JP6823420B2 - Biosensor - Google Patents

Description

The present invention relates to a disposable biosensor.

Conventional disposable biosensors are used in which a reagent containing an enzyme and a mediator is applied to an electrode system on a substrate, and a spacer and a cover form a capillary for drawing in a liquid sample (for example, Patent Document 1). ). By applying a voltage to the biosensor, the concentration of the substance to be measured in the sample can be measured by the diffusion current generated by the mediator.

Japanese Patent No. 2901678

In a disposable biosensor, for example, a glucose sensor, a sample (liquid sample) drawn into a capillary dissolves and diffuses a reagent, and detects a current generated by an enzymatic reaction as a response current. Therefore, the sensitivity of the glucose sensor is affected by the diffusion status of the substance in the reagent. The size (particularly height) and volume of the capillary are specified so that the diffusion status of the reagent by the sample does not vary among the sensors.

However, the formation of capillaries requires parts such as spacers and covers to be laminated on the substrate, and the presence of these parts is a factor that increases the manufacturing cost of the biosensor. Further, in a biosensor having a capillary, the sample is brought close to the suction port of the capillary and the sample is introduced into the capillary. However, depending on how the user handles the biosensor, an appropriate amount of the sample may not be introduced into the capillary, and the concentration of the substance to be measured in the sample may not be measured appropriately.

An object of the present invention is to provide a biosensor capable of facilitating operation at the time of measurement while reducing the manufacturing cost.

One aspect of the present invention is a detection layer containing a plurality of electrodes including an working electrode and an enzyme fixed on the working electrode and transferring electrons between the cross-linking agent, the conductive polymer, and the working electrode. It is a biosensor including the above-mentioned working electrode, which is characterized in that it is an open system.

The biosensor according to the present invention preferably adopts a configuration in which the contact area of the detection layer with the working electrode is defined by a predetermined area.

According to the present invention, it is possible to provide a biosensor that can be easily operated at the time of measurement while reducing the manufacturing cost.

FIG. 1 is a diagram showing a configuration example of a biosensor according to an embodiment. FIG. 2 is a diagram showing a configuration example of the measuring device. FIG. 3 is a flowchart showing a processing example in the measuring device. FIG. 4 is a graph showing a calibration curve based on the result of test 1 and the measurement result of glucose concentration using the chronoamperometry method. FIG. 5 is a graph showing the result of Test 2, that is, the calibration curve based on the measurement result of glucose concentration by the chronoamperometry method. FIG. 6 is a graph showing the S / B (sample / blank) ratio based on the measurement result of the glucose concentration using the biosensor of Example 1. FIG. 7 is a graph showing the S / B ratio based on the measurement results by the CV method and the CA method using the biosensor of Comparative Example 1. FIG. 8 is a diagram showing a configuration example of the biosensor according to the second embodiment.

Hereinafter, the enzyme electrode according to the embodiment of the present invention will be described with reference to the drawings. The configurations of the embodiments described below are examples, and the present invention is not limited to the configurations of the embodiments.

[First Embodiment]
<Biosensor configuration>
FIG. 1 is a diagram schematically showing a configuration example of a disposable biosensor according to an embodiment. In FIG. 1, the biosensor 1 includes an insulating substrate 2, electrodes 3 and 4, an insulating layer 5, and a reagent layer (hereinafter, referred to as “detection layer”) 6. The biosensor 1 does not have a capillary (sometimes without a cover) formed by a spacer or a cover. That is, at least the working electrode is an open system.

<< Insulation Substrate >>
The insulating substrate 2 is formed in a rectangular flat plate shape having a length direction and a width direction. The insulating substrate 2 is a thermoplastic resin such as polyetherimide (PEI), polyethylene terephthalate (PET), polyethylene (PE), a polyimide resin, various resins (plastics) such as epoxy resin, glass, ceramics, and paper. It can be formed of an insulating material such as.

<< Electrode >>
The electrodes 3 and 4 are formed on one side 2a of the insulating substrate 2. The electrode 3 has one end 3a and the other end 3b, and the other end 3b extends in the width direction of the insulating substrate 2 and is used as a working electrode. The one end portion 3a is used as a lead portion (electrode extraction portion). The electrode 4 has one end 4a and the other end 4b, and the other end 4b is provided with a predetermined gap from the other end 3b (working electrode) in the width direction of the insulating substrate 2. It extends to and is used as a counter electrode. One end portion 4a is also used as a lead portion (electrode extraction portion).

Each of the electrodes 3 and 4 is formed using, for example, a metal material such as gold (Au), platinum (Pt), silver (Ag), palladium, or a carbon material such as carbon. For example, electrode 3,
No. 4 can be formed as a metal layer having a desired thickness by forming a metal material by physical vapor deposition (PVD, for example, sputtering) or chemical vapor deposition (CVD). Alternatively, the electrodes 3 and 4 can be formed by printing an ink containing a carbon material on the insulating substrate 2 by screen printing. Alternatively, the electrodes 3 and 4 can be formed as silver / silver chloride electrodes formed by screen printing. In addition, a silver / silver chloride electrode, a carbon electrode formed by screen printing, or a three-electrode system using a metal electrode formed by physical vapor deposition (PVD, for example, sputtering) or chemical vapor deposition (CVD) as a reference electrode is used. May be good.

Any known material can be applied to the electrode material forming the electrodes 3 and 4 and the material of the insulating substrate 2. The size and thickness of the electrodes 3 and 4 and the insulating substrate 2 can be appropriately set. The combination of the insulating substrate 2 and the electrodes 3 and 4 is sometimes called a "base material". In the embodiment, a two-pole electrode system having a working electrode and a counter electrode is illustrated, but a three-pole electrode system including a reference electrode may be used.

<< Insulation layer >>
The insulating layer 5 is provided on the base material in a state where an opening 5a for exposing the other ends 3b and 4b of the electrodes 3 and 4 is formed. Further, one ends 3a and 4a of the electrodes 3 and 4 are also provided so as to be exposed. The other end 3b exposed at the opening 5a is used as the working electrode, and the other end 4b is used as the counter electrode. Hereinafter, the other end portion 3b may be referred to as "acting pole 3b", and the other end portion 4b may be referred to as "counter electrode 4b".

The insulating layer 5 can also be formed by, for example, screen printing of resist ink. Alternatively, it can also be formed by adhering a resin plate on which the opening 5a is formed onto a base material. In some cases, the insulating layer 5 may not be provided. The insulating layer 5 is for suppressing the mixing of noise current, and the step of the opening 5a formed by the insulating layer 5 is the distribution (spreading) of the sample adhered on the electrodes (working electrode 3b, counter electrode 4b). ) It does not specify the range. As described above, the biosensor 1 does not have a capillary (cover or spacer) that covers the electrodes (working electrode 3b, counter electrode 4b).

<< Detection layer >>
The detection layer 6 is fixed on the working electrode 3b (exposed other end 3b). The detection layer 6 contains an enzyme that contacts the working electrode 3b, a conductive polymer, and a cross-linking agent 7, and does not contain an electron transfer mediator. The detection layer 6 may contain at least one of sugar and conductive particles.

What is measured using the biosensor according to the embodiment is not a current that depends on the diffusion of the substance to be measured, but a charge transfer rate-determining current based on the movement of electrons derived from the substance to be measured to the electrode. This is a current generated by the movement of electrons from the enzyme to the electrode due to the reaction between the enzyme and the substance to be measured. The charge transfer rate-determining current is a time-independent steady-state current, preferably a steady-state current after a transient current is generated by charging the electric double layer.

In order to measure the charge transfer rate-determining current, the working electrode is a "direct electron transfer type enzyme electrode". Here, the "direct electron transfer type enzyme electrode" means that electrons generated by an enzymatic reaction in the reagent layer are directly or via a conductive polymer without the involvement of a redox substance such as an electron transfer mediator. It is a type of enzyme electrode in which electron transfer is directly performed between the enzyme and the electrode by being transmitted to the electrode.

Even when the electron transfer mediator is used, the charge transfer rate-determining current can be measured when the electron transfer mediator is fixed and does not diffuse.

In the detection layer 6, the enzyme molecules have a structure in which they are crosslinked by a cross-linking agent and further intricately entangled by a conductive polymer. The electrons generated by the enzymatic reaction can move directly or through the conductive polymer having conductivity to the electrode (working electrode 3b). As described above, in the biosensor according to the embodiment, electron transfer is performed between the enzyme and the working electrode 3b by direct electron transfer in the detection layer 6.

It is said that the limit distance at which electron transfer occurs directly in the physiological reaction system is 1-2 nm, and even in the electron transfer in the electrochemical reaction system composed of electrodes and enzymes, at a longer distance than this, the mediator Unless it is accompanied by movement (for example, movement by diffusion), it becomes difficult to detect electron transfer on the electrode. Therefore, in the detection layer 6, the active site of the enzyme (the site where electrons are generated by the enzyme reaction) and the conductive site of the conductive polymer have a suitable distance relationship for electron transfer, that is, the degree to which suitable electron transfer occurs. The conductive site is close to the active site.

(enzyme)
As the enzyme, for example, redox enzyme is applied. Oxidoreductases include, for example, glucose oxidase (GOD), galactose oxidase, birylbin oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthine oxidase, sarcosin oxidase, L-lactic acid oxidase. , Ascorbic acid oxidase, alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase (GDH), fructose dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, malic acid dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase , Glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaholase, titochrome oxidoreductase, catalase, peroxidase, glutathione reductase and the like. Among them, it is preferably a saccharide oxidoreductase, and examples of the saccharide oxidoreductase include glucose oxidase (GOD), galactose oxidase, glucose dehydrogenase (GDH), fructose dehydrogenase, and sorbitol dehydrogenase. ..

In addition, the oxidoreductase can contain at least one of pyrroloquinoline quinone (PQQ) and flavin adenine dinucleotide (FAD) as a catalytic subunit and a catalytic domain. For example, PQQ glucose dehydrogenase (PQQGDH) is mentioned as an oxidoreductase containing PQQ, and titochrom glucose dehydrogenase (Cy-GDH) and glucose oxidase (GOD) having an α-subunit containing FAD are examples of oxidoreductase containing FAD. ).

In addition, the oxidoreductase can include an electron transfer subunit or an electron transfer domain. Examples of the electron transfer subunit include a subunit having a heme having an electron transfer function. Examples of the oxidoreductase containing a subunit having this heme include those containing cytochrome, and for example, glucose dehydrogenase and a fusion protein of PQQGDH and cytochrome can be applied.

Examples of the enzyme containing an electron transfer domain include cholesterol oxidase and quinohem ethanol dehydrogenase (QHEDH (PQQ Ethanol dh). Further, the electron transfer domain includes a domain containing thitochrome having a heme having an electron transfer function. It is preferred to apply, for example, "QHGDH" (fusion enzyme; GDH with heme domain of QHGDH), sorbitol dehydrogenase (Sorbitol DH), D-fructose dehydrogenase (Fructose DH), glucose-3-dehydrogenase from Agrobacterium tumefasience ( Glucose-3-Dehydrogenase) (G3DH from Agrobacterium tumefasience), cellobiose dehydrogenase. The fusion protein of PQQGDH and cytochrome, which is an example of the above-mentioned subunit containing cytochrome, and the cytochrome domain of PQQGDH, which is an example of the domain containing cytochrome, are disclosed in, for example, WO 2005/030807. ..

Further, as the oxidoreductase, it is preferable to apply an oligomeric enzyme composed of at least a catalytic subunit and a subunit containing cytochrome having a heme having an electron acceptor function.

As the enzyme, an oxidoreductase having no electron transfer subunit can be applied. One example is cytochrome glucose dehydrogenase (Cy-GDH). Cy-GDH has an electron transfer subunit β, a catalyst subunit α, and a catalyst subunit γ. As the enzyme contained in the detection layer 6, Cy-GDH having no electron transfer subunit β (having catalytic subunits α and γ) can be applied.

Cy-GDH having no electron transfer subunit β can be obtained at a lower cost than Cy-GDH having an electron transfer subunit β, a catalyst subunit α and a catalyst subunit γ. Therefore, by applying Cy-GDH having no electron transfer subunit β as an enzyme, the manufacturing cost of the enzyme electrode can be suppressed.

Further, Cy-GDH having no electron transfer subunit β has higher stability as a substance than Cy-GDH having an electron transfer subunit β, a catalyst subunit α and a catalyst subunit γ. This means that the enzyme electrode (biosensor) to which Cy-GDH having no electron transfer subunit β is applied has Cy-GDH having electron transfer subunit β, catalyst subunit α and catalyst subunit γ applied. It means that it can be stored for a longer period of time than an enzyme electrode (biosensor). Therefore, a charge transfer rate-determining measurement type enzyme electrode (biosensor) having a long product life can be obtained.

(Conductive polymer)
Examples of the conductive polymer include polypyrrole, polyaniline, polystyrene sulfonate, polythiophene, polyisothianaphene, polyethylenedioxythiophene (poly (3,4-ethylenedioxythiophene) poly (styrene sulfonate)), or a combination thereof. Can be mentioned. Examples of these commercially available products include, for example, polypyrrole, "SSPY" (ethyl 3-methyl-4-pyrrole carboxylate) (manufactured by Kaken Sangyo Co., Ltd.) and the like. Further, as the polyaniline, for example, "AquaPASS 01-x" (manufactured by TA Chemical Co., Ltd.) and the like. Further, as polystyrene sulfonate, for example, "Polynas" (manufactured by Tosoh Organic Chemical Co., Ltd.) and the like are available. Examples of polythiophene include "Espacer 100" (manufactured by TA Chemical Co., Ltd.). Examples of polyisotianaphthenic acid include "Espacer 300" (manufactured by TA Chemical Co., Ltd.). Examples of polyethylene dioxythiophene (poly (3,4-ethylenedioxythiophene) poly (styrene sulfonate)) include "PEDOT-PSS" (Polyscience Inc.,) and the like.

Further, a conductive polymer having various attributes (for example, water solubility) can be applied. The functional group of the conductive polymer preferably has a hydroxyl group or a sulfo group.

(sugar)
The detection layer 6 can contain sugar in addition to the enzyme, the cross-linking agent, and the conductive polymer. The sugar is a sugar that does not serve as a substrate for the enzyme, and the number of constituent sugars of the sugar is, for example, 1 to 6, preferably 2 to 6. These may be either D-form or L-form, or may be a mixture thereof, and one kind alone or two or more kinds may be used in combination as appropriate. However, when a sugar such as glucose is used as the measurement target, a sugar different from the sugar to be measured and which does not serve as a substrate for the enzyme is used as the sugar.

Disaccharides include xylobiose, agarobiose, carabiose, maltose, isomaltose, sophorose, cellobiose, trehalose, neotrehalose, isotorehalose, inulobiose, bicianose, isoprimeberos, sambubios, primeberos, sorabios, melibioce, Examples thereof include cellobiose, sucrose, trehalose, maltose, lactose, epigentibiose, robinobiose, silanobiose, and rutinose. Examples of trisaccharides include glucosyltrehalose, cellotriose, cacotriose, gentianose, isomalttriose, isopanose, maltotriose, manninotriose, merenjitos, panose, planteose, raffinose, soratorioce, and umbelliferose.

Examples of the tetrasaccharide include maltosyltrehalose, maltotetraose, stachyose and the like. Examples of pentasaccharides include maltotriosyltrehalose, maltopentaose, and velva course. Examples of the hexasaccharide include maltohexaose and the like.

(Crosslinking agent)
As a type of cross-linking agent, specifically, as an aldehyde group-containing compound, glutaaldehyde, formaldehyde, malonaldehyde, terephthalaldehyde, isobutylaldehyde, barrelaldehyde, isobarrelaldehyde, cinnamaldehyde, nicotyaldehyde, glyceraldehyde, glycoaldehyde. , Succin aldehyde, adipaldehyde, isophthalaldehyde, terephthalaldehyde and the like. As carbodiimide group-containing compounds, hexamethylene diisocyanate, hydrogenated xylylene diisocyanate, xylylene diisocyanate, 2,2,4-trimethylhexamethylene diisocyanate, 1,12-diisocyanate dodecane, norbornan diisocyanate, 2,4-bis- (8-). Isocyanate octyl) -1,3-dioctylcyclobutane, 4,4'-dicyclohexylmethane diisocyanate, tetramethylxylylene diisocyanate, isophorone diisocyanate and the like can be mentioned. The carbodiimide group-containing compounds are carbodilite V-02, carbodilite V-02-L2, carbodilite V-04, carbodilite V-06, carbodilite E-02, carbodilite V-01, carbodilite V-03, carbodilite V-05, and carbodilite V. It is commercially available as -07, Carbodilite V-09 (both trade names, manufactured by Nisshinbo Holdings, Inc.) and the like.

Maleimide group-containing compounds include m-maleimidebenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl 4- (p-maleimidephenyl) butyrate, m-maleimidebenzoylsulfosuccinimide ester, N-γ-maleimidebutyryloxysuccinimide ester. , Succinimidyl 4- (N-maleimidemethyl) cyclohexane1-carboxylate, N-succinimidyl-2-maleimideacetic acid, N-succinidyl-4-maleimidebutyric acid, N-succinidyl-6-maleimidehexanoic acid, N-succinidyl-4- Maleimidemethylcyclohexane-1-carboxylic acid, N-sulfosuccinidyl-4-maleimidemethylcyclohexane-1-carboxylic acid, N-succinidyl-4-maleimidemethylbenzoic acid, N-succinidyl-3-maleimidebenzoic acid, N- Scusinidyl-4-maleimidephenyl-4-butyric acid, N-sulfosuccinidyl-4-maleimidephenyl-4-butyric acid, N, N'-oxydimethylene-dimaleimide, N, N'-o-phenylene-dimaleimide, N , N'-m-phenylene-dimaleimide, N, N'-p-phenylene-dimaleimide, N, N'-hexamethylene-dimaleimide, N-succinidylmaleimidecarboxylic acid and the like. In addition, commercial products such as Sanfel BM-G (manufactured by Sanshin Chemical Industry Co., Ltd.) can also be mentioned.

As the oxazoline group-containing compound, 2,2'-bis- (2-oxazoline), 2,2'-methylene-bis- (2-oxazoline), 2,2'-ethylene-bis- (2-oxazoline), 2 , 2'-Trimethylene-bis- (2-oxazoline), 2,2'-tetramethylene-bis- (2-oxazoline), 2,2'-hexamethylene-bis- (2-oxazoline), 2,2' -Octamethylene-bis- (2-oxazoline), 2,2'-ethylene-bis- (4,4'-dimethyl-2-oxazoline), 2,2'-p-phenylene-bis- (2-oxazoline) , 2,2'-m-phenylene-bis- (2-oxazoline),
Oxazolines such as 2,2'-m-phenylene-bis- (4,4'-dimethyl-2-oxazoline), bis- (2-oxazolinylcyclohexane) sulfide, bis- (2-oxazolinyl norbornane) sulfide Examples include compounds.

As addition-polymerizable oxazoline compounds, 2-vinyl-2-oxazoline, 2-vinyl-4-methyl-2-oxazoline, 2-vinyl-5-methyl-2-oxazoline, 2-isopropenyl-2-oxazoline, 2 -Isopropenyl-4-methyl-2-oxazoline, 2-isopropenyl-5-ethyl-2-oxazoline and the like can be mentioned, and one or more of these compounds can be polymerized or copolymerized. is there.

The oxazoline group-containing compounds are Epocross WS-500, Epocross WS-700, Epocross K-1010E, Epocross K-1020E, Epocross K-1030E, Epocross K-2010E, Epocross K-2020E, Epocross K-2030E, Epocross RPS. It is commercially available as -1005, Epocross RAS-1005 (all manufactured by Nippon Shokubai Co., Ltd.), NK Linker FX (manufactured by Shin-Nakamura Chemical Industry Co., Ltd.), and the like.

Specific examples of the epoxy group-containing compound include sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, ethylene glycol diglycidyl ether, and polyethylene glycol diglycidyl. Examples thereof include ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and the like, and two or more of these compounds can be used in combination. The epoxy group-containing compounds include Denacol EX-611, Denacol EX-612, Denacol EX-614, Denacol EX-614B, Denacol EX-512, Denacol EX-521, Denacol EX-421, Denacol EX-313, and Denacol EX-. 314, Denacol EX-321, Denacol EX-810, Denacol EX-811, Denacol EX-850, Denacol EX-851, Denacol EX-821, Denacol EX-830, Denacol EX-832, Denacol EX-841, Denacol EX- 861, Denacol EX-911, Denacol EX-941, Denacol EX-920, Denacol EX-145, Denacol EX-171 (trade name, manufactured by Nagase ChemteX Corporation), SR-PG, SR-2EG, SR- 8EG, SR-8EGS, SR-GLG, SR-DGE, SR-4GL, SR-4GLS, SR-SEP (trade name, manufactured by Sakamoto Pharmaceutical Co., Ltd.), Epolite 200E, Epolite 400E, Epolite 400P (all) It is commercially available as Kyoeisha Chemical Co., Ltd.).

The type of the cross-linking agent is not limited to the above compounds and commercially available products, and may be a compound containing at least one functional group of an aldehyde group, a maleimide group, a carbodiimide group, an oxazoline group and an epoxy group. Further, the form of the cross-linking agent is not limited, and may be any form of a monomer and a polymer.

(Conductive particles)
The detection layer 6 can further contain conductive particles. As the conductive particles, metal particles such as gold, platinum, silver and palladium, or higher-order structures made of carbon can be applied. The higher-order structure can contain, for example, one or more fine particles (carbon fine particles) selected from conductive carbon black, Ketjen black (registered trademark), carbon nanotubes (CNT), and fullerenes.

The surface of the detection layer 6 may be covered with an outer layer film such as cellulose acetate. Examples of the raw material of the outer layer film include polyurethane, polycarbonate, polymethylmethacrylate, butylmethacrylate, polypropylene, polyetheretherketone and the like.

(Manufacturing method of biosensor)
The biosensor 1 described above is manufactured (manufactured) as follows, for example. That is, a metal layer that functions as electrodes 3 and 4 is formed on one side 2a of the insulating substrate 2. For example, a metal material is deposited on one side 2a of a film-like insulating substrate 2 having a predetermined thickness (for example, about 100 μm) by physical vapor deposition (PVD, for example, sputtering) or chemical vapor deposition (CVD). As a result, the metal layer (electrodes 3 and 4) having a desired thickness (for example, about 30 nm)
Is formed. Electrodes 3 and 4 can also be formed by, for example, screen printing a carbon material instead of the metal layer.

Next, the insulating layer 5 having a predetermined exposure pattern (having an opening 5a) is formed by screen printing the resist ink on the single surface 2a on which the electrodes 3 and 4 are formed.

Next, the detection layer 6 is formed on the working electrode 3b. For example, a solution (reagent) containing at least an enzyme, a conductive polymer, and a cross-linking agent is prepared. When sugar is added to the reagent, the sugar concentration is preferably 0.1 to 2% by weight, more preferably 0.2 to 2% by weight. The solution (reagent) is dropped on the surface of the working electrode 3b. When the solution (reagent) is solidified by drying on the working electrode 3b, the detection layer 6 is formed on the working electrode 3b. In this way, a biosensor 1 having an open system on the working electrode 3b can be obtained.

By using the biosensor 1 according to the embodiment, the concentration of the substance to be measured contained in the sample (sample) can be measured based on the charge transfer rate-determining current. Here, the substance to be measured is not particularly limited as long as it is a substance that can be measured by the measurement method using the biosensor 1, but it may be a substance derived from a living body and can be an index of a disease or a health condition. preferable. Examples of the substance include glucose and cholesterol. The sample is not particularly limited as long as it contains the substance to be measured. However, biological samples are preferable. The biological sample is, for example, blood, urine, or the like.

(measuring device)
Next, a measuring device for measuring the concentration of a substance using the biosensor 1 according to the embodiment will be described. Here, a glucose measuring device using a glucose sensor, which is an example of the biosensor 1, will be illustrated. However, the measuring device is not limited to the glucose measuring device, and the application of the measuring device varies depending on the substance to be measured by the biosensor 1.

FIG. 2 shows a configuration example of a main electronic component housed in the measuring device B. The control computer 18, the potentiostat 19, and the power supply device 11 are provided on the substrate 20 housed in the housing. In terms of hardware, the control computer 18 includes a processor such as a CPU (Central Processing Unit) and a memory (RAM (Random Access).
It includes a recording medium such as Memory) and ROM (Read Only Memory), and a communication unit.

When the processor loads the program stored in the recording medium (for example, ROM) into the RAM and executes the program, the control computer 18 functions as a device including an output unit 10, a control unit 12, a calculation unit 13, and a detection unit 14. To do. The control computer 18 may include an auxiliary storage device for storing programs and data, such as a semiconductor memory (EEPROM, flash memory) or a hard disk.

The control unit 12 controls the timing of voltage application, the applied voltage value, and the like. The power supply device 11 has a battery 16 and supplies electric power for operation to the control computer 18 and the potentiometer 19. The power supply device 11 can also be placed outside the housing.

The potentiostat 19 is a device that makes the potential of the working electrode constant with respect to the reference electrode, and is controlled by the control unit 12. The potentiostat 19 applies a predetermined voltage between the counter electrode and the working electrode of the glucose sensor 17 using the terminals CR and W, measures the response current of the working electrode obtained at the terminal W, and measures the response current. The result is sent to the detection unit 14.

The calculation unit 13 calculates the concentration of the substance to be measured from the detected current value and stores it. The output unit 10 performs data communication with the display unit 15 and transmits the calculation result of the concentration of the substance to be measured by the calculation unit 13 to the display unit 15. The display unit 15 can display, for example, the calculation result of the glucose concentration received from the measuring device B on the display screen in a predetermined format.

FIG. 3 is a flowchart showing an example of glucose concentration measurement processing by the control computer 18. The CPU (control unit 12) of the control computer 18 receives an instruction to start measuring the glucose concentration. Then, the control unit 12 controls the potentiostat 19 to apply a predetermined voltage to the working electrode, and starts measuring the response current from the working electrode (step S01). The detection of the attachment of the sensor to the measuring device may be used as a concentration measurement start instruction.

Next, the potentiostat 19 has a response current obtained by applying a voltage, that is, a charge transfer rate-determining current based on the transfer of electrons derived from the substance to be measured (here, glucose) in the sample to the electrode, preferably an electric double layer. After the transient current is generated by charging the multiple layers, for example, the steady current 1 to 20 seconds after the voltage is applied is measured and sent to the detection unit 14 (step S02).

The calculation unit 13 performs calculation processing based on the current value to calculate the glucose concentration (step S03). For example, the calculation unit 13 of the control computer 18 holds in advance the glucose concentration calculation formula or the glucose concentration calibration curve data corresponding to the glucose dehydrogenase arranged on the electrode, and these calculation formulas or calibrations are performed. Calculate the glucose concentration using the line.

The output unit 10 transmits the calculation result of the glucose concentration to the display unit 15 through a communication link formed between the output unit 10 and the display unit 15 (step S04). After that, the control unit 12 detects the presence or absence of a measurement error (step S05), ends the measurement if there is no error, and displays the glucose concentration on the display unit. If there is an error, an error is displayed and then the process according to the flow shown in FIG. 3 is terminated. It is also possible to save the calculation result in the storage medium, recall the calculation result later, display it on the display unit 15, and confirm it. In the example of FIG. 3, the measurement error detection (step S05) is performed by the control unit 12 after the calculation result is transmitted to the display unit 15 (step S04), but the order of these steps can be changed. Is.

[Example]
Hereinafter, examples of the enzyme electrode will be described.

<Test 1>
A plurality of biosensors having capillaries with different spacer thicknesses were prepared and glucose was measured.
(Manufacturing of biosensor)
It consists of 3 electrodes by forming a working electrode and a counter electrode on one side of an insulating substrate by screen printing using carbon ink, and forming a reference electrode by screen printing of silver / silver chloride (Ag / AgCl). An electrode system was formed. A reagent solution according to the following formulation was added dropwise on the working electrode and solidified by drying to form a detection layer. Further, by forming a capillary using a spacer and a cover on the insulating substrate on which the electrode system was formed, biosensors having spacer thicknesses of 150 μm, 300 μm, and 450 μm were obtained, respectively.
[Reagent prescription]
-Phosphate buffer (pH 5.8): 10 mM
・ Sucrose: 0.5%
-Enzyme (Cy-GDH: γαβ): 4.5 mg / mL
-Conductive polymer: Sulfonized polyaniline aqueous solution (trade name: aquaPASS-01x, manufactured by Mitsubishi Rayon Co., Ltd.): 0.4%
-Oxazoline group-containing polymer Epocross WS-700 (Nippon Shokubai): 6.0%
・ Lion Paste W-311N (manufactured by Lion Corporation): 2.42%
In addition, "%" indicates the weight% concentration of the reagent contained in the reagent solution.
(sample)
As a sample (sample), a 20 mM HEEPES solution (pH 7.0) having a glucose concentration in the sample of 0,100 mg / dL, 200 mg / dL, 400 mg / dL, and 800 mg / dL was prepared. 23 ±
Glucose concentration was measured using each biosensor by the chronoamperometry method (+60 mV vs. Ag / AgCl) in an atmosphere of 1 ° C.

FIG. 4 is a graph showing a calibration curve based on the result of test 1 and the measurement result of glucose concentration using the chronoamperometry method. According to the result of FIG. 4, a calibration curve having a similar inclination was obtained regardless of the thickness of the spacer. This means that the amount of sample attached to the electrode does not affect the response current value.

<Test 2>
Next, as a biosensor having a detection layer containing an electron transfer mediator, a biosensor having a capillary and a biosensor having no capillary were prepared and measured by a chronoamperometry method.
(Reagent prescription)
・ Enzyme (FAD-GDH): 1U
-Mediator (Ru complex, 1-methoxy-PMS): 200 mM
-ACES buffer (pH 7.5): 0.17 mM
・ Smectite (Lucentite SWN): 0.2%
(sample)
As a sample (sample), whole blood having glucose concentrations in the sample of 100 mg / dL, 321 mg / dL, and 624 mg / dL, respectively, was prepared. The glucose response current was measured using each biosensor by the chronoamperometry method (+200 mV vs. Ag / AgCl) in an atmosphere of 24 ± 1 ° C.

FIG. 5 is a graph showing the result of Test 2, that is, the calibration curve based on the measurement result of glucose concentration by the chronoamperometry method. As shown in FIG. 5, when there is no capillary, the response current value is higher than when there is a capillary, and it can be seen that the absence of the capillary affects the response current value.

<Test 3>
(Example 1)
Next, the biosensor according to Example 1 was created as follows. Two electrodes, an working electrode 3b formed by screen printing of carbon ink and a counter electrode 4b formed by screen printing of silver / silver chloride ink (ercon), were formed on one side 2a of the insulating substrate 2. No capillary was provided on the electrode, and the working electrode 3b and the counter electrode 4b were used as an open system.

The detection layer 6 was formed by printing a reagent ink having the following formulation on the working electrode 3b by screen printing and solidifying it by drying.
[Prescription]
-Phosphate buffer (pH 5.8): 10 mM
・ Sucrose: 0.5%
・ GDH: 7 mg / mL
-Conductive polymer: Sulfonized polyaniline aqueous solution (trade name: aquaPASS-01x, manufactured by Mitsubishi Rayon Co., Ltd.): 0.40%
-Oxazoline group-containing polymer Epocross WS-700 (Nippon Shokubai): 5.0%
・ Ketjen Black: 0.8%
In addition, "%" indicates the weight% concentration of the reagent contained in the reagent solution.

As a sample (sample), whole blood having a glucose concentration of 0 (blank: B) and 336 mg / dL (sample: S) was used. Each sample was dispensed (dotted) on the electrodes of each biosensor, and the glucose concentration was measured by the chronoamperometry method in an atmosphere of 25 ° C. The concentration was measured by preparing a plurality of the above biosensors and changing the instillation amount of the sample to 1 μL, 2 μL, and 4 μL.

FIG. 6 is a graph showing the S / B (sample / blank) ratio based on the measurement result of the glucose concentration using the biosensor of Example 1. As shown in FIG. 6, the S / B ratio was at a level of about 20 in all of 1 μL, 2 μL, and 4 μL, and no dependence on the sample amount was found.

<Test 4>
(Comparative Example 1)
As Comparative Example 1, a biosensor of Example 1 provided with a capillary was prepared. Biosensors having capillary heights of 10 μm, 30 μm, 50 μm, 100 μm, and 200 μm were prepared. The formulation of the detection layer 6 was the same as in Example 1. As a sample (sample), an aqueous solution of 0,300 mg / dL was used, and the electrode was impregnated into the sample to bring the sample into contact with the electrode.

The electrode response characteristics of the biosensor according to Comparative Example 1 were examined using the cyclic voltammetry method (CV method) and the chronoamperometry method (CA method) in an atmosphere of 25 ° C. FIG. 7 is a graph showing the S / B ratio based on the measurement results by the CV method and the CA method using the biosensor of Comparative Example 1. As shown in FIG. 7, it was confirmed that the S / B ratio did not depend on the height of the capillary.

According to Tests 1 to 4, it was confirmed that the response current value did not depend on the sample amount and the height of the capillary. Therefore, at least according to the biosensor 1 in which the working electrode is an open system, it is possible to measure the concentration with high accuracy without adjusting or controlling the sample amount by the capillary. As described above, since no capillary is required, it is possible to reduce the manufacturing cost of the biosensor by reducing the number of parts. Further, since it does not depend on the sample amount, it is possible to avoid a difference in the measurement result depending on how the user of the biosensor 1 handles the biosensor 1. That is, the user's biosensor can be easily handled. Since the biosensor according to the embodiment does not need to cover the electrode with a capillary (open system), the biosensor is provided on the puncture needle, and the sample can be spotted on the electrode at the same time as the puncture. The configuration can be applied.

[Second Embodiment]
The second embodiment will be described below. Since the second embodiment has the same configuration as the first embodiment, the differences will be mainly described, and the common points will be omitted.

The charge transfer rate-determining current described in the first embodiment is represented by the following equation (1). From equation (1), it can be seen that the current is proportional to the concentration of the substrate and the enzyme reaction rate constant. Equation (1) can be expanded into equation (2), where X is the constant term. Although not shown in the equations (1) and (2), the constant term X may include a correction coefficient or the like.

Equation (1) was derived by considering the equations for the initial rate of the enzyme reaction and the equation for the electron transfer rate from the enzyme to the electrode, and developing the equations in a state where they are equivalent. The biosensor according to the first embodiment measures, for example, the concentration of the substance (substrate) to be measured by using the above formulas (1) and (2).

Equation (1) is a current equation by charge transfer rate-determining that does not include the diffusion coefficient included in the Cottrell current in the initial rate equation of the enzyme reaction. As can be seen from equation (1), the current is proportional to the enzyme reaction rate constant. In the method for measuring a substance to be measured according to the first embodiment, electrons move to the electrode without going through a redox reaction by a mediator such as an electron acceptor. Therefore, it can be seen that the charge transfer rate-determining current is not affected by the diffusion of substances and is not time-dependent.

It should be noted that the fact that the electrode system is charge transfer rate-determining can be confirmed by examining the presence or absence of a peak and the increasing tendency of the current depending on the voltage sweeping direction by cyclic voltammetry or the like.

The biosensor according to the second embodiment also includes a "direct electron transfer type enzyme electrode" that measures a charge transfer rate-determining current. Further, in the biosensor according to the second embodiment, a detection layer in which the contact area with the working electrode is defined by a predetermined area is formed on the working electrode. That is, the contact area of the detection layer with the working electrode is defined by a predetermined area. The contact area between the detection layer and the working electrode can be defined by applying (applying) the detection layer material only to the working electrode among the plurality of electrodes, instead of simply dropping the liquid detection layer material. Thereby, "A: electrode area" and "n: number of reaction electrons" in the formula (1) can be made constant. Therefore, it is possible to suppress the variation in the response current between the individual biosensors and improve the simultaneous reproducibility.

Further, in order to define the contact area between the detection layer and the working electrode, it can be exemplified that an insulating layer is formed on an insulating base material on which an electrode is formed. The insulating layer is formed so that the working electrode is exposed on the bottom surface thereof, and has an opening in which the detection layer material is filled. The opening has a continuous inner wall surface, and the diffusion range of the detection layer material filled in the opening is restricted in the opening. The filled detection layer material is solidified by drying to form a detection layer filled in the opening (filling the opening). The bottom area of the opening is defined in a size that matches the contact area between the detection layer and the working electrode, and the opening is filled with at least an amount of the detection layer material that fills the bottom of the opening. As a result, a detection layer in which the contact area with the working electrode is defined as a predetermined area (contacting with the working electrode in a predetermined area) can be easily formed on the working electrode. The insulating layer can be formed, for example, by screen printing of non-conductor ink (also referred to as resist ink). Further, the insulating layer can be formed by using various resists such as photoresist and processing with insulating tape, in addition to screen printing, as long as an opening capable of defining the contact area between the working electrode and the detection layer can be formed. ..

As another method of use, the contact area between the detection layer and the working electrode can be determined according to the reaction rate of the enzyme. The reaction rate of the enzyme may vary between lots with different specific activities. Therefore, the area of the detection layer and the working electrode according to the reaction rate of the enzyme is obtained in advance by an experiment or the like, and the detection layer is formed with the contact area according to the reaction rate for each lot of the enzyme. As a result, it is possible to suppress variations in simultaneous reproducibility between lots.

FIG. 8 is a diagram showing a configuration example of the biosensor 1A according to the second embodiment. The biosensor 1A has a configuration different from that of the biosensor 1 (FIG. 1) in the first embodiment in the following points.

The insulating layer 5 has an opening 5b and an opening 5c instead of the opening 5a (FIG. 1). The opening 5b is formed so that the counter electrode 4b and the upper surface of the insulating substrate 2 around the counter electrode 4b are exposed.

On the other hand, the opening 5c is formed on the working pole 3b, and the working pole 3b is covered with the insulating layer 5 except for the portion exposed by the opening 5c. The opening 5c has a predetermined bottom area and has a continuous inner wall surface. In the example shown in FIG. 8, the opening 5c has a circular inner wall surface having a cylindrical side surface shape.

The size (bottom area) of the opening 5c is formed in the working pole 3b and is formed according to the contact area of the detection layer 6 in contact with the working pole 3b. The detection layer 6 is formed by filling the detection layer material so as to fill the inside of the opening 5c. In the example of FIG. 8, the detection layer 6 is a columnar shape that fills the opening 5c.

In this way, by forming the detection layer 6 using the opening 5c having a defined bottom area, the contact area between the working electrode 3b and the detection layer 6 is defined. The components of the detection layer 6 are as described in the first embodiment.

In the example of FIG. 8, the planar shape of the opening 5c is circular, and the inner wall surface is a side surface of a cylinder. However, the opening 5c may be formed in a tapered shape, and the inner wall surface may be formed in the shape of a side surface of a truncated cone. The planar shape of the opening 5c and the shape of the inner wall surface can be appropriately set as long as a continuous inner wall surface that regulates the diffusion range of the detection layer material to be filled can be formed. For example, the planar shape of the opening 5c may be an ellipse, a triangle, a quadrangle, or a polygon consisting of five or more sides. Further, the shape of the inner wall surface of the opening 5c may be the side surface shape of the pillar corresponding to the plane shape or the side surface shape of the pillar not corresponding to the plane shape. That is, the shape of the opening 5c can be appropriately set as long as the contact area between the detection layer 6 and the working electrode 3b can be defined as a predetermined area.

As described above, according to the biosensor in the second embodiment, since the working extremity is an open system (has no capillary), it is easy to spot the sample. Further, the detection layer 6 is in contact with the working pole 3b in a predetermined area, that is, the contact area between the detection layer 6 and the working pole 3b is defined. Thereby, the CV value (simultaneous reproducibility) can be improved. That is, the error between the biosensors can be reduced and the accuracy can be stabilized.

1 ... Biosensor 2 ... Insulating substrate 3, 4 ... Electrode 3b ... Working electrode 4b ... Counter electrode 5 ... Insulating layer 6 ... Detection layer (reagent layer)
B ... Measuring device 10 ... Output unit 11 ... Power supply device 12 ... Control unit 13 ... Calculation unit 14 ... Detection unit 15 ... Display unit 16 ... Battery 18.・ ・ Control computer 19 ・ ・ ・ Potentiometer

Claims (1)

With multiple electrodes including working electrodes,
It comprises a detection layer that is immobilized on the working electrode and contains a cross-linking agent, a conductive polymer, and an enzyme that transfers electrons to and from the working electrode.
The best of action is an open system,
The enzyme comprises a catalytic subunit and a catalytic domain and an electron transfer subunit or electron transfer domain.
The working electrode is covered with an insulating layer leaving one opening where the working electrode is exposed, and the detection layer is filled in the opening and is in contact with the working electrode of the detection layer. A biosensor characterized in that the area is defined by the opening as a predetermined area.

Images