JP2013108791A - Method for manufacturing biosensor, and biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique capable of forming a reaction layer without segmenting enzyme and mediator to an end part of a cavity.SOLUTION: A reaction layer 106 is formed by mixing and gelatinizing gelatinizer 201 to be gelatinized by reaction with ions ionized from a predetermined ionic material and reagents 202, 203 in which the ionic material is dissolved in a cavity 103. Since the reaction layer 106 is gelatinized, a dispersion state of enzyme and mediator contained in the reaction layer 106 is fixed in the reaction layer 106 and a shape of the gelatinized reaction layer 106 is maintained, segmentation of the enzyme and the mediator to the end part of the cavity 103 can be prevented when drying the gelatinized reaction layer 106, and the reaction layer 106 in which the enzyme and the mediator are uniformly dispersed can be formed.

Description

  The present invention includes an electrode layer provided with an electrode system including a working electrode and a counter electrode, a spacer layer in which a slit for forming a cavity is formed and stacked on the electrode layer, and an air hole communicating with the cavity. The present invention relates to a biosensor manufacturing method and a biosensor comprising a cover layer laminated on a spacer layer and a reaction layer provided on a working electrode and a counter electrode.

  As shown in the conventional biosensor of FIG. 4, a bio having an electrode system including a working electrode 501 and a counter electrode 502 and a detection electrode 503, and a reaction layer (not shown) including an enzyme that specifically reacts with a measurement target substance. Oxidation obtained by applying a voltage between the working electrode 501 and the counter electrode 502 to oxidize the reducing substance generated by the reaction between the measurement target substance contained in the sample and the reaction layer using the sensor 500. A method for measuring a substance that quantifies a substance to be measured by measuring an electric current is known (see, for example, Patent Document 1).

  A biosensor 500 shown in FIG. 4 is a sensor for quantifying glucose contained in a sample, and includes an electrode layer 504 formed by providing an electrode on an insulating substrate such as polyethylene terephthalate or polyimide, and a cover layer. 506 and a spacer layer 505 disposed between the electrode layer 504 and the cover layer 506 are stacked. In addition, the spacer layer 505 is provided with a slit 505a for forming a cavity 507 to which a sample is supplied. The cover layer 506 is laminated and bonded to the electrode layer 504 with the spacer layer 505 interposed therebetween. The cavity 507 to which the sample is supplied is formed by the electrode layer 504, the slit 505a portion of the spacer layer 505, and the cover layer 506. Then, a sample is supplied to the cavity 507 from the sample inlet formed on the side surface of the biosensor 500 by the opening portion of the slit 505a. The cover layer 506 has an air hole 506a communicating with the terminal portion of the cavity 507 in order to smoothly supply the sample to the cavity 507 by capillary action.

  The electrode layer 504 is provided with a working electrode 501, a counter electrode 502, and a detection electrode 503, and electrode patterns 501a to 503a that are electrically connected to the electrodes 501 to 503, respectively, are provided on the electrode layer 504. An electrode system is formed. In addition, a reaction layer 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 507 formed in the biosensor 500. 504 is provided.

  Accordingly, when a liquid sample is supplied to the cavity 507 from the sample inlet, the electrodes 501 to 503 exposed to the cavity 507 and the reaction layer come into contact with the sample, and the reaction layer dissolves in the sample. Further, when the sample contacts the detection electrode 503, it is detected that the sample is supplied to the cavity 507.

  The reaction layer provided on the working electrode 501 and the counter electrode 502 contains glucose oxidase that specifically reacts with glucose contained in the sample and potassium ferricyanide as a mediator (electron acceptor). The ferricyanide ions produced by the dissolution of potassium ferricyanide in the sample are reduced to ferrocyanide ions, which are reduced, by electrons released when glucose is oxidized to gluconolactone by reacting with glucose oxidase. The Therefore, when a sample containing glucose is supplied to the cavity 507 formed in the biosensor 500 from the sample introduction port, ferricyanide ions are reduced by the 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 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 sample. By measuring the current, glucose contained in the sample can be quantified.

JP-A-8-320304 (paragraphs 0011 to 0017, FIG. 1, etc.)

  By the way, in general, the reaction layer is formed as follows. That is, the reaction layer is formed by dropping a reagent in which an enzyme or a mediator is dissolved into a cavity 507 formed by stacking the spacer layer 505 on the electrode layer 504. However, when the reaction layer is formed in this way, when the dropped reagent is dried in the cavity 507, there is a possibility that the enzyme or mediator contained in the reagent is segregated at the end of the cavity 507. When the enzyme or mediator is segregated at the end of the cavity 507 to form a reaction layer, when a sample such as blood is supplied to the cavity 507, the enzyme or mediator contained in the reaction layer is uniformly supplied to the sample. Therefore, the quantitative accuracy of the measurement target substance using the biosensor 500 deteriorates.

  This invention is made | formed in view of the said subject, and it aims at providing the technique which can form a reaction layer, without an enzyme and a mediator segregating to the edge part of a cavity.

  In order to achieve the above-described object, the biosensor manufacturing method of the present invention includes an electrode layer provided with an electrode system including a working electrode and a counter electrode on one surface of an insulating substrate, and a slit, and the slit Is disposed on the tip side of the working electrode and the counter electrode and is laminated on the one surface of the electrode layer, a cavity formed by the electrode layer and the slit and supplied with a sample, and a cavity A cover layer that is formed in a communicating air hole so as to cover the cavity and is laminated on the spacer layer, and an enzyme that is provided on the front end side of the working electrode and the counter electrode exposed to the cavity and reacts with the measurement target substance And a reaction layer containing a mediator in a method for producing a biosensor, a predetermined ionic substance reacts with ions resulting from ionization and gels That a gelling agent, and a reagent in which the ionic substance is dissolved, is characterized by forming said reaction layer by mixing in said cavity (claim 1).

  The gelling agent is a carrageenan or alginic acid compound (claim 2).

  The reaction layer includes a mediator layer forming step of mixing the reagent containing the mediator functioning as the ionic substance and the gelling agent in the cavity to form a mediator layer, the ionic substance and It is characterized by being formed by an enzyme layer forming step of forming the enzyme layer by mixing the reagent containing the enzyme and the gelling agent on the mediator layer (Claim 3).

  In addition, the reaction layer functions as the ionic substance, and an enzyme layer forming step in which the reagent containing the ionic substance and the enzyme and the gelling agent are mixed in the cavity to form an enzyme layer. It is formed by a mediator layer forming step of forming the mediator layer by mixing the reagent containing the mediator and the gelling agent on the enzyme layer (claim 4).

  Further, the ionic substance is any one of a ferricyanide compound, sodium chloride, potassium chloride and calcium chloride (Claim 5).

  Further, the biosensor of the present invention includes an electrode layer provided with an electrode system including a working electrode and a counter electrode on one surface of an insulating substrate, and a slit, and the slit is on the tip side of the working electrode and the counter electrode. A spacer layer disposed on the one surface of the electrode layer, a cavity formed by the electrode layer and the slit and supplied with a sample, and an air hole communicating with the cavity. And a reaction layer containing an enzyme and a mediator that are provided on the tip side of the working electrode and the counter electrode exposed to the cavity and react with the measurement target substance. In the sensor, the reaction layer includes a gelling agent that reacts with ions resulting from ionization of a predetermined ionic substance, and the ionic substance. There the reagent dissolved, is characterized in that it is formed by mixing in the cavity (claim 6).

  The reaction layer includes a mediator layer formed by mixing the reagent containing the mediator functioning as the ionic substance and the gelling agent; the reagent containing the ionic substance and the enzyme; and An enzyme layer formed by mixing with a gelling agent is provided (claim 7).

  According to the first and sixth aspects of the present invention, a predetermined ionic substance is ionized in the reaction layer provided on the tip side of the working electrode and the counter electrode exposed to the cavity and including the enzyme and the mediator that reacts with the measurement target substance. A gelling agent that reacts with ions generated by the gelation and a reagent in which an ionic substance is dissolved are mixed in the cavity to form a gel. Therefore, since the reaction layer is gelled, the dispersed state in the reaction layer of the enzyme and mediator contained in the reaction layer is fixed, and the shape of the reaction layer when gelled is maintained. When the gelled reaction layer is dried, the enzyme and mediator can be prevented from segregating at the end of the cavity, and a reaction layer in which the enzyme and mediator are uniformly dispersed can be formed. .

  In addition, since the enzyme and mediator are uniformly dispersed in the reaction layer, the enzyme and mediator are uniformly dissolved in the sample when the reaction layer dissolves in the sample supplied to the cavity. The quantitative accuracy of the target substance can be improved.

  Further, when forming the reaction layer, it is only necessary to individually drop the gelling agent and the reagent in which the ionic substance is dissolved into the cavity, and the reaction layer is substantially the same as the conventional method for forming the reaction layer. Is very practical.

  According to invention of Claim 2, a biosensor can be manufactured by a practical structure by comprising a gelatinizer with a carrageenan or an alginate compound.

  According to the third, fourth, and seventh aspects of the present invention, the reaction layer includes a mediator layer formed by mixing a reagent containing a mediator that functions as an ionic substance and a gelling agent, an ionic substance, and an enzyme. Since the enzyme layer formed by mixing the reagent and the gelling agent is separated, it prevents the enzyme contained in the reaction layer from reacting with the mediator in the storage state of the biosensor. be able to. Therefore, when the enzyme and mediator react in the storage state of the biosensor and the enzyme and mediator contained in the reaction layer decrease and the sample is supplied to the cavity, the measurement target substance contained in the sample and redox Since it can prevent that the quantity of the enzyme and mediator which react is reduced, it can suppress that the fixed_quantity | quantitative_assay precision of the measuring object substance using a biosensor deteriorates.

  According to the invention of claim 5, the gelling agent can be surely gelled by the cation generated by the ionization of the ionic substance comprising any of the ferricyanide compound, sodium chloride, potassium chloride and calcium chloride. So it is practical.

  In addition, when a ferricyanide compound is used as an ionic substance that gels the gelling agent, the ferricyanide compound functions as a mediator, so the mediator and the ionic substance can be composed of the same substance. The manufacturing cost of the biosensor can be reduced.

  In addition, when calcium oxide is employed as an ionic substance for gelling the gelling agent, the gelling agent can be reliably gelled by divalent cations due to the ionization of calcium oxide to the sample. is there.

It is a figure which shows the biosensor concerning one Embodiment of this invention, Comprising: (a) is a disassembled perspective view, (b) is a perspective view. It is a cross-sectional view of the cavity part of the biosensor of FIG. It is a figure which shows the manufacturing method of the biosensor concerning one Embodiment of this invention, Comprising: (a)-(e) shows a different process, respectively. It is a figure which shows the conventional biosensor.

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

  1A and 1B show a biosensor according to an embodiment of the present invention, in which FIG. 1A is an exploded perspective view and FIG. 1B is a perspective view. FIG. 2 is a cross-sectional view of the cavity portion of the biosensor of FIG. FIG. 3 is a diagram showing a method of manufacturing a biosensor according to an embodiment of the present invention, and (a) to (e) show different processes.

  The biosensor 100 of the present invention includes an electrode system including a working electrode 101 and a counter electrode 102, and a reaction layer 106 including an enzyme that reacts with a mediator and a substance to be measured, and is used by being attached to a measuring instrument (not shown). It is what is done. That is, a substance to be measured such as glucose contained in a sample such as blood supplied to a cavity 103 provided on the tip side of the biosensor 100 attached to the measuring instrument, and a reaction layer 106 provided on 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 of the reaction, the quantitative determination of the measurement target substance contained in the sample Is done.

  That is, as shown in FIGS. 1 and 2, the biosensor 100 includes a working electrode 101 and a counter electrode 102 formed of an insulating material such as ceramic, glass, plastic, paper, biodegradable material, and polyethylene terephthalate, respectively. An electrode layer 110 provided with an electrode system including a cover layer 130 in which an air hole 105 communicating with the cavity 103 is formed, and a slit 104 for forming the cavity 103 is formed, and the electrode layer 110 and the cover layer 130 are formed. The spacer layer 120 is sandwiched between the two layers, and the spacer layer 120 is formed by being laminated and bonded in a state where the tip side where the sample introduction port 103a is provided is aligned. A reaction layer 106 containing an enzyme that reacts with a measurement target substance such as glucose contained in a sample is provided on the working electrode 101 and the counter electrode 102. Then, the biosensor 100 is attached to the measuring device 2 by being inserted and attached to a predetermined insertion port of the measuring device 2 from the rear end side.

  In this embodiment, the electrode layer 110 is formed of an insulating substrate made of an insulating material such as polyethylene terephthalate. In addition, a conductive layer made of a noble metal such as platinum, gold, palladium, or a conductive material such as carbon, copper, aluminum, titanium, ITO, or ZnO is formed on one surface of the insulating substrate that forms the electrode layer 110 by screen printing or It is formed by sputtering vapor deposition. Then, the conductive layer formed on one surface of the insulating substrate is subjected to patterning by laser processing or photolithography, so that the working electrode 101 and the counter electrode 102 and the biosensor sensor chip 100 are mounted on the measuring instrument. Then, an electrode system including electrode patterns 101a and 102a that electrically connect each of the working electrode 101 and the counter electrode 102 to the measuring instrument is formed.

  In addition, the working electrode 101 and the counter electrode 102 are arranged so that their respective distal end sides are exposed to the cavity 103. In addition, electrode patterns 101a and 102a on the rear end side of each of the working electrode 101 and the counter electrode 102 are edges of the electrode layer 110 on the opposite side to the sample introduction port 103a, and the electrode layer 110 on which the spacer layer 120 is not stacked. It is formed by extending to the edge.

  Next, the spacer layer 120 is laminated on the electrode layer 110 formed as described above. The spacer layer 120 is formed of a substrate made of an insulating material such as polyethylene terephthalate, and a slit 104 for forming the cavity 103 is formed at substantially the center of the front end edge of the substrate. Then, the slit 104 is disposed on the distal end side of the working electrode 101 and the counter electrode 102, and the spacer layer 120 is partially covered and laminated on one surface of the electrode layer 110, so that the electrode layer 110 and the slit 104 are stacked. A cavity 103 to which a sample is supplied is formed.

  Subsequently, after the cavity 103 formed by stacking the spacer layer 120 on the electrode layer 110 is cleaned with plasma, the reaction layer 106 is formed. 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. 3A to 3E, the reaction layer 106 is formed on the tip side of the working electrode 101 and the counter electrode 102 exposed to the cavity 103 before the cover layer 130 is laminated on the spacer layer 120. Gelling agent 201 that gels by reacting with ions resulting from ionization of the ionic substance, ionic substance that gels the gelling agent, thickeners such as carboxymethylcellulose and gelatin, enzymes, mediators, amino acids, It is formed by dropping and mixing reagents 202 and 203 containing an additive such as an organic acid. In addition, a hydrophilic agent such as a surfactant or phospholipid is applied to the inner wall of the cavity 104 in order to smoothly supply a sample such as blood to the cavity 104.

  Specifically, the reaction layer 106 includes a mediator layer 107 formed by mixing a reagent 202 containing at least a mediator functioning as an ionic substance and a gelling agent 201, and a reagent 203 containing at least an ionic substance and an enzyme. And the enzyme layer 108 formed by mixing the gelling agent 201 and formed as follows.

  That is, as shown in FIG. 3A, a predetermined amount of gelling agent 201 containing carrageenan or alginic acid compound is dropped from the dropping device 200 into the cavity 103. Next, as shown in FIG. 3 (b), a reagent 202 that functions as an ionic substance that gels the gelling agent 201, for example, at least a mediator made of a ferricyanide compound is transferred from the dropping device 200 to the cavity 103. As a result, the gelling agent 201 and the reagent 202 are mixed in the cavity 103 to form the mediator layer 107 (mediator layer forming step).

  Subsequently, as shown in FIG. 3C, a predetermined amount of the gelling agent 201 is dropped from the dropping device 200 into the cavity 103. As shown in FIG. 3 (d), a reagent 203 containing at least an ionic substance such as sodium chloride, potassium chloride, or calcium chloride and an enzyme is dropped from the dropping device 200 into the cavity 103 by a predetermined amount. The reaction layer 106 is formed by mixing the agent 201 and the reagent 203 on the mediator layer 107 to form the enzyme layer 108 (FIG. 3E, enzyme layer forming step).

  In the above example, the enzyme layer 108 is formed on the mediator layer 107 after the mediator layer 107 is formed on the cavity 103, but the enzyme layer 108 is formed on the enzyme layer 108 after the enzyme layer 108 is formed on the cavity 103. A mediator layer 107 may be formed. In the above example, after the gelling agent 201 is dropped into the cavity 103, the reagents 202 and 203 containing the ionic substance that gels the gelling agent 201 are dropped into the cavity 103. The gelling agent 201 may be dropped into the cavity 103 after the 202 and 203 containing the liquid are dropped into the cavity 103.

  Examples of 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, and 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 blood sample, and alcohol oxidase or alcohol dehydrogenase can be used to form an alcohol sensor that detects ethanol in a blood sample. For example, a lactic acid sensor for detecting lactic acid in a blood 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, methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, polyethyleneimine DEAE cellulose, dimethylaminoethyl dextran, 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 buffering agent such as phosphoric acid may be provided in order to reduce variation in the concentration of ions contained in the sample.

  Next, after the reaction layer 106 is formed in the cavity 103, the cover layer 130 formed of a substrate made of an insulating material such as polyethylene terephthalate is laminated on the spacer layer 120, whereby the biosensor 100 is formed. The As shown in FIGS. 1A and 1B, the cover layer 130 has an air hole 105 that communicates with the cavity 103 when laminated on the spacer layer 120, and the cover layer 130 has the cavity 103. And is laminated on the spacer layer 120.

  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. Is a mediator that is reduced by electrons generated by the reaction between glucose and FAD-GDH, which is a measurement object, and becomes a reducing substance, including GDH (glucose dehydrogenase) (hereinafter referred to as FAD-GDH). The reaction layer 106 containing potassium ferricyanide is provided on the tip side of the working electrode 101 and the counter electrode 102 exposed to the cavity 103.

  In the biosensor 100 configured as described above, a sample made of blood is brought into contact with the sample introduction port 103a at the tip, whereby the sample is sucked toward the air hole 105 by a capillary phenomenon and supplied to the cavity 103. . Then, when the reaction layer 106 is dissolved in the sample supplied to the cavity 103, electrons are released by an enzyme reaction between glucose as a measurement target substance in the sample and FAD-GDH, and ferricyanization is performed by the emitted electrons. The ions are reduced to produce ferrocyanide ions, which are reducing substances. Then, by applying a voltage between the working electrode 101 and the counter electrode 102 of the biosensor 100 to electrochemically oxidize the reducing substance generated by the redox reaction caused by the reaction layer 106 being dissolved in the sample. By measuring the oxidation current flowing between the working electrode 101 and the counter electrode 102, glucose in the sample is quantified in the measuring device.

  As described above, according to this embodiment, the reaction layer 106 provided on the tip side of the working electrode 101 and the counter electrode 102 exposed to the cavity 103 and including an enzyme and a mediator that reacts with the measurement target substance has a predetermined ionicity. A gelling agent 201 that gels by reacting with ions resulting from ionization of the substance and reagents 202 and 203 in which the ionic substance is dissolved are gelled by mixing in the cavity 103.

  Therefore, since the reaction layer 106 is gelled, the dispersion state of the enzymes and mediators contained in the reaction layer 106 in the reaction layer 106 is fixed, and the shape of the reaction layer 106 when gelled is maintained. Therefore, for example, when the gelled reaction layer 106 is dried, the enzyme and mediator can be prevented from segregating at the end of the cavity 103, and the reaction layer in a state where the enzyme and mediator are uniformly dispersed can be prevented. 106 can be formed.

  In addition, since the enzyme and the mediator are uniformly dispersed in the reaction layer 106, when the reaction layer 106 is dissolved in the sample supplied to the cavity 103, the enzyme and the mediator are uniformly dissolved in the sample. It is possible to improve the accuracy of quantification of the measurement target substance using.

  In addition, it is not necessary to drop the highly viscous reagents 202 and 203 into the cavity 103 in order to prevent segregation of the enzyme and mediator in the cavity 103. Therefore, by dropping the low-viscosity reagents 202 and 203 into the cavity 103, variation in the amount of the reagent 202 and 203 dropped into the cavity 103 can be reduced, so that the amount of enzyme and mediator included in the reaction layer 106 However, it is possible to reduce the variation among the biosensors 100. Therefore, the quantitative accuracy of the measurement target substance using the biosensor 100 can be improved.

  Further, when forming the reaction layer 106, it is only necessary to individually drop the gelling agent and the reagent in which the ionic substance is dissolved into the cavity 103, which is almost the same as the conventional method for forming the reaction layer 106. Since the reaction layer 106 can be formed, it is very practical.

  Moreover, a biosensor can be manufactured with a practical configuration by configuring a gelling agent with a carrageenan or alginic acid compound.

  The reaction layer 106 includes a mediator layer 107 formed by mixing a reagent 202 containing a mediator functioning as an ionic substance and a gelling agent 201, a reagent 203 containing an ionic substance and an enzyme, and a gelling agent 201. Is formed in a state in which the enzyme layer 108 formed by mixing is separated from the enzyme layer 108, so that the enzyme contained in the reaction layer 106 and the mediator can be prevented from reacting in the storage state of the biosensor 100. it can. Accordingly, when the biosensor 100 is stored, when the enzyme and mediator react to reduce the enzyme and mediator contained in the reaction layer 106 and the sample is supplied to the cavity 103, the reaction layer 106 converts the sample into the sample. Since it can prevent that the quantity of the enzyme and mediator which carries out an oxidation reduction reaction with the to-be-measured substance contained is reduced, it can suppress that the fixed_quantity | quantitative_assay precision of the to-be-measured substance using the biosensor 100 deteriorates.

  Further, the gelling agent can be reliably gelled by a cation resulting from ionization of an ionic substance composed of any of a ferricyanide compound, sodium chloride, potassium chloride and calcium chloride, which is practical.

  In addition, when a ferricyanide compound is used as an ionic substance that gels the gelling agent, the ferricyanide compound functions as a mediator, so the mediator and the ionic substance can be composed of the same substance. In addition, the manufacturing cost of the biosensor 100 can be reduced.

  In addition, when calcium oxide is employed as an ionic substance for gelling the gelling agent, the gelling agent can be reliably gelled by divalent cations due to the ionization of calcium oxide to the sample. is there.

  Note that 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 thereof. For example, the reaction of the above-described biosensor 100 is possible. 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 layer 106.

  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, a blood sample is placed in the cavity 103 by monitoring a current flowing between the working electrode 101 and the counter electrode 102 by applying a predetermined voltage between the working electrode 101 and the counter electrode 102. Although it is detected that the sample has been supplied, a detection electrode for detecting that the sample has been supplied to the cavity 103 may be further provided. In this case, by applying a predetermined voltage between the counter electrode 102 and the detection electrode, the current flowing between the counter electrode 102 and the detection electrode is monitored to detect that the sample is supplied to the cavity 103. do it.

  In addition, of the electrode layer 110, the spacer layer 120, and the cover layer 130 that form the biosensor 100, at least the cover layer 130 is formed of a transparent member so that it can be visually confirmed that the blood sample has been supplied to the cavity 103. Is desirable.

  The present invention can also be applied to various biosensor manufacturing methods and biosensors.

DESCRIPTION OF SYMBOLS 100 Biosensor 101 Working electrode 102 Counter electrode 103 Cavity 104 Slit 105 Air hole 106 Reaction layer 107 Mediator layer 108 Enzyme layer 110 Electrode layer 120 Spacer layer 130 Cover layer 201 Gelling agent 202, 203 Reagent

Claims (7)

An electrode layer provided with an electrode system including a working electrode and a counter electrode on one surface of an insulating substrate;
A spacer layer formed on the one surface of the electrode layer, the slit being formed on the tip side of the working electrode and the counter electrode;
A cavity formed by the electrode layer and the slit and supplied with a sample;
An air hole communicating with the cavity to cover the cavity and be laminated on the spacer layer;
In a method for producing a biosensor comprising a reaction layer provided on a tip side of the working electrode and the counter electrode exposed to the cavity, and comprising an enzyme that reacts with a measurement target substance and a mediator,
A gelling agent that reacts with ions resulting from ionization of a predetermined ionic substance and a reagent in which the ionic substance is dissolved are mixed in the cavity to form the reaction layer. A method for manufacturing a biosensor.   The method for producing a biosensor according to claim 1, wherein the gelling agent is a carrageenan or alginic acid compound. The reaction layer is
A mediator layer forming step of forming a mediator layer by mixing the reagent containing the mediator functioning as the ionic substance and the gelling agent in the cavity;
3. The enzyme layer forming step of forming the enzyme layer by mixing the reagent containing the ionic substance and the enzyme and the gelling agent on the mediator layer. A method for producing the biosensor as described in 1. The reaction layer is
An enzyme layer forming step of forming the enzyme layer by mixing the reagent containing the ionic substance and the enzyme and the gelling agent in the cavity;
The mediator layer forming step of forming the mediator layer by mixing the reagent containing the mediator functioning as the ionic substance and the gelling agent on the enzyme layer. Or the manufacturing method of the biosensor of 2.   5. The method for producing a biosensor according to claim 1, wherein the ionic substance is any one of a ferricyanide compound, sodium chloride, potassium chloride, and calcium chloride. An electrode layer provided with an electrode system including a working electrode and a counter electrode on one surface of an insulating substrate;
A spacer layer formed on the one surface of the electrode layer, the slit being formed on the tip side of the working electrode and the counter electrode;
A cavity formed by the electrode layer and the slit and supplied with a sample;
An air hole communicating with the cavity to cover the cavity and be laminated on the spacer layer;
In a biosensor comprising a reaction layer provided on a tip side of the working electrode and the counter electrode exposed to the cavity, and containing an enzyme and a mediator that reacts with a measurement target substance,
The reaction layer is
A gelling agent that gels by reacting with ions generated by ionization of a predetermined ionic substance and a reagent in which the ionic substance is dissolved are mixed and formed in the cavity. Biosensor. The reaction layer is
A mediator layer formed by mixing the reagent containing the mediator functioning as the ionic substance and the gelling agent;
The biosensor according to claim 6, further comprising: an enzyme layer formed by mixing the reagent containing the ionic substance and the enzyme and the gelling agent.

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