0, 45, 330, 800 and 1500 (all Hct 42%)

[0210] Number of evaluations: n = 10 (for each GLU concentration)

[0211] As a result, when the average height of the reagent layer is about 2 μm (less than 4 μm), the reagent in the reagent layer diffuses due to the sample, and sufficient reagent required for the measurement is no longer present on the working electrode. As a result, there is a problem of not being able to obtain a sufficient measurement current value in a sample with a high glucose concentration (for example, 800 mg / dl), and a problem of the current value increasing depending on the situation, resulting in unstable measurement and deterioration of measurement performance.

Claims

1. 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, which is placed on the working electrode and the counter electrode; and a flow path for allowing the sample to flow from the sample supply port to the reagent layer, The reagent layer is formed to cover the entire area of ​​the working electrode in the flow path, extends at least 50 μm from the edges of the upstream and downstream sides of the working electrode, and is formed to cover an area of ​​the counter electrode in the flow path that is equivalent to at least 70% of the area of ​​the working electrode in the flow path, The average height of the reagent layer is 4 μm to 12 μm.

2. 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, which is placed on the working electrode and the counter electrode; and a flow path for allowing the sample to flow from the 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 extends from the edge of the upstream side of the working electrode to the upstream side, and extends from the edge of the downstream side of the working electrode to the downstream side, and the area extending to the upstream side and the area extending to the downstream side are respectively at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, and the coverage area of ​​the reagent layer at the counter electrode is equivalent to at least 70% of the surface area of ​​the working electrode in the flow path, The average height of the reagent layer is 4 μm to 12 μm.

3. The biosensor according to claim 1 or 2, wherein: The area of ​​the reagent layer extending from the downstream edge of the working electrode is larger than the area of ​​the reagent layer extending from the upstream edge of the working electrode.

4. The biosensor according to claim 1 or 2, wherein: The working electrode and the counter electrode extend in a direction perpendicular to a supply direction of the sample in the flow path. The working electrode is formed adjacent to the counter electrode on the upstream side of the counter electrode.

5. The biosensor according to claim 1 or 2, wherein: The reagent layer extends from the edge of the working electrode within a range of 100 μm to less than 150 μm.

6. The biosensor according to claim 1 or 2, wherein: The reagent layer is formed by dot matrix printing.

7. A method for manufacturing a biosensor, the biosensor comprising a conductive portion and a reagent layer in a flow path, wherein: The method for manufacturing the biosensor comprises the following steps: preparing a substrate having the conductive portion including an electrode pair formed on one main surface thereof, the electrode pair including 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 comprises the following steps: The reagent liquid is dot-printed so that the entire area of ​​the working electrode in the flow path, an area of ​​at least 50 μm from the respective edges of the upstream and downstream sides of the working electrode, and an area of ​​the counter electrode in the flow path equivalent 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.

8. A method for manufacturing a biosensor, the biosensor comprising a conductive portion and a reagent layer in a flow path, wherein: The method for manufacturing the biosensor comprises the following steps: preparing a substrate having the conductive portion including an electrode pair formed on one main surface thereof, the electrode pair including 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 comprises the following steps: The reagent liquid is dot-printed so that 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 extends from the edge of the upstream side of the working electrode to the upstream side, and extends from the edge of the downstream side of the working electrode to the downstream side, the area extending to the upstream side and the area extending to the downstream side are respectively at least 12.5% ​​of the surface area of ​​the working electrode in the flow path, the coverage area of ​​the reagent layer at the counter electrode is equivalent to at least 70% of the surface area of ​​the working electrode in the flow path, and the average height of the reagent layer is 4μm to 12μm.

Images