JP2006308458A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly sensitive biosensor which prevents a decrease in enzyme activity and suppresses a decrease in electrode sensitivity.
SOLUTION: Electrodes 2, 3 and 4, a cavity 33 including the electrode therein, and a measurement reagent 32 including an enzyme disposed in the cavity, wherein the measurement reagent 32 includes the The biosensor is disposed in the cavity 33 and in a region other than the surfaces of the electrodes 2, 3, and 4. In this case, it is more preferable that the electrode is arranged on the surface of the cavity, and the measuring reagent is a surface of the cavity, and the biosensor is dry-supported on the surface portion where no electrode is formed.
[Selection] Figure 3

Description

  The present invention relates to a biosensor that electrochemically measures the concentration of a biological substance in a measurement sample using an enzyme reaction.

  A biosensor is a sensor that uses a molecular identification function of a living body to detect the presence of a specific chemical substance. In particular, a technology that uses an enzyme that acts as a catalyst for biological reaction has attracted particular attention because of its high selectivity for a measurement target substance. Many biosensors such as a glucose sensor, an alcohol sensor, a uric acid sensor, and an amylase sensor have been used so far. Has been put to practical use.

  A biosensor using an enzyme includes at least an enzyme corresponding to a measurement target substance and an electrode that converts a substance obtained by the enzyme reaction into an electrical signal. Here, since an enzyme is generally expensive, it is immobilized on the surface of an electrode so that it can be used repeatedly. Examples of the technique for immobilizing the enzyme on the electrode surface include, for example, a technique for containing the enzyme using a polymer electrolyte or collagen gel (for example, Patent Document 1), or a technique for binding the enzyme to the electrode surface using glutaraldehyde or the like ( For example, Patent Document 2) has been proposed. However, these techniques have problems such as a decrease in enzyme activity due to immobilization and a complicated immobilization method.

  Therefore, a disposable biosensor that does not repeatedly use an enzyme has been proposed (for example, Patent Document 3). In general, in a biosensor, an electrode system including a measurement electrode, a counter electrode, and a reference electrode is formed on an insulating substrate by a method such as screen printing, and a hydrophilic polymer and an oxidoreductase are further formed on the electrode system. And an electron reaction layer including an electron mediator. Here, in the disposable type, since the enzyme is disposable in one measurement, it is not necessary to form the enzyme reaction layer using the above-described immobilization method. For example, the enzyme or the like is simply attached on the electrode. Can be formed.

Similarly, as a technique for holding an enzyme in a biosensor without using the above-described immobilization method, a biosensor that fixes a fibrous carrier carrying an enzyme in the vicinity of an electrode has been proposed (for example, a patent). Reference 4). In this biosensor, the fibrous carrier is fixed by using an adhesive or by providing a carrier fitting protrusion.
Japanese Patent Publication No.52-18270 JP-A-5-215710 Japanese Patent Laid-Open No. 2-62952 JP 2001-201479 A

  However, the biosensor described in Patent Document 3 has a problem in that the detection signal ratio of the reaction current is not stable because the enzyme adsorption on the electrode surface becomes excessive and the electrode sensitivity may decrease. Further, in the biosensor described in Patent Document 4, it is necessary to use a carrier carrying an enzyme, and it is necessary to use an adhesive or to provide a fitting projection for fixing the carrier. There is a problem that the number of processes and the number of processes required for manufacturing are large.

  Accordingly, an object of the present invention is to provide a biosensor that prevents a decrease in enzyme activity, prevents a decrease in electrode sensitivity, and reduces the number of components.

  The present invention includes a sensor substrate including an insulating substrate and two or more electrodes formed on the insulating substrate, a cavity formed on the sensor substrate and including the electrodes therein, An injection hole for supplying a measurement sample into the cavity, and a measurement reagent containing a measurement enzyme held in the cavity, wherein the measurement reagent is in the cavity other than the surface of the electrode. A biosensor is provided that is retained in an area.

  The present invention also provides a sensor substrate including an insulating substrate and two or more electrodes formed on the insulating substrate, and a measurement formed on the sensor substrate and including the electrodes therein. A reaction cavity, one or more reaction cavities formed on the sensor substrate, at least one flow path connecting the reaction cavity and the measurement cavity, and at least one reaction cavity. An injection hole for supplying a measurement sample, and a measurement reagent containing a measurement enzyme is held in at least one region in the reaction cavity and is not held on the surface of the electrode. A biosensor is provided.

  According to the present invention, a decrease in enzyme activity can be prevented, and a decrease in electrode sensitivity can be suppressed, so that a biosensor with excellent sensitivity can be provided.

  In the biosensor of the present invention, the measurement reagent can be dry-supported in a region in the cavity.

  In the biosensor of the present invention, the measurement reagent can be dried and supported on the surface of the sensor substrate.

  In the biosensor of the present invention, the cavity can be formed by the sensor substrate and the housing substrate, and the measurement reagent can be dried and supported on the surface of the housing substrate.

  In the biosensor of the present invention, the measurement enzyme may include an oxidoreductase. The oxidoreductase can include diaphorase.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following embodiments, and can take various forms as long as the gist of the present invention is not changed.

<Embodiment 1>
FIG. 1 is a perspective view showing an example of a sensor substrate 7 in the biosensor of the present invention. The sensor substrate 7 includes an insulating substrate 1, a measurement electrode 2, a counter electrode 3, and a reference electrode 4 formed on the insulating substrate 1. On the insulating substrate 1, a conductive pattern 5 and a terminal portion 6 that is a connection portion to an external circuit are provided. The conductive pattern 5 is provided so that each electrode is electrically connected to the corresponding terminal portion 6. These conductive members can be provided on the surface of one main surface of the sensor substrate 7, for example.

  Each electrode material may be a known conductive material such as gold, platinum, palladium, or other noble metal, or carbon, but from the viewpoint of improving the stability of the electrode surface, gold is preferably used as the material. . In addition, it is preferable that the reference electrode 4 is a reference electrode that exhibits nonpolarization properties, for example, from the viewpoint of increasing potential stability in a solution to be measured. For example, considering the ease of handling, a silver / silver chloride electrode is preferable. As a method of forming a silver / silver chloride electrode, for example, a method of applying a voltage in an aqueous NaCl solution after applying silver plating to the surface of an electrode pattern formed of gold, platinum or the like, or using a silver / silver chloride paste For example, a known forming method such as a method of forming a silver paste into an electrode pattern and then bringing the surface into contact with an aqueous solution such as sodium hypochlorite can be used.

  As a material for the conductive pattern 5 and the terminal portion 6, a known conductive material can be used, but from the viewpoint of facilitating the manufacture of the sensor substrate, it is preferable to use the same material as that for the electrode. As a method for forming electrodes, conductive patterns, and terminal portions on the insulating substrate 1, for example, a conductive material is printed, sputtered or vapor-deposited on the insulating substrate to form a coating, and then the coating is formed by etching or laser irradiation. A known forming method such as a method of removing a predetermined portion or a method of directly sputtering to a predetermined pattern using a mask can be used.

  As a material of the insulating substrate 1, for example, a known insulating substrate material such as a semiconductor such as silicon or germanium, glass such as quartz glass, lead glass or borosilicate glass, ceramic or resin can be used. In the case of a biosensor, it is preferable to use a resin material. This is because the substrate can be easily processed and the raw materials can be kept relatively inexpensive. The resin material is not particularly limited. For example, polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyimide (PI), polytetrafluoroethylene (PTFE), polyphenylene sulfide (PPS), polyetheretherketone Known resins such as (PEEK (registered trademark)), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyethylene-2,6-naphthalate (PEN) can be appropriately used depending on the purpose. In the case of forming a gold electrode by sputtering, it is preferable to use polyethylene terephthalate from the viewpoint of improving adhesion with gold.

  The thickness of the insulating substrate 1 is preferably in the range of 0.1 mm to 2.0 mm, and more preferably in the range of 0.188 mm to 0.5 mm, from the viewpoint of easy handling.

  As shown in FIG. 1, the sensor substrate 7 may have a mode in which the conductive pattern 5 is exposed, or as shown in FIG. 2, the sensor substrate 7 covers the surface of the conductive pattern 5 so as to cover the surface of the insulating substrate 1. It is good also as an aspect in which the insulating film 21 was formed on it. By providing the insulating film 21, the exposed area of each electrode can be easily controlled, and the conductive pattern 5 can be insulated and protected from mechanical damage. As a material of the insulating film 21, polyimide or the like can be used. In the present specification, the “surface of the sensor substrate” means an exposed surface on the side where the conductive member is provided. For example, in the embodiment shown in FIG. 1, it means the surface of the insulating substrate. In an embodiment in which an insulating film is provided on the surface of the insulating substrate as indicated by 2, this means the exposed surface of this insulating film.

  FIG. 3 is a perspective view showing an example of the biosensor of the present invention. This biosensor includes a sensor substrate 7 shown in FIG. 1 and a housing substrate 31 shown in FIG. The housing substrate 31 is provided with a recess 36, and the electrodes (measurement electrode 2, counter electrode 3, reference electrode 4) of the sensor substrate 7 are arranged in the hollow portion of the recess 36, A housing substrate 31 is laminated. In this way, the cavity 33 is formed by the sensor substrate 7 and the recess 36 of the housing substrate 31.

  In the region inside the cavity 33, the measuring reagent 32 is arranged in a portion other than the surface of each electrode. The measurement reagent 32 can contain an oxidoreductase or an electron mediator. As an oxidoreductase, a well-known thing can be used suitably according to the measurement objective. For example, NADH oxidase (for example, diaphorase), alcohol oxidase, glucose oxidase, lactate oxidase, cholesterol oxidase and the like can be used. As the electron mediator, a known compound that can mediate electron transfer between the enzyme and the electrode can be appropriately used depending on the purpose of measurement. For example, potassium ferricyanide, p-benzoquinone, phenazine methosulfate, ferrocene derivatives, etc. can be used. Can do.

  The measurement reagent 32 may be carried on the surface of the sensor substrate 7 corresponding to the bottom surface of the cavity 33 as shown in FIG. 3, or the surface of the housing substrate 31 corresponding to the side surface or top surface of the cavity 33, for example. You may make it carry on. As an aspect of carrying, for example, dry carrying can be used.

  For the lamination of the housing substrate 31 and the sensor substrate 7, for example, an adhesive such as acrylic, epoxy, or silicon, an adhesive sheet such as a double-sided tape, or an organic solvent such as chloroform may be used. May be heat-sealed. Note that the measurement reagent is preferably carried prior to the lamination of the sensor substrate 7 and the housing substrate 31.

  The housing substrate 31 is provided with at least one through hole that allows the inside of the cavity 33 to vent to the outside of the biosensor. This through hole can be used as an injection hole 34 for injecting a measurement sample. When two or more through holes are provided, at least one is preferably used as the air hole 35. This is because the air hole 35 functions as an air escape path inside the cavity 33 when the measurement sample is injected, so that the sample can be injected quickly. The cavity 33 is preferably subjected to a known hydrophilic treatment. This is because the injection rate of the measurement sample can be increased. After the measurement sample is injected, the through holes such as the injection hole 34 and the air hole 35 may be sealed with an adhesive tape or the like, or may not be sealed for a short time measurement.

  The material of the housing substrate 31 is preferably a material having low reactivity with the measurement sample. For example, a semiconductor such as silicon or germanium, a glass such as quartz glass, lead glass, or borosilicate glass, a ceramic, a resin, or the like. Can be used. In the case of a disposable biosensor, it is preferable to use a resin material. This is because the substrate can be easily processed and the raw materials can be kept relatively inexpensive. The resin material is not particularly limited. For example, polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyimide (PI), polytetrafluoroethylene (PTFE), polyphenylene sulfide (PPS), polyetheretherketone (PEEK (registered trademark)), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyethylene-2,6-naphthalate (PEN), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS), etc. Known resins can be appropriately used depending on the purpose. For example, when polymethyl methacrylate or polycarbonate is used, the state of the internal sample can be confirmed by its excellent transparency, and fine cutting and the like are facilitated. Moreover, when polycarbonate is used, heat resistance can also be improved.

  The method of forming the recess 36 in the housing substrate 31 is not particularly limited. For example, when the housing substrate is made of a resin material, it can be formed by a method such as cutting, molding with a mold, or embossing by thermal transfer. . Moreover, you may form the housing substrate containing a recessed part by sticking together the sheet | seat containing a through-hole, and the sheet | seat which does not contain a through-hole.

<Embodiment 2>
FIG. 5 is a perspective view showing another example of the biosensor of the present invention. This biosensor includes a sensor substrate 7 similar to that of the first embodiment and a housing substrate 51. The housing substrate 51 is provided with a first recess and a second recess, and the electrodes (measurement electrode 2, counter electrode 3, reference electrode 4) of the sensor substrate 7 are arranged in the hollow portion of the first recess. Thus, the sensor substrate 7 and the housing substrate 51 are laminated. Here, a measurement cavity 41 is formed by the first recess of the housing substrate and the sensor substrate 7, and a reaction cavity 42 is formed by the second recess of the housing substrate and the sensor substrate 7. A measuring reagent 32 is arranged on the surface of the sensor substrate 7 corresponding to the bottom surface of the reaction cavity 42. The measuring reagent 32 may be disposed on the surface of the housing substrate, for example, other than the surface of the sensor substrate 7 as long as it is an area inside the reaction cavity 42.

  The measurement cavity 41 and the reaction cavity 42 are connected by a flow path 43. As means for moving the measurement sample (solution) from the reaction cavity 42 to the measurement cavity 41, an external pump such as a syringe pump, a plunger pump, or a peristaltic pump, or a micro pump that is integrated on a biosensor is used. Can do. Furthermore, a centrifugal force generated by rotating the biosensor at a position where the reaction cavity is on the center side and the measurement cavity is on the outside may be used.

  Similar to the biosensor shown in FIG. 3, the housing substrate 51 is also provided with two through holes that allow the inside of the cavity to vent to the outside of the biosensor, and one through hole is measured in the reaction cavity 42. It is preferable to use it as an injection hole 34 for injecting a sample for use and use another through hole as the air hole 35. Here, from the viewpoint of facilitating the injection of the measurement sample, a through hole provided at the end of the measurement cavity 41 and away from the reaction cavity 42 is used as the air hole 35. It is preferable.

  In this embodiment, for example, even when the reaction time between the measurement sample and the measurement reagent takes a long time, the measurement cavity 41 is measured after a sufficient reaction time is left in the reaction cavity 42. Since the measurement sample can be moved, the contact time between the electrode necessary for measurement and the measurement sample can be shortened. Thereby, since it can prevent that the protein in a sample adsorb | sucks to the electrode surface, the precision of a measurement can be improved.

  Two or more reaction cavities may be provided by forming two or more second recesses. A plurality of reaction cavities may be connected by providing flow paths.

  Hereinafter, the biosensor of the present invention will be described more specifically, but the present invention is not limited to the following examples.

Example 1
Example 1 is an experimental example in which a current derived from the activity of nicotinamide adenine dinucleotide (hereinafter sometimes referred to as NADH), which is a small molecule in a living body, was measured with a biosensor.

The biosensor used in Example 1 was produced as follows. First, a polyethylene terephthalate sheet having a thickness of 188 μm was prepared as an insulating substrate, and then a 700 Å (70 nm) gold thin film was formed on the surface thereof by sputtering. Next, this gold thin film was etched to form a measurement electrode, a counter electrode, a reference electrode, a conductive pattern and a terminal as shown in FIG. Thereafter, the insulating substrate was cut out to a predetermined size to produce a sensor substrate. Two such sensor substrates were produced. The electrode area of the measurement electrode is 2.0 mm ² .

  Subsequently, after applying a silver chloride paste (DB2275 manufactured by Japan Atchison Co., Ltd.) to the surface of the reference electrode in each sensor substrate, this was dried at 80 ° C. for 30 minutes, and the silver / silver chloride electrode was applied to the surface of the reference electrode Formed.

  Then, the measurement reagent was dried and supported on one sensor substrate as follows. A part where cavities are formed in a later process, and a mixture of 0.4 μl of 100 mM potassium ferricyanide and 0.5 μl of 1000 U / ml diaphorase on the surface of the sensor substrate other than the surfaces of the measurement electrode, counter electrode and reference electrode After the solution was added dropwise, it was dried at 40 ° C. for 10 minutes. Hereinafter, this substrate is referred to as a reagent carrying sensor substrate A.

  Further, the above-mentioned mixed solution was used for the other sensor substrate in the same manner, except that the measurement reagent was dried and supported on the surface of the measurement electrode. Hereinafter, this substrate is referred to as a reagent carrying sensor substrate B.

  Finally, two housing substrates each having a recess formed by cutting a predetermined portion of the polymethyl methacrylate resin plate are prepared, and the above-described reagent-carrying sensor substrates A and B are made using silicon resin (KE44 manufactured by Shin-Etsu Silicone Co., Ltd.). The biosensor as shown in FIG. 3 was completed. In the following, the biosensor provided with the reagent-carrying sensor substrate A (example sample 1) is referred to as biosensor A, and the biosensor provided with the reagent-carrying sensor substrate B (comparative sample 1) is referred to as biosensor B.

  Here, as a measurement sample, 40 μl of 400 μM NADH / 50 mM Tris-HCl buffer was injected from the injection holes of the biosensors A and B, respectively, and then a potential of +400 mV was applied to the reference electrode in an atmosphere at 30 ° C. In this case, the value of the current flowing through the measurement electrode was measured.

  Table 1 shows the current values measured 5 minutes after the injection of the measurement sample in each biosensor.

  As shown in Table 1, biosensor B detected only a weak current value of 39 nA, but biosensor A was able to detect a current value as large as 468 nA.

(Example 2)
Example 2 is an experimental example in which a current derived from the activity of lactate dehydrogenase (hereinafter sometimes referred to as LDH), which is an enzyme contained in blood or the like in a living body, was measured with a biosensor.

The biosensor used in Example 2 was produced as follows. First, after preparing a polycarbonate plate having a thickness of 0.5 mm as an insulating substrate, a 2000 angstrom (200 nm) gold thin film was formed on the surface thereof by sputtering. Next, this gold thin film was etched to form a measurement electrode, a counter electrode, a reference electrode, a conductive pattern and a terminal as shown in FIG. Thereafter, the insulating substrate was cut out to a predetermined size to produce a sensor substrate. Two such sensor substrates were produced. The electrode area of the measurement electrode is 2.0 mm ² .

  Subsequently, a conductive paste (D-550 manufactured by Fujikura Kasei Co., Ltd.) was applied to the surface of the reference electrode in each sensor substrate, and then dried at 25 ° C. for 3 hours to form a composite containing silver. . Thereafter, 5% sodium hypochlorite and 0.5% sodium hydroxide are dropped on the surface of the composite and left for 10 minutes to make the composite surface silver chloride. The surface of the reference electrode is silver / silver chloride. An electrode was formed.

Then, the measurement reagent was dried and supported on one sensor substrate as follows. On the surface of the sensor substrate where reaction cavities are formed in the subsequent step, 0.4 μl of 100 mM potassium ferricyanide, 0.5 μl of 1000 U / ml diaphorase, 0.4 μl of 1M lithium lactate, and 100 mM nicotinamide A mixed solution of 0.4 μl of oxidized dinucleotide (NAD ⁺ ) was added dropwise and dried in vacuum at room temperature (25 ° C.) for 1 hour. Hereinafter, this substrate is referred to as a reagent carrying sensor substrate C.

  Further, the above-mentioned mixed solution was used for the other sensor substrate in the same manner, except that the measurement reagent was dried and supported on the surface of the measurement electrode. Hereinafter, this substrate is referred to as a reagent carrying sensor substrate D.

  Finally, two housing substrates each having a recess formed by cutting a predetermined portion of the polycarbonate plate are prepared, and the above-described reagent-carrying sensor substrates C and D are prepared using silicon resin (Silex (R) clear manufactured by Konishi Co., Ltd.). The biosensors as shown in FIG. 5 were completed by bonding them to each other. In the following, the biosensor provided with the reagent-carrying sensor substrate C (example sample 2) is referred to as biosensor C, and the biosensor provided with the reagent-carrying sensor substrate D (comparative example sample 2) is referred to as biosensor D.

  Here, as a measurement sample, 40 μl of 400 U / L LDH / 50 mM Tris-HCl buffer was injected from the injection holes of the biosensors C and D, and reacted in an atmosphere at 30 ° C. for 5 minutes. After the reaction solution was moved to the measurement cavity, a potential of +400 mV was applied to the reference electrode, and the value of the current flowing through the measurement electrode was measured.

  Table 2 shows current values measured 5 minutes after injection of the measurement sample in each biosensor.

  As shown in Table 2, Biosensor D detected only a weak current value of 32 nA, but Biosensor C was able to detect a current value as large as 375 nA.

  INDUSTRIAL APPLICABILITY The present invention can be applied to provide a high-sensitivity biosensor in which a decrease in enzyme activity is prevented and a decrease in electrode sensitivity is suppressed. In particular, the sensitivity of a disposable biosensor to which μTAS is applied is improved. It is particularly preferable.

It is a perspective view which shows an example of the sensor board | substrate in the biosensor of this invention. It is a perspective view which shows another example of the sensor board | substrate in the biosensor of this invention. It is a perspective view which shows an example of the biosensor of this invention. It is a perspective view which shows an example of the housing substrate in the biosensor of this invention. It is a perspective view which shows another example of the biosensor of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Insulating substrate 2 Measuring electrode 3 Counter electrode 4 Reference electrode 5 Conductive pattern 6 Terminal part 7 Sensor substrate 21 Insulating film 31 Housing substrate 32 Reagent for measurement 33 Cavity 34 Injection hole 35 Air hole 36 Recessed part 41 Measurement cavity 42 Reaction cavity 43 Flow path 51 Housing substrate

Claims (7)

A sensor substrate including an insulating substrate and two or more electrodes formed on the insulating substrate;
A cavity formed on the sensor substrate and including the electrode therein;
An injection hole for supplying a measurement sample into the cavity;
A measuring reagent containing a measuring enzyme held in the cavity;
Including
The measurement reagent is held in a region in the cavity other than the surface of the electrode,
Biosensor.   The biosensor according to claim 1, wherein the measurement reagent is dried and supported in a region in the cavity.   The biosensor according to claim 2, wherein the measurement reagent is dried and supported on the surface of the sensor substrate. The cavity is formed by the sensor substrate and the housing substrate;
The biosensor according to claim 2, wherein the measurement reagent is dried and supported on a surface of the housing substrate.   The biosensor according to claim 2, wherein the measurement enzyme includes an oxidoreductase.   The biosensor according to claim 5, wherein the oxidoreductase contains diaphorase. A sensor substrate including an insulating substrate and two or more electrodes formed on the insulating substrate;
A measurement cavity formed on the sensor substrate and including the electrode therein;
One or more reaction cavities formed on the sensor substrate;
A flow path connecting at least one of the reaction cavity and the measurement cavity;
An injection hole for supplying a measurement sample into at least one of the reaction cavities;
Including
A measuring reagent containing a measuring enzyme is held in at least one region in the reaction cavity and is not held on the surface of the electrode;
Biosensor.

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