JPWO2014064978A1 - Biosensor and manufacturing method thereof - Google Patents

Abstract

Provided is a biosensor capable of reliably supplying a liquid sample brought into contact with an introduction port from the front into a cavity. Since the notch 108 is connected to the introduction port 104a and is narrower than the introduction port 104a, the cavity 104 communicated with the outside by the introduction port 104a is formed even if the notch 108 connected to the introduction port 104a is formed. The liquid sample that has been maintained in a tubular shape and has entered the cavity 104 from the inlet 104a and the notch 108 is promoted to enter the cavity 104 by the hydrophilizing agent provided in the cover layer, and becomes a hole due to capillary action. It is sucked toward. Therefore, the liquid sample brought into contact with the introduction port 104a from the front can be reliably supplied to the cavity 104.

Description

  The present invention relates to a cavity formed by an inner surface of a slit and a main surface on the slit side of the first and second substrates by bonding the first and second substrates through a spacer formed with a slit. The present invention relates to a biosensor in which a liquid sample is introduced by capillary action and a manufacturing method thereof.

  As shown in an example of the conventional biosensor of FIG. 14, a bio having an electrode system including a working electrode 501, a counter electrode 502, and a detection electrode 503, and a reaction layer 504 including an enzyme that specifically reacts with a measurement target substance. Using the sensor 500, the reducing substance generated by the reaction between the measurement target substance contained in the liquid sample and the reaction layer 504 is oxidized by applying a voltage between the working electrode 501 and the counter electrode 502. A method for measuring a substance that quantifies a substance to be measured by measuring the oxidation current generated is known (see, for example, Patent Document 1).

  A biosensor 500 shown in FIG. 14 is a sensor for quantifying glucose contained in a liquid sample, for example, and an electrode layer 505 formed by providing an electrode on an insulating substrate such as polyethylene terephthalate or polyimide; A cover layer 507 and a spacer layer 506 disposed between the electrode layer 505 and the cover layer 507 are stacked. The spacer layer 506 is provided with a slit 506a for forming a cavity 508 to which a liquid sample is supplied, and a cover layer 507 is laminated and bonded to the electrode layer 505 with the spacer layer 506 interposed therebetween. Thus, the cavity 508 to which the liquid sample is supplied is formed by the inner surface of the slit 506a of the spacer layer 506 and the main surface of the electrode layer 505 and the cover layer 507 on the slit 506a side. Then, the liquid sample is supplied to the cavity 508 from the liquid sample inlet 509 formed in the slit 506 a by the edge of the tip portion of the electrode layer 505 by capillary action. The cover layer 507 has an air hole 507a that communicates with the terminal portion of the cavity 508 so that the liquid sample is smoothly supplied to the cavity 508 by capillary action.

  The electrode layer 505 is provided with a working electrode 501, a counter electrode 502, and a detection electrode 503, and an electrode pattern electrically connected to each of these electrodes 501 to 503 is provided, whereby an electrode system is provided in the electrode layer 505. Is formed. A reaction layer 504 is provided on the working electrode 501 and the counter electrode 502, and the working electrode 501, the counter electrode 502, and the detection electrode 503 are each exposed to a cavity 508 formed in the biosensor 500. Layer 505 is provided.

  Therefore, when the liquid sample is supplied from the inlet 509 to the cavity 508, the electrodes 501 to 503 exposed to the cavity 508 and the reaction layer 504 come into contact with the liquid sample, and the reaction layer 504 is dissolved in the liquid sample. Further, when the liquid sample comes into contact with the detection electrode 503, it is detected that the liquid sample is supplied to the cavity 508.

  The reaction layer 504 provided on the working electrode 501 and the counter electrode 502 includes, for example, glucose oxidase that specifically reacts with glucose contained in a liquid sample and potassium ferricyanide as a mediator (electron acceptor). It is. The ferricyanide ions produced by the dissolution of potassium ferricyanide in the liquid sample are reduced to ferrocyanide ions, which are reduced by the electrons released when glucose is oxidized to gluconolactone by reacting with glucose oxidase. Is done. Accordingly, when a liquid sample containing glucose is supplied to the cavity 508 formed in the biosensor 500 from the inlet 509, ferricyanide ions are reduced by electrons released by the oxidation of glucose. Ferrocyanide ions, which are reduced forms of ferricyanide ions, are generated in an amount corresponding to the concentration of glucose contained in the sample and oxidized by the enzymatic reaction.

  In the biosensor 500 configured as described above, the oxidation current obtained by oxidizing the reduced form of the mediator resulting from the enzyme reaction on the working electrode 501 has a magnitude depending on the glucose concentration in the liquid sample. By measuring this oxidation current, glucose contained in the liquid sample can be quantified.

  In addition, in the biosensor 500 of FIG. 14, the electrode layer 505 and the cover layer 507 forming the cavity 508 are supplied to the cavity 508 so that the liquid sample can be easily introduced into the cavity 508 from the introduction port 509. In the vicinity of the introduction port 509, the end portions viewed in plan view are pasted so as to be arranged at different positions. That is, the electrode layer 505 and the cover layer 507 have the same shape at the tip portion near the inlet 509 of the cavity 508, but the cover layer 507 and the spacer layer 506 protrude from the inlet 509 in the tip direction. The electrode layer 505 is shifted in the direction of the introduction port 509.

JP 2002-168821 A (paragraphs 0018 to 0045, FIG. 1, abstract, etc.)

  In the conventional biosensor 500 described above, the cover layer 507 and the spacer layer 506 are disposed so as to protrude in the direction of the introduction port 509 with respect to the electrode layer 505. Therefore, when the liquid sample is supplied to the cavity 508, it is necessary to bring the liquid sample into contact with the inlet 509 from a slightly diagonally downward direction toward the paper surface of FIG. Therefore, there is a demand for a technique that can reliably supply the liquid sample to the cavity 508 even if the liquid sample is brought into contact with the inlet 509 from the front.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a biosensor capable of reliably supplying a liquid sample brought into contact with the inlet from the front to the cavity and a method for manufacturing the biosensor. And

  In order to achieve the above-described object, a biosensor of the present invention includes a first substrate, a spacer formed with a slit and stacked on the first substrate, and a second substrate stacked on the spacer. A cavity formed by an inner surface of the slit and a main surface on the slit side of the first and second substrates and supplied with a liquid sample; and formed in the slit by communicating the cavity and the outside. In the biosensor comprising the liquid sample inlet and the air hole formed in one of the first and second substrates so as to communicate with the cavity, the first and second substrates The spacers are arranged with the end surfaces on the introduction port sides aligned, and each of the first and second substrates has a notch formed narrower than the introduction port, connected to the introduction port, Said Hydrophilizing agent is characterized by being provided and on the other of said major surface forming a cavity of the second substrate.

  In the invention configured as described above, the first and second substrates and the spacers are arranged with their end faces on the inlet side aligned, but one of the first and second substrates is connected to the inlet. A notch formed narrower than the introduction port is provided, and a hydrophilizing agent is provided on the main surface forming the other cavity of the first and second substrates. Therefore, when the liquid sample is brought into contact with the introduction port from the front, the liquid sample enters the cavity from the introduction port and the notch formed continuously therewith, but the hydrophilizing agent is formed on the main surface of the substrate facing the notch. Therefore, the penetration of the liquid sample from the introduction port into the cavity is promoted.

  In addition, a cutout connected to the introduction port is formed on one of the first and second substrates, but since the cutout is formed narrower than the introduction port, the side of the cutout viewed from the introduction port is The substrate is maintained in a tubular shape by being sandwiched between the main surface of the portion of the substrate on which the notch is formed, the side of the notch and continuing to the introduction port, and the main surface of the other substrate facing the main surface. Therefore, the liquid sample that has entered the cavity from the inlet and the notch is promoted to enter the cavity by the hydrophilizing agent, and is supplied into the cavity by capillary action through the tubular portion on the side of the notch. .

  That is, since the notch is formed to be narrower than the introduction port so as to be continuous with the introduction port, the cavity communicated with the outside by the introduction port is maintained in a tubular shape even if the notch continuing to the introduction port is formed. The liquid sample that has entered the cavity from the inlet and the cutout is sucked toward the air hole by capillary action while being promoted to enter the cavity by the hydrophilizing agent. Therefore, the liquid sample brought into contact with the introduction port from the front can be reliably supplied to the cavity.

  The notch is formed in the first substrate, the air holes are formed in the second substrate, and the hydrophilizing agent is provided on a main surface forming the cavity of the second substrate. An electrode layer formed by providing an electrode system including a working electrode and a counter electrode on a main surface of the first substrate on the slit side, and the slit is disposed on one end side of the working electrode and the counter electrode. The spacer is formed by laminating the spacer on the first substrate, and the second substrate is disposed on the end of the cavity opposite to the introduction port. It is preferable to further include a cover layer formed by laminating and a reaction layer provided on one end side of the working electrode and the counter electrode exposed to the cavity.

  With this configuration, a reducing substance is generated by an enzyme that specifically reacts with a specific measurement target substance contained in the reaction layer reacts with the measurement target substance contained in the liquid sample supplied to the cavity. Is done. Therefore, by measuring the oxidation current obtained when the generated reducing substance is oxidized by applying a voltage between the working electrode and the counter electrode, the substance to be measured can be quantified. A biosensor having a configuration can be provided.

  Moreover, it is good for the said notch to be formed in the center position in the width direction of the said inlet.

  If comprised in this way, since the liquid sample introduced from the inlet and the notch can penetrate | invade in a cavity along the both sides of a notch, a liquid sample can be efficiently supplied to a cavity.

  Further, the biosensor manufacturing method of the present invention includes a first substrate, a spacer in which a slit is formed and stacked on the first substrate, a second substrate stacked on the spacer, and the slit. The cavity formed by the inner surface and the slit-side main surface of the first and second substrates and supplied with the liquid sample, and the liquid formed in the slit by communicating the cavity and the outside In a biosensor manufacturing method including a sample introduction port and an air hole formed in the second substrate so as to communicate with the cavity, a preparation step of preparing the first substrate provided with a through hole And a first laminating step of laminating the spacer formed with the slit wider than the through-hole on the first substrate so that the through-hole is disposed inside the slit in a plan view; , The second substrate having a hydrophilizing agent provided on the main surface forming a tee is laminated on the spacer so that the air hole is disposed at a position inside the slit and different from the through hole. A second laminating step; and a cutting step of cutting the laminated body of the first and second substrates and the spacer along a preset cutting line into a predetermined shape. The introduction port is set at a position passing through the through hole and the slit, and is formed at an end edge of the slit after cutting, and is narrower than the introduction port by being connected to the introduction port by the through hole after cutting. It is characterized in that a notch is formed.

  In the invention configured as described above, a first substrate provided with a through hole is prepared, and a spacer in which a slit wider than the through hole is formed is arranged inside the slit in a plan view. As described above, the second substrate, which is laminated on the first substrate and provided with the hydrophilizing agent on the main surface forming the cavity, has an air hole at a position different from the through hole inside the slit. Is laminated on the spacer. Then, the laminate of the first and second substrates and the spacer is cut into a predetermined shape by cutting along a preset cutting line at a position passing through the through hole and the slit, so that the slit after cutting. An introduction port is formed at the end edge of the slab, and a cutout having a width narrower than that of the introduction port is formed by the through hole after cutting. Therefore, for example, the first and second substrates in the aggregate substrate state and the laminated body of the spacers are cut together to provide a notch formed narrower than the introduction port. Since a plurality of biosensors can be efficiently manufactured collectively, the productivity is high and the manufacturing cost of the biosensor can be reduced.

  According to the present invention, the notch is formed so as to be narrower than the introduction port connected to the introduction port that communicates the cavity and the outside. Therefore, even if the notch that is connected to the introduction port is formed, The cavity that communicates with the liquid is maintained in a tubular shape, and the liquid sample that has entered the cavity from the inlet and the notch is sucked toward the air hole by capillary action while being promoted to enter the cavity by the hydrophilizing agent. The Therefore, the liquid sample brought into contact with the introduction port from the front can be reliably supplied to the cavity.

It is a top view of the insulating substrate in a certain manufacturing process of the biosensor concerning one Embodiment of this invention. It is a top view which shows the state in which the conductive layer was provided over the whole one main surface of the insulating board | substrate in the different manufacturing process of the biosensor concerning one Embodiment of this invention. It is a top view which shows the state by which the through-hole was penetrated by the predetermined position of the insulating board | substrate in the further different manufacturing process of the biosensor concerning one Embodiment of this invention. It is a top view which shows the state by which the cut was formed in the conductive layer in the further different manufacturing process of the biosensor concerning one Embodiment of this invention. It is a principal part enlarged view of FIG. It is a top view which shows the state by which the spacer layer was laminated | stacked on the electrode layer in the further different manufacturing process of the biosensor concerning one Embodiment of this invention. It is a principal part enlarged view of FIG. It is a top view which shows the state by which the reaction layer was provided in the cavity in the further different manufacturing process of the biosensor concerning one Embodiment of this invention. It is a principal part enlarged view of FIG. It is a top view which shows the state punched by the aggregate | assembly of several biosensors. It is a top view which shows the biosensor concerning one Embodiment of this invention. It is a figure for demonstrating the supply method of the liquid sample to a cavity, Comprising: It is a figure which shows the state before a liquid sample is supplied in a cavity. It is a figure for demonstrating the supply method of the liquid sample to a cavity, Comprising: It is a figure which shows the state by which the liquid sample was supplied in the cavity. It is a figure which shows the conventional biosensor.

  A configuration of a biosensor according to an embodiment of the present invention and a manufacturing method thereof will be described with reference to FIGS.

  1 to 11 are diagrams showing a method of manufacturing a biosensor according to an embodiment of the present invention, in which FIG. 1 is a plan view of an insulating substrate, and FIG. 2 is an entire surface of one main surface of the insulating substrate. 3 is a plan view showing a state in which a conductive layer is provided, FIG. 3 is a plan view showing a state in which a through hole is provided at a predetermined position of the insulating substrate, and FIG. 4 is a view in which a cut is formed in the conductive layer. FIG. 5 is a plan view showing a state in which a spacer layer is stacked on the electrode layer, FIG. 6 is a plan view showing the main part of FIG. 5, and FIG. FIG. 9 is an enlarged view of a main part of FIG. 8, and FIG. 10 is a plan view showing a state in which a laminated body of insulating substrates is punched into an assembly of a plurality of biosensors. FIG. 11 is a plan view of the biosensor. 12 and 13 are diagrams for explaining a method of supplying a liquid sample to the cavity. FIG. 12 shows a state before the liquid sample is supplied into the cavity, and FIG. 13 shows a liquid in the cavity. It is a figure which shows the state in which the sample was supplied.

  In addition, the dotted line in FIGS. 1-3, FIG.4, FIG.6, FIG. 8 shows the cutting line CL at the time of dividing the biosensor 100 into pieces. 5, FIG. 7, and FIG. 9 are enlarged views of essential parts showing regions surrounded by the cutting line CL in FIG. 4, FIG. 6, and FIG. 12 and 13 are cross-sectional views taken along line AA in FIG.

  The biosensor 100 of the present invention has substantially the same configuration except for the conventional biosensor 500 described with reference to FIG. 14 and the structure in the vicinity of the inlet 104a, and the working electrode 101 and the counter electrode 102 are used for detection. It has an electrode system including the electrode 103 and a reaction layer 107 containing an enzyme that reacts with the substance to be measured, and is used by being attached to a measuring instrument (not shown). That is, a measurement target substance such as glucose contained in a liquid sample such as blood supplied to the cavity 104 provided on the distal end side of the biosensor 100 attached to the measuring instrument via the introduction port 104a, and the biosensor 100 By measuring the oxidation current obtained by applying a voltage between the working electrode 101 and the counter electrode 102 to oxidize the reducing substance produced by the reaction with the reaction layer 107 provided, a liquid sample is obtained. The measurement target substance contained in is quantified.

  That is, as shown in FIGS. 5, 7, 9, 11, 12, and 13, the biosensor 100 has insulating properties such as ceramic, glass, plastic, paper, biodegradable material, and polyethylene terephthalate. The electrode layer 110, the spacer layer 120, and the cover layer 130, which are made of a material, are formed by being laminated and bonded together with the end surfaces on the front end side where the liquid sample introduction port 104a is provided aligned. The In the electrode layer 110 (corresponding to the “first substrate” of the present invention), an electrode system including the working electrode 101, the counter electrode 102, and the detection electrode 103 is provided on the main surface on the slit 105 side. The cover layer 130 (corresponding to the “second substrate” of the present invention) has an air hole 106 communicating with the cavity 104, and a hydrophilizing agent is provided on the main surface forming the cavity 104. The spacer layer 120 (corresponding to the “spacer” of the present invention) is formed with a slit 105 for forming the cavity 104, and the electrode layer 110 and the electrode layer 110 so that the slit 105 is disposed on one end side of the working electrode 101 and the counter electrode 102. It is disposed between the cover layers 130.

  The cavity 104 is formed by the inner surface of the slit 105 and the main surface of the electrode layer 110 and the cover layer 130 on the slit 105 side, and is supplied with a liquid sample. The cavity 104 communicates with the outside. A liquid sample inlet 104 a is formed in the slit 105. The cover layer 130 is laminated on the spacer layer 120 so that the air hole 106 is disposed at the end of the cavity 104 opposite to the introduction port 104a. At the tip of the electrode layer 110, a notch 108 that is continuous with the introduction port 104a and is narrower than the introduction port 104 is provided. The notch 108 is formed at the center position in the width direction of the introduction port 104a. As shown in FIG. 9, a reaction layer 107 containing an enzyme that reacts with a substance to be measured such as glucose contained in a liquid sample is provided on one end side of the working electrode 101 and the counter electrode 102. Then, the biosensor 100 is attached to a measuring instrument (not shown) by being inserted into a predetermined insertion port of the measuring instrument 2 from the rear end side.

  In this embodiment, the electrode layer 110 is prepared as follows.

  That is, first, as shown in FIGS. 1 and 2, platinum, gold, palladium, or the like is formed on one main surface of an insulating substrate 111 made of an insulating material such as polyethylene terephthalate by screen printing or sputtering deposition. A conductive layer 112 made of a conductive material such as noble metal, carbon, copper, aluminum, titanium, ITO, or ZnO is formed. Next, as shown in FIG. 3, a through-hole 109 is formed on the cutting line CL at a position corresponding to the introduction port 104a of the biosensor 100 of the insulating substrate 111 (preparation step). Note that the conductive layer 112 may be formed after the through-hole 109 is formed in the insulating substrate 111.

  Subsequently, as shown in FIGS. 4 and 5, the conductive layer 112 is subjected to patterning by laser processing or photolithography to form a notch 113, whereby the working electrode 101, the counter electrode 102, and the detection electrode are formed. An electrode system including 103 is formed. Further, as shown in FIGS. 6 to 9, the working electrode 101, the counter electrode 102, and the detection electrode 103 are arranged so that one end sides thereof are exposed to the cavity 104. The other end of each of the working electrode 101, the counter electrode 102, and the detection electrode 103 is an edge of the electrode layer 110 on the side opposite to the introduction port 104a, and the edge of the electrode layer 110 on which the spacer layer 120 is not laminated. To be formed.

  As shown in FIGS. 1 to 4, the insulating substrate 111 is formed into an aggregate substrate state by forming a plurality of electrode systems constituting the plurality of biosensors 100 on one main surface thereof. Yes. Then, as will be described later, after the spacer layer 120 and the cover layer 130 are laminated on the insulating substrate 111 (electrode layer 120), it is cut along the cutting line CL and cut into a predetermined shape, thereby forming individual biosensors 110. It is divided into pieces.

  Next, the spacer layer 120 is stacked on the electrode layer 110 prepared as described above (first stacking step). As shown in FIGS. 6 and 7, the spacer layer 120 is formed of an insulating substrate 121 made of an insulating material such as polyethylene terephthalate, and the insulating substrate 121 has a slit 105 for forming the cavity 104. It is formed wider than the hole 109. In addition, the insulating substrate 121 includes a rear end portion of the biosensor 100 when separated into the biosensor 100, that is, a working electrode 101, a counter electrode 102, and a detection electrode 103, which are inserted into a measuring instrument. A position corresponding to the other end side of is cut out. The slit 105 is disposed on one end side of the working electrode 101, the counter electrode 102, and the detection electrode 103, and the through hole 109 is disposed on the inner side of the slit 105 in a plan view, so that the insulating substrate 121 (spacer layer 120). Is partially covered and laminated on one main surface of the insulating substrate 111 (electrode layer 110), whereby a cavity 104 to which a liquid sample is supplied is formed by the electrode layer 110 and the slit 105.

  Subsequently, after the cavity 104 formed by stacking the spacer layer 120 on the electrode layer 110 is cleaned with plasma, the reaction layer 107 is formed in the cavity 104. As the plasma used in the plasma cleaning process, various plasmas used in metal activation treatment by plasma such as oxygen plasma, nitrogen plasma, and argon plasma can be used. Plasma may be used.

  As shown in FIGS. 8 and 9, the reaction layer 107 is formed on one end side of the working electrode 101 and the counter electrode 102 and the detection electrode 103 exposed to the cavity 104 before the cover layer 130 is laminated on the spacer layer 120. It is formed by dropping a reagent containing a thickener such as carboxymethyl cellulose or gelatin, an enzyme, a mediator, an additive such as an amino acid or an organic acid. Further, in order to smoothly supply a liquid sample such as blood to the cavity 104, a hydrophilizing agent such as a surfactant or phospholipid is applied to the inner wall of the cavity 104.

  Enzymes include glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, glucose dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, hydroxybutyrate dehydrogenase, cholesterol esterase, creatininase, creatinase DNA polymerase or the like can be used, and various sensors can be formed by selecting these enzymes according to the substance to be measured.

  For example, glucose oxidase or glucose dehydrogenase can be used to form a glucose sensor that detects glucose in a liquid sample, and alcohol oxidase or alcohol dehydrogenase can be used to form an alcohol sensor that detects ethanol in a liquid sample. For example, a lactic acid sensor for detecting lactic acid in a liquid sample can be formed, and a total cholesterol sensor can be formed by using a mixture of cholesterol esterase and cholesterol oxidase.

  As the mediator, potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes, ruthenium complexes, and the like can be used.

  As the thickener, carboxymethyl cellulose, carboxyethyl cellulose, polyethyleneimine DEAE cellulose dimethylaminoethyl dextran, carrageenan sodium alginate, dextran and the like can be used. As the hydrophilizing agent, a surfactant such as Triton X100 (manufactured by Sigma Aldrich), Tween 20 (manufactured by Tokyo Chemical Industry Co., Ltd.), sodium bis (2-ethylhexyl) sulfosuccinate, and a phospholipid such as lecithin can be used.

  Further, a buffer such as phosphoric acid may be provided in order to reduce variation in the concentration of ions contained in the liquid sample.

  Next, after the reaction layer 107 is formed in the cavity 104, a cover layer 130 formed of a substrate made of an insulating material such as polyethylene terephthalate is stacked (second stacking step). As shown in FIGS. 10 and 11, the cover layer 130 is formed with an air hole 106 that communicates with the cavity 104 when laminated on the spacer layer 120. Further, the cover layer 130 covers the cavity 104 and is laminated on the spacer layer 120 so that the air holes 106 are arranged in positions different from the through holes 109 inside the slits 105 of the spacer layer 120 in plan view. Is done. The main surface of the cover layer 130 forming the cavity 104 is provided with a hydrophilizing agent such as a surfactant or phospholipid in order to smoothly supply a liquid sample such as blood to the cavity 104. .

  Then, an assembly (laminated body) of biosensors 110 formed by laminating the cover layer 130 is cut into the biosensor 100 having a predetermined shape by being cut along the cutting line CL (cutting process). ). Specifically, as shown in FIG. 10, the assembly of biosensors 110 is punched along the cutting line CL, so that a plurality of biosensors 100 are first connected in the lateral direction. Aggregates are formed. Then, as shown in FIG. 11, the assembly of biosensors 100 connected in the horizontal direction is cut along the cutting line CL and separated into individual pieces of biosensors 100. Then, the biosensor 100 is formed in which the introduction port 104a formed by the opening portion of the slit 105 formed in the tip surface portion of the spacer layer 120 is provided at the tip.

  That is, the cutting line CL is set at a position passing through the through hole 109 and the slit 105, and an introduction port 104 a that communicates the cavity 104 with the outside is formed at the edge of the slit 105 after cutting. A notch 108 that is narrower than the introduction port 104a is formed continuously with the introduction port 104a.

  In this embodiment, the biosensor 100 is formed for the purpose of quantifying glucose in blood, and FAD (flavin adenine dinucleotide) is used as an enzyme that specifically reacts with glucose as a measurement target substance. A mediator that contains GDH (glucose dehydrogenase) (hereinafter referred to as FAD-GDH), which is reduced as a result of the reaction between glucose, which is a measurement object, and FAD-GDH, and becomes a reducing substance The reaction layer 107 containing potassium ferricyanide is provided on one end side of the working electrode 101 and the counter electrode 102 exposed to the cavity 104.

  In the biosensor 100 configured as described above, a liquid sample made of blood is brought into contact with the introduction port 104a formed at the tip thereof, whereby the liquid sample is sucked toward the air hole 106 due to capillary action and is brought into the cavity 104. A liquid sample is supplied. Specifically, the electrode layer 110, the cover layer 130, and the spacer layer 120 are arranged with their end faces on the introduction port 104a side aligned. However, the electrode layer 110 includes a notch 108 that is continuous with the introduction port 104a and is narrower than the introduction port 104a, and a hydrophilizing agent is provided on the main surface of the cover layer 130 that forms the cavity 104. Therefore, as shown in FIG. 12, when the liquid sample is brought into contact with the inlet 104a from the front, in the region surrounded by the dotted line in the same figure, from the inlet 104a and the notch 108 formed continuously therewith. A liquid sample enters the cavity 104. At this time, since the hydrophilizing agent is provided on the main surface of the cover layer 130 facing the notch 108, the penetration of the liquid sample from the inlet 104a into the cavity 104 is promoted.

  In addition, a notch 108 that is continuous with the introduction port 104a is formed in the electrode layer 110. Since the notch 108 is formed narrower than the introduction port 104a, the side of the notch 108 viewed from the introduction port 104a is The electrode layer 110 in which the notch 108 is formed is maintained in a tubular shape by being sandwiched between the main surface of the portion that is lateral to the notch 108 and continuous with the introduction port 104a and the main surface of the cover layer 130 that faces the main surface. Has been. Therefore, as shown in FIG. 13, the liquid sample that has entered the cavity 104 from the inlet 104a and the notch 108 is promoted to enter the cavity 104 by the hydrophilizing agent provided in the cover layer 130, and the notch It is fed into the cavity 104 by capillary action along the side tubular portion of 108.

  Then, when the reaction layer 107 is dissolved in the liquid sample supplied to the cavity 104, electrons are released by an enzyme reaction between glucose as a measurement target substance in the liquid sample and FAD-GDH, and ferricia is emitted by the emitted electrons. The ferric ion is reduced to produce ferrocyanide ion as a reducing substance. Then, the reducing substance generated by the oxidation-reduction reaction caused by the reaction layer 107 dissolving in the liquid sample is electrochemically oxidized by applying a voltage between the working electrode 101 and the counter electrode 102 of the biosensor 100. Thus, by measuring the oxidation current flowing between the working electrode 101 and the counter electrode 102, glucose in the liquid sample is quantified in the measuring device.

  As described above, according to this embodiment, the notch 108 is continuous with the introduction port 104a and is formed to be narrower than the introduction port 104a. Therefore, even if the notch 108 connected to the introduction port 104a is formed, the cavity 104 communicated with the outside by the introduction port 104a is maintained in a tubular shape. Therefore, the liquid sample that has entered the cavity 104 from the inlet 104 a and the notch 108 is sucked toward the air hole 106 by capillary action while being promoted to enter the cavity 104 by the hydrophilizing agent provided in the cover layer 130. Is done. Therefore, the liquid sample brought into contact with the introduction port 104a from the front can be reliably supplied to the cavity 104.

  In addition, a reducing substance is generated when an enzyme that specifically reacts with a specific measurement target substance contained in the reaction layer 107 reacts with the measurement target substance contained in the liquid sample supplied to the cavity 104. Therefore, the measurement target substance can be quantified by measuring the oxidation current obtained by oxidizing the generated reducing substance by applying a voltage between the working electrode 101 and the counter electrode 102.

  Further, since the notch 108 is formed at the center position in the width direction of the inlet 104a, the liquid sample introduced from the inlet 104a and the notch 108 can enter the cavity 104 along both sides of the notch 108. . Therefore, the liquid sample can be efficiently supplied to the cavity 104.

  The biosensor 100 is manufactured as follows. That is, the electrode layer 110 provided with the through hole 109 is prepared, and the spacer layer 120 in which the slit 105 wider than the through hole 109 is formed is arranged so that the through hole 109 is disposed inside the slit 105 in a plan view. The electrode layer 110 is laminated. Further, a cover layer 130 provided with a hydrophilizing agent on the main surface forming the cavity 104 is laminated on the spacer so that the air holes 106 are arranged at positions different from the through holes 109 inside the slit 105. The

  Next, the laminated body of the electrode layer 110, the cover layer 130, and the spacer layer 120 is cut along a cutting line CL set in advance at a position passing through the through-hole 109 and the slit 105, thereby obtaining a biomaterial having a predetermined shape. The sensor 100 cuts out. Then, an introduction port 104a is formed at the edge of the slit 105 after cutting, and a notch 108 having a width narrower than that of the introduction port 104a is formed by the through-hole 109 after cutting so as to be connected to the introduction port 104a. Therefore, for example, the laminated body of the electrode layer 110 and the cover layer 130 in the aggregate substrate state and the spacer layer 120 is cut together to form a narrower width than the introduction port 104a continuously to the introduction port 104a. A plurality of biosensors 100 having the cutouts 108 can be efficiently manufactured collectively, so that productivity is high and the manufacturing cost of the biosensor 100 can be reduced.

  The present invention is not limited to the above-described embodiment, and various modifications other than those described above can be made without departing from the spirit of the present invention. For example, the notch 108 is covered with the cover layer 130 ( A hydrophilizing agent may be provided on the main surface of the electrode layer 110 (first substrate) where the cavity 104 is formed.

  Further, an ethanol sensor, a lactic acid sensor, or the like may be formed by changing a combination of an enzyme and a mediator included in the reaction layer 107 of the biosensor 100 described above. In addition, the reaction layer 107 does not necessarily include a mediator. In this case, an oxidation current generated by oxidation of a reducing substance such as hydrogen peroxide or a reduced form of the enzyme generated by an enzyme reaction of a measurement target substance such as glucose is generated. Just measure.

  In the above-described embodiment, the biosensor 100 is formed in a bipolar electrode structure having the working electrode 101 and the counter electrode 102. However, the biosensor 100 is formed in a tripolar electrode structure by further providing a reference electrode. May be. In this case, a predetermined potential based on the counter electrode 102 may be applied to the working electrode 101 in a state where the counter electrode 102 is grounded and a reference potential is applied to the reference electrode by the voltage output unit.

  In the above-described embodiment, by applying a predetermined voltage between the counter electrode 102 and the detection electrode 103, the current flowing between the counter electrode 102 and the detection electrode 103 is monitored, so that the liquid is supplied to the cavity 104. Although it is configured to detect that the sample has been supplied, the detection electrode 103 is not necessarily provided. In this case, by applying a predetermined voltage between the working electrode 101 and the counter electrode 102, the current flowing between the working electrode 101 and the counter electrode 102 is monitored to confirm that the liquid sample is supplied to the cavity 104. What is necessary is just to detect.

  Further, at least the cover layer 130 of the electrode layer 110, the spacer layer 120, and the cover layer 130 forming the biosensor 100 is formed of a transparent member so that it can be visually recognized that the liquid sample is supplied to the cavity 104. Is desirable.

  Further, the configuration of the electrode system and the like provided in the biosensor 100 is not limited to the above-described example, and the configuration may be changed as appropriate according to the measurement target substance.

  The present invention can be applied to various biosensors and methods for producing the biosensors.

DESCRIPTION OF SYMBOLS 100 Biosensor 101 Working electrode 102 Counter electrode 103 Detection electrode 104 Cavity 104a Inlet 105 Slit 106 Air hole 107 Reaction layer 108 Notch 109 Through-hole 110 Electrode layer (1st board | substrate)
120 Spacer layer (spacer)
130 Cover layer (second substrate)
CL cutting line

Claims (4)

A first substrate;
A spacer formed with a slit and stacked on the first substrate;
A second substrate stacked on the spacer;
A cavity that is formed by an inner surface of the slit and a main surface on the slit side of the first and second substrates to be supplied with a liquid sample;
An inlet for the liquid sample formed in the slit through the cavity and the outside;
In a biosensor comprising an air hole formed in one of the first and second substrates in communication with the cavity,
The first and second substrates and the spacers are arranged with their end faces on the inlet side aligned,
One of the first and second substrates is provided with a notch formed narrower than the introduction port and connected to the introduction port.
A biosensor characterized in that a hydrophilizing agent is provided on a main surface forming the other cavity of the first and second substrates. The notch is formed in the first substrate, the air holes are formed in the second substrate, and the hydrophilizing agent is provided on the main surface forming the cavity of the second substrate,
An electrode layer formed by providing an electrode system including a working electrode and a counter electrode on a main surface on the slit side of the first substrate;
A spacer layer formed by laminating the spacer on the first substrate such that the slit is disposed on one end side of the working electrode and the counter electrode;
A cover layer formed by laminating the second substrate on the spacer so that the air hole is disposed at an end of the cavity opposite to the introduction port;
The biosensor according to claim 1, further comprising: a reaction layer provided on one end side of the working electrode and the counter electrode exposed to the cavity.   The biosensor according to claim 1, wherein the notch is formed at a central position in the width direction of the introduction port. A first substrate;
A spacer formed with a slit and stacked on the first substrate;
A second substrate stacked on the spacer;
A cavity that is formed by an inner surface of the slit and a main surface on the slit side of the first and second substrates to be supplied with a liquid sample;
An inlet for the liquid sample formed in the slit through the cavity and the outside;
In the biosensor manufacturing method comprising the air hole formed in the second substrate so as to communicate with the cavity,
A preparation step of preparing the first substrate provided with a through hole;
A first laminating step of laminating the spacer formed with the slit wider than the through hole on the first substrate so that the through hole is disposed inside the slit in a plan view;
Laminating the second substrate provided with a hydrophilizing agent on the main surface forming the cavity on the spacer so that the air hole is arranged at a position different from the through hole inside the slit. A second laminating step,
A cutting step of cutting the first and second substrates and the spacer stack along a predetermined cutting line into a predetermined shape;
The cutting line is set at a position passing through the through hole and the slit, the introduction port is formed at an edge of the slit after cutting, and the introduction port is connected to the introduction port by the cut through hole. A method for producing a biosensor, characterized in that a narrower notch is formed.

Images