JP2012208101A - Biosensor with multilayer structure - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biosensor having a superior storage stability and capable of measuring a concentration of a specific component such as neutral fat in a sample.SOLUTION: The biosensor according to the present invention includes: an insulation substrate; an electrode system formed on the insulation substrate and including at least a working electrode and a counter electrode; and a sample supply section formed on the electrode system. The sample supply section includes an intermediate layer formed on the electrode system, and a reaction layer formed on the intermediate layer. The intermediate layer includes a surfactant layer containing surfactant, and a hydrophilic polymer layer containing hydrophilic polymer. The reaction layer includes, as a prosthetic group, at least oxidoreductase including pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), and lipolytic enzyme.

Description

  The present invention relates to a biosensor that quantifies the concentration of a specific component in a specific sample such as a biosensor or a biological sample using an enzyme reaction. In particular, the present invention relates to a biosensor for measuring neutral fat concentration, and relates to a biosensor for measuring neutral fat concentration having excellent storage stability.

  In recent years, biosensors have been applied in fields such as medicine. Biosensors are various chemical substances ranging from low molecules to macromolecules, and biosensors having various functions are being developed according to the measurement objects.

  2. Description of the Related Art Conventionally, biosensors that can easily quantify specific components (substrates) contained in biological samples and foods without dilution or stirring are known. For example, an enzyme reaction layer in which an electrode system having at least a working electrode and a counter electrode is formed on an insulating substrate, and an oxidoreductase and an electron acceptor are immobilized on the electrode system with a fixing agent such as a hydrophilic polymer. Next, a biosensor has been proposed in which a filtration layer (blood cell removal layer) is provided on the enzyme reaction layer, and a cover is covered from the filtration layer.

  This biosensor quantifies the substrate concentration in a sample by the following method. First, a sample solution such as blood is dropped onto the filtration layer, and the filtrate penetrates into the enzyme reaction layer. Thereby, the oxidoreductase and the electron acceptor are dissolved in the sample solution, and the enzyme reaction proceeds between the substrate and the enzyme. This enzymatic reaction oxidizes the substrate and simultaneously reduces the electron acceptor. After the completion of the enzyme reaction, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the sample solution is obtained from the oxidation current value obtained at this time.

  For example, as a method for measuring neutral fat with a biosensor, a method for quantifying neutral fat in a sample as described below is known. First, the neutral fat contained in the sample solution is decomposed into free fatty acid and glycerol by, for example, lipoprotein lipase (LPL). Glycerol produced here is glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) or glycerol-3-phosphate dehydrogenase (GPDH) as shown in the following formulas (1) and (2). Can be quantified. That is, glycerol can be quantified by measuring the decrease of the oxidized electron acceptor, the increase of the reduced electron acceptor or the amount of dihydroxyacetone phosphate represented by the following formula. In particular, glycerol can be quantified by electrochemically measuring the increased amount of reduced electron acceptor.

  However, the three types of enzymes, lipoprotein lipase (LPL), glycerol kinase (GK), and glycerol-3-phosphate oxidase (GPO), used for the measurement of neutral fat are all expensive.

  In order to solve such a problem, a biosensor that reduces the cost of an enzyme by using two types of enzymes, ie, a neutral lipolytic enzyme and glycerol dehydrogenase (GLDH), as an enzyme used for a neutral lipolytic reaction. (Patent Document 1). However, the biosensor of Patent Document 1 cannot be said to have sufficient measurement time and accuracy, and further higher accuracy and faster measurement are desired.

  On the other hand, a method using NAD + -dependent glycerol dehydrogenase (NAD-GLDH) is known as a method using one kind of enzyme without being affected by dissolved oxygen, as shown in the following formula (3).

  However, this reaction requires the addition of expensive NAD +.

As a method for quantifying glycerol more easily and cheaply, there is a method using polyol dehydrogenase (PQQ-PDH) containing pyrroloquinoline quinone as a prosthetic group.
Since this method is carried out by the reaction of the following formula (4), there are merits that it is not affected by dissolved oxygen, the reaction is simple and it is not necessary to use a plurality of enzymes, and it is not necessary to add expensive NAD +. is there.

  In view of the above, a biosensor in which neutral lipolytic enzyme and glycerol dehydrogenase are arranged in different layers has been reported for the purpose of a biosensor capable of measuring neutral fat in a short time and with high accuracy (Patent Document 2, 3). Here, the biosensor of Patent Document 2 has a structure in which two reaction layers of a polymer layer containing GLDH and a hydrophilic polymer and a filter paper layer supporting neutral lipolytic enzyme are sequentially stacked on an electrode. Have. In addition, the biosensor of Patent Document 3 has a structure in which two reaction layers of a polymer layer containing GLDH and a hydrophilic polymer and a nonwoven fabric layer carrying a neutral lipolytic enzyme are sequentially laminated on the electrode. It is characterized by that.

  However, the biosensor as described above may be manufactured in a disposable form because, for example, the method of use is simple. Since such a biosensor may be stored for a long time in a state containing an enzyme, storage stability is required. Therefore, in order to exhibit sufficient measurement accuracy even after long-term storage, further storage stability of the biosensor is required.

International Publication No. 2006/104077 Pamphlet JP 2009-244013 A JP 2009-244014 A

  However, the biosensors described in Patent Documents 1 to 3 have variations in storage stability and still have room for improvement.

  Therefore, the present invention has been made to solve the above-mentioned problems, and is a biosensor exhibiting excellent storage stability that can withstand long-term storage, particularly useful for measuring neutral fat concentration, and storage stability. An object of the present invention is to provide an excellent biosensor.

  The present inventors have intensively studied to solve the above problems. As a result, the biosensor has a structure in which an intermediate layer and a reaction layer are sequentially laminated on the electrode system, that is, by interposing the intermediate layer between the electrode and the reaction layer containing the enzyme, It has been found that the storage stability is improved. Based on such knowledge, the present inventors have completed the present invention.

That is, the above-described object is to provide a bio, which has an insulating substrate, an electrode system including at least a working electrode and a counter electrode formed on the insulating substrate, and a sample supply unit formed on the electrode system. A sensor,
The sample supply unit is
An intermediate layer formed on the electrode system, and a reaction layer formed on the intermediate layer,
The intermediate layer includes a surfactant layer containing a surfactant and a hydrophilic polymer layer containing a hydrophilic polymer,
A biosensor in which the reaction layer includes at least an oxidoreductase containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group, and a lipolytic enzyme Achieved by:

  The biosensor of the present invention exhibits excellent storage stability after long-term storage. By improving the storage stability, it is not necessary to increase the amount of the enzyme in consideration of the enzyme that is inactivated by storage, and the cost for manufacturing the biosensor can be reduced.

It is a disassembled perspective view which shows one Embodiment of the biosensor of this invention. It is sectional drawing of the 2-2 direction of the biosensor of FIG. It is a disassembled perspective view which shows another embodiment of the biosensor of this invention. FIG. 4 is a cross-sectional view of the biosensor of FIG. 3 in the 4-4 direction. It is a disassembled perspective view which shows another embodiment of the biosensor of this invention. FIG. 6 is a cross-sectional view of the biosensor of FIG. 5 in the 6-6 direction. It is a graph which shows the enzyme activity after the preservation | save of the oxidoreductase of Example 1, the comparative example 1, and the comparative example 2. FIG. It is a graph which shows the enzyme activity after the preservation | save of a lipolytic enzyme of Example 1, Comparative Example 1, and Comparative Example 2.

  The present invention is a biosensor having an insulating substrate, an electrode system including at least a working electrode and a counter electrode formed on the insulating substrate, and a sample supply unit formed on the electrode system. The sample supply unit includes an intermediate layer formed on the electrode system and a reaction layer formed on the intermediate layer, and the intermediate layer includes a surfactant. And a hydrophilic polymer layer containing a hydrophilic polymer, and the reaction layer contains at least pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group A biosensor comprising an oxidoreductase and a lipolytic enzyme is provided.

  There are various reasons why biosensors deteriorate due to long-term storage, and they are complicated and difficult to grasp simply. The present inventors have improved the storage stability of a biosensor by interposing an intermediate layer containing a surfactant layer and a hydrophilic polymer layer between a reaction layer containing an enzyme and an electrode. Successful. This is reflected in the improvement of the residual activity of the enzyme, as specifically shown in the examples described later. Enzymes are generally sensitive to environmental changes, are easily inactivated by heat, pH, and other compounds that come into contact with them, and a certain amount of enzyme is required for accurate measurement with a biosensor. Yes. In the biosensor of the present invention, the mechanism for achieving storage stability is not clear in detail, but an oxidoreductase is fixed to the electrode by providing a surfactant layer as an intermediate layer between the electrode and the electrode. This is considered to be due to the fact that the storage stability of the biosensor was further improved as a result of which it was possible to significantly suppress / prevent this, thereby suppressing / preventing electrode passivation and deactivation of the immobilized enzyme. At that time, it is important that the intermediate layer includes a surfactant layer and a hydrophilic polymer layer, and the order of the two layers can obtain the effect of storage stability regardless of which is the upper layer. Further, by adding a hydrophilic polymer, sugar, and protein to the reaction layer containing the enzyme, the storage stability of the enzyme is improved, and thus the storage stability of the biosensor is improved. As a mechanism for improving the storage stability by these additives, hydrophilic polymers prevent the inactivation by maintaining the three-dimensional structure of the enzyme by comprehensively immobilizing the enzyme, and sugar is the water present in the vicinity of the enzyme. As the water is removed during the drying process, deactivation is prevented by maintaining the enzyme's three-dimensional structure as a surrogate water, and the protein is concentrated into the enzyme by water molecules and other contaminants by increasing the protein concentration of the entire system. It is thought that the inactivation of the enzyme is prevented by mitigating the inactivation action.

  Hereinafter, embodiments of the biosensor of the present invention will be specifically described with reference to the drawings. In addition, this invention is not limited to the following embodiment, unless it deviates from the concept prescribed | regulated by the following claim. In addition, the dimensional ratios in the drawings are exaggerated for convenience of explanation, and may be different from the actual ratios.

  FIG. 1 is an exploded perspective view showing one embodiment (hereinafter, also referred to as a first embodiment) of the biosensor of the present invention. 2 is a cross-sectional view of the biosensor of FIG. 1 in the 2-2 direction.

  As shown in FIGS. 1 and 2, an electrode system including a working electrode 2, a reference electrode 3 and a counter electrode 4 is formed on an insulating substrate 1 (also simply referred to as “substrate” in the present specification). In addition, the said electrode system should just contain a working electrode and a counter electrode at least. For this reason, the reference electrode 3 can be omitted. In addition, the adhesive 6 is installed at the end on the insulating substrate 1. The working electrode 2, the reference electrode 3 and the counter electrode 4 function as means for electrically connecting the biosensor. The working electrode 2, the reference electrode 3, and the counter electrode 4 can form electrodes having a desired pattern by appropriately referring to or combining conventionally known knowledge such as screen printing / sputtering method.

  An insulating layer 5 is formed on the working electrode 2, the reference electrode 3 and the counter electrode 4 formed on the insulating substrate 1 so as to expose the electrode system. The insulating layer 5 functions as an insulating means for preventing a short circuit between the electrodes. There is no restriction | limiting in particular also about the formation method of an insulating layer, It can form by conventionally well-known methods, such as a screen printing method and an adhesion method.

  Further, a working electrode working part 2-1, a reference electrode working part 3-1, and a counter electrode working part 4-1 are formed so as to sandwich the insulating layer 5. An intermediate layer 13 is formed on the working electrode working part 2-1, the reference electrode working part 3-1, and the counter electrode working part 4-1. The intermediate layer 13 is composed of a surfactant layer 11 and a hydrophilic polymer layer 12. Furthermore, a reaction layer 10 containing an enzyme involved in the measurement is formed on the intermediate layer 13. In the embodiment of FIG. 1, the intermediate layer 13, the reaction layer 10, and the space S located between the reaction layer 10 and the cover 7 form a sample supply unit.

  Of the intermediate layer 13, the surfactant layer 11 includes a surfactant and is formed to cover the working electrode working part 2-1, the reference electrode working part 3-1, and the counter electrode working part 4-1. The hydrophilic polymer layer 12 includes a hydrophilic polymer and is formed on the surfactant layer 11. In the embodiment shown in FIGS. 1 and 2, the intermediate layer 13 has a configuration in which the surfactant layer 11 and the hydrophilic polymer layer 12 are laminated in this order on the electrode system. The molecular layer 12 may be laminated in reverse.

  The reaction layer 10 is an oxidoreductase containing at least pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group (hereinafter also referred to as “the oxidoreductase of the present invention”). ) And lipolytic enzymes.

  In the biosensor of this embodiment, it is preferable that the sample supply unit further includes an electron carrier. The electron carrier may be present in the sample supply unit in any form. Specifically, an electron carrier may be included in the reaction layer 10, or a layer including the electron carrier may be separated from the reaction layer 10 and provided on the cover 7 side. Among these, since the electron carrier may be reduced upon contact with the oxidoreductase during storage, the electron carrier is separated from the reaction layer 10 and arranged in the sample supply unit, that is, including the electron carrier. The layer is preferably provided on the cover 7 side separately from the reaction layer 10.

  The action part (2-1, 3-1, 4-1) detects a current flowing in the potential applying means and a potential applying means for applying a potential to the sample in the reaction layer 10 when the biosensor is used. Functions as current detection means. In addition, the working part (2-1, 3-1, 4-1) may be referred to as the working electrode 2, the reference electrode 3, and the counter electrode 4. The working electrode 2 and the counter electrode 4 are paired when the biosensor is used, and function as current measuring means for measuring an oxidation current (response current) that flows when a potential is applied to the sample in the reaction layer 10. When the biosensor is used, a predetermined potential is applied between the counter electrode 4 and the working electrode 2 with the reference electrode 3 as a reference.

  The biosensor of the present embodiment is configured by bonding a cover 7 so as to cover the reaction layer 10 via an adhesive (double-sided tape) 6 installed on the substrate 1. The adhesive (double-sided tape) 6 may be installed on the electrode side, may be installed only on the cover 7 side, or may be installed on both. The biosensor of the first embodiment has an advantage that it can be easily manufactured.

FIG. 3 is an exploded perspective view showing another embodiment (hereinafter also referred to as a second embodiment) of the biosensor of the present invention. 4 is a cross-sectional view of the biosensor of FIG. 3 in the 4-4 direction. The second embodiment is
The reaction layer 10 is the same as the embodiment shown in FIGS. 1 and 2 except that the reaction layer 10 includes the first reaction layer 8 and the second reaction layer 9. In the present embodiment, the reaction layer 10 is referred to as a first reaction layer 8 and a second reaction layer 9 sequentially from the electrode side. In the present embodiment, the first reaction layer 8 and the second reaction layer 9 constituting the reaction layer 10 and the space S positioned between the second reaction layer 9 and the cover 7 are the sample supply unit. Form. Among the reaction layers 10, the first reaction layer 8 includes an oxidoreductase, and the second reaction layer 9 includes a lipolytic enzyme.

  The first reaction layer 8 comprises at least an oxidoreductase (hereinafter referred to as “oxidoreductase of the present invention”) containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group. ").

  The second reaction layer 9 is formed on the first reaction layer 8 and is formed by applying a solution containing at least a lipolytic enzyme. In the present specification, “the second reaction layer is formed by applying a solution containing a lipolytic enzyme” means that the lipid-decomposing enzyme is not supported on a carrier such as a filter paper or a non-woven fabric. It means that a coating film is formed by applying a solution containing a degrading enzyme directly to the first reaction layer and drying.

  In the biosensor of this embodiment, the second reaction layer is directly formed by applying a solution containing a lipolytic enzyme on the first reaction layer containing the oxidoreductase. Usually, lipolytic enzymes are more soluble than oxidoreductases. Therefore, when a sample containing fat such as neutral fat passes through the sample supply unit, the lipolytic enzyme in the second reaction layer dissolves quickly by contact with the sample, and decomposes and releases the fat in the sample. Produces fatty acids and glycerol. This reaction is completed in about the same time as the dissolution of the lipolytic enzyme is completed. Next, since the oxidoreductase exists as the first reaction layer between the second reaction layer and the electrode, the lipolytic enzyme in the second reaction layer starts to dissolve after the dissolution is completed. The sample from which the lipid has been decomposed quickly penetrates into the first reaction layer, dissolves the oxidoreductase, and produces a reduced electron carrier from the produced glycerol. The biosensor electrochemically measures the increase amount of the reduced electron carrier. Therefore, the biosensor of this embodiment can quantify fat in the sample in a shorter time.

  Also in the biosensor of this embodiment, it is preferable that the sample supply unit further includes an electron carrier. The electron carrier in the present embodiment may be present in the sample supply unit in any form. Specifically, (a) the first reaction layer 8 includes an electron carrier, (b) the second reaction layer 9 includes an electron carrier, and (c) the layer including the electron carrier reacts. The form which isolate | separates from the layer 10 (the 1st reaction layer 8, the 2nd reaction layer 9), and is provided in the cover 7 side is mentioned. Any of these forms (a) to (c) may be applied, or two or more of the above forms (a) to (c) may be applied in combination. Among these, since the electron carrier may be reduced upon contact with the oxidoreductase during storage, the electron carrier and the oxidoreductase are not included in the same layer, that is, (A) or (C) Is more preferable. According to such a form, the electron carrier can be prevented from being reduced by contacting the oxidoreductase during storage, which can contribute to the storage stability of the biosensor.

  In the biosensor of this embodiment, the cover 7 is provided so as to cover the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) via the adhesive (double-sided tape) 6 installed on the substrate 1. It is configured by bonding. The adhesive (double-sided tape) 6 may be installed on the electrode side, may be installed only on the cover 7 side, or may be installed on both.

  FIG. 5 is an exploded perspective view showing another preferred embodiment (hereinafter also referred to as a third embodiment) of the biosensor of the present invention. 6 is a cross-sectional view of the biosensor of FIG. 5 in the 6-6 direction.

  The biosensor of the third embodiment is the same as the biosensor shown in FIGS. 3 and 4 except that the upper surfactant layer 14 is provided, as shown in FIGS. In this embodiment, the intermediate layer 13, the first reaction layer 8, the second reaction layer 9, the upper surfactant layer 14, and between the second reaction layer 9 and the upper surfactant layer 14. And the space part S arranged in the above form a sample supply part.

  In the biosensor of this embodiment, the upper surfactant layer 14 is separated from the first reaction layer 8 and the second reaction layer 9 and is formed in the sample supply section on the cover 7 side. The upper surfactant layer 14 is formed in a gap between both ends on the cover 7 on which adhesive (double-sided tape) 6a is installed at both ends. When the upper surfactant layer 14 is formed on the cover 7 side, it is more familiar from the viewpoint of spreading and wettability with a sample such as whole blood than when the cover 7 is in direct contact with the sample, and the sample is supplied to the sample. It is preferable because it can be quickly introduced into the part.

  Also in the biosensor of this embodiment, it is preferable that the sample supply unit further includes an electron carrier. The electron carrier in the present embodiment may be present in the sample supply unit in any form. Specifically, (a) the first reaction layer 8 includes an electron carrier, (b) the second reaction layer 9 includes an electron carrier, and (c) the upper surfactant layer 14 includes electrons. The form etc. which are provided with the layer containing a transmission body or containing an electron transmission body further are mentioned. Any of these forms (a) to (c) may be applied, or two or more of the above forms (a) to (c) may be applied in combination. Of the above forms, (i) or (c) is more preferred. Among these, since the electron carrier may be reduced upon contact with the oxidoreductase during storage, the electron carrier and the oxidoreductase are not included in the same layer, that is, (A) or (C) Is more preferable. According to such a form, it is possible to prevent the electron carrier from being reduced by contact with the oxidoreductase during storage, which can contribute to the improvement of the measurement accuracy of the biosensor.

  The biosensor of this embodiment includes an adhesive (double-sided tape) 6b adhered to the substrate 1 on which the intermediate layer 13 and the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) are formed, An adhesive (double-sided tape) 6a adhered to the cover 7 on which the upper surfactant layer 14 is formed is configured to be bonded together. The adhesive (double-sided tape) 6 may be installed only on the substrate 1 side, or may be installed only on the cover 7 side.

  Hereinafter, each component will be described in detail. In addition, as for the structure common to the biosensors of the first to third embodiments or the structure common to the second and third embodiments, unless otherwise specified, the specific description of each component is the same structure. The present invention is also applied to the biosensor according to the embodiment.

  In addition, the term “1 sensor” may be used in describing the content of each constituent element. In this specification, “1 sensor” refers to the size of a general biosensor, sample supply. It is assumed that the sample supplied to the section is “0.1 to 20 μl (preferably about 1 μl)”. Therefore, in a biosensor that is smaller or larger than that, the present invention can be applied by appropriately adjusting the content of each constituent element.

<Insulating substrate>
The insulating substrate 1 used in the present invention is not particularly limited, and a conventionally known substrate can be used. An example is plastic, paper, glass, ceramics, and the like. Further, the shape and size of the insulating substrate 1 are not particularly limited.

  There is no restriction | limiting in particular also as a plastic, A conventionally well-known thing can be used. For example, polyethylene terephthalate (PET), polyester, polystyrene, polypropylene, polycarbonate, polyimide, acrylic resin, etc. can be used.

<Electrode>
The electrode of the present invention includes at least a working electrode 2 and a counter electrode 4.

  The electrode of the present invention is not particularly limited as long as it can electrochemically detect the reaction between the sample (object to be measured) and the oxidoreductase of the present invention. For example, a carbon electrode, a gold electrode, a silver electrode, A platinum electrode, a palladium electrode, etc. are mentioned. From the viewpoint of corrosion resistance and cost, a carbon electrode is preferred.

  In the present invention, a two-electrode system including only the working electrode 2 and the counter electrode 4 or a three-electrode system further including the reference electrode 3 may be used. Note that the three-electrode method can be preferably used rather than the two-electrode method from the viewpoint that the potential is controlled with higher sensitivity. In addition, a sensing electrode for sensing the amount of liquid may be included.

  Moreover, the part (action part) which contacts a sample supply part may differ from an electrode part other than that and a constituent material. For example, when the reference electrode 3 is made of carbon, the reference electrode action part 3-1 may be made of silver / silver chloride. In addition, since a biosensor is generally disposable, it is good to use a disposable electrode as an electrode.

<Insulating layer>
Although the material which comprises the insulating layer 5 is not restrict | limited in particular, For example, it can comprise with resist ink, resin, such as PET and polyethylene, glass, ceramics, paper, etc. Preferably, it is PET.

<Sample supply unit>
In the biosensor according to the first embodiment of the present invention, the sample supply unit includes the intermediate layer 13 including the surfactant layer 11 and the hydrophilic polymer layer 12, the reaction layer 10, and the space S into which the sample is introduced. Consists of. In the biosensor of the second embodiment, the sample supply unit includes the intermediate layer 13 including the surfactant layer 11 and the hydrophilic polymer layer 12, the first reaction layer 8 including the oxidoreductase, and the lipolytic enzyme. The second reaction layer 9 and the space S are included. In the biosensor of the third embodiment, the sample supply unit includes an intermediate layer 13 including a surfactant layer 11 and a hydrophilic polymer layer 12, a first reaction layer 8 including an oxidoreductase, and a lipolytic enzyme. It has the 2nd reaction layer 9 containing, the upper surface active agent layer 14, and the space part S.

  In the biosensor of the present invention, the intermediate layer 13 (surfactant layer 11 and hydrophilic polymer layer 12) includes the electrode system described above and a reaction layer 10 (first reaction layer 8, It is interposed between the second reaction layer 9). That is, the intermediate layer 13 separates the electrode and the enzyme involved in the measurement and prevents both from coming into direct contact. The presence of the intermediate layer improves the storage stability of the biosensor of the present invention.

  The intermediate layer 13 includes a hydrophilic polymer layer 12 containing a hydrophilic polymer and a surfactant layer 11 containing a surfactant. The hydrophilic polymer layer 12 and the surfactant layer 11 are not limited in the order of lamination, and either may be on the electrode side, but the surfactant layer 11 exists between the electrode and the hydrophilic polymer layer 12. Is more preferable.

  In the biosensor of the present invention, the thicknesses of the surfactant layer 11 and the hydrophilic polymer layer 12 are not particularly limited and may be the same or different from each other and can be appropriately selected. The surfactant layer 11 is preferably 0.01 to 15 μm, more preferably 0.025 to 10 μm, and particularly preferably 0.05 to 8 μm. The thickness of the hydrophilic polymer layer 12 is preferably 0.01 to 15 μm, more preferably 0.025 to 10 μm, and particularly preferably 0.05 to 8 μm. Furthermore, the total thickness of the entire intermediate layer is preferably 0.02 to 30 μm, more preferably 0.05 to 20 μm, and particularly preferably 0.1 to 16 μm. Although there is no restriction | limiting in particular as the thickness control method in this case, For example, it can control by adjusting suitably the application quantity (for example, dripping quantity) of the solution containing a predetermined component.

  In the biosensor of the first embodiment, the thickness of the reaction layer 10 is not particularly limited, but is preferably 0.01 to 50 μm, more preferably 0.05 to 40 μm, and particularly preferably 0.1 to 25 μm. Good. Although there is no restriction | limiting in particular as a thickness control method in this case, For example, it can control by adjusting the dripping quantity suitably. In the form which has one reaction layer like the biosensor of 1st embodiment, it is preferable at the point which can produce a biosensor simply. Moreover, it is preferable at the point that the manufacturing cost at the time of mass production becomes cheap.

  In the biosensors of the second and third embodiments, the thicknesses of the first reaction layer 8 and the second reaction layer 9 are not particularly limited and can be appropriately selected so that the total becomes the thickness of the normal reaction layer. . The first reaction layer 8 is preferably 0.01 to 25 μm, more preferably 0.025 to 10 μm, and particularly preferably 0.05 to 8 μm. The thickness of the second reaction layer 9 is preferably 0.01 to 25 μm, more preferably 0.025 to 10 μm, and particularly preferably 0.05 to 8 μm. Furthermore, the total thickness of the first reaction layer 8 and the second reaction layer 9 is preferably 0.02 to 50 μm, more preferably 0.05 to 20 μm, and particularly preferably 0.1 to 16 μm. . Although there is no restriction | limiting in particular as the thickness control method in this case, For example, it can control by adjusting suitably the application quantity (for example, dripping quantity) of the solution containing a predetermined component.

  In the biosensor of the third embodiment, the upper surfactant layer 14 formed on the cover 7 side separately from the reaction layer 10 (first reaction layer 8 and second reaction layer 9) Although there is no restriction | limiting in particular in thickness, 0.01-25 micrometers is preferable, More preferably, it is 0.025-10 micrometers, More preferably, it is 0.05-8 micrometers. The separation distance between the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) and the upper surfactant layer 14 is preferably 0.1 to 250 μm, more preferably 1 to 200 μm, and still more preferably 10 ~ 150 μm. If it is the said range, a capillary phenomenon will occur easily and a sample will be easy to be introduced into a sample supply part. The separation distance can be controlled by controlling the thickness of the adhesive. That is, the adhesive also serves as a spacer that separates the second reaction layer 9 and the upper surfactant layer 14.

  Moreover, in the biosensor of the first embodiment, when the layer containing the electron carrier is formed on the cover 7 side by being separated from the reaction layer 10, the thickness is not particularly limited, but 0.01 to 30 micrometers is preferable, More preferably, it is 0.025-25 micrometers, More preferably, it is 0.05-8 micrometers. The separation distance between the reaction layer 10 and the layer containing the electron carrier is preferably 0.1 to 250 μm, more preferably 1 to 200 μm, and still more preferably 10 to 150 μm. If it is the said range, a capillary phenomenon will occur easily and a sample will be easy to be introduced into a sample supply part.

(Surfactant)
In the biosensor of the present invention, the intermediate layer 13 includes a surfactant layer 12 including a surfactant layer. In the biosensor of the first embodiment, the reaction layer 10 may contain a surfactant, or may be contained in a layer including an electron carrier formed separately from the reaction layer 10, An upper surfactant layer 14 containing a surfactant may be separated from the reaction layer 10 and formed on the cover 7 side. In the biosensors of the second and third embodiments, at least one of the first reaction layer 8 and the second reaction layer 9 may contain a surfactant. Moreover, in 3rd embodiment, it isolate | separates from the 2nd reaction layer 9, and the upper surface active agent layer 14 is formed in the cover 7 side.

  Since the enzyme is composed of a protein, there is a possibility that the electrode surface is passivated when it is attached to the electrode surface. However, in the present invention, by providing the surfactant layer 11 as the intermediate layer 13 between the reaction layer 10 (first reaction layer 8) and the electrode, the oxidoreductase is significantly fixed to the electrode. It is possible to suppress / prevent, thereby suppressing / preventing electrode passivation and deactivation of the immobilized enzyme, and as a result, the storage stability of the biosensor is further improved. Furthermore, it is possible to improve the conversion efficiency of the oxidized electron mediator to the reduced electron mediator by the oxidoreductase in the vicinity of the electrode, in other words, to increase the correlation with the substrate concentration in the sample solution. it can. In addition, when the upper surfactant layer 14 is formed on the cover 7 side, it is more familiar from the viewpoint of spreading and wettability with a sample such as whole blood than when the cover 7 is in direct contact with the sample. This is preferable because it can be quickly introduced into the sample supply section.

  The surfactant used in the present invention is not particularly limited as long as the enzyme activity of the oxidoreductase of the present invention to be used is not lowered. For example, a nonionic surfactant, an amphoteric surfactant, a positive surfactant is used. An ionic surfactant, an anionic surfactant, a natural surfactant and the like can be appropriately selected and used. These may be used alone or in the form of a mixture. Preferably, it is at least one of a nonionic surfactant and an amphoteric surfactant from the viewpoint of not affecting the enzyme activity of the oxidoreductase of the present invention.

  Although it does not restrict | limit especially as a nonionic surfactant, From a viewpoint that it does not affect the enzyme activity of the oxidoreductase of this invention, it is preferable that it is a polyoxyethylene type | system | group or an alkylglycoside type | system | group.

  The polyoxyethylene-based nonionic surfactant is not particularly limited, but polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-octylphenol; Triton (registered) Trademark) X-100)], polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Polyoxyethylene sorbitan monopalitanate; Tween oxyethylene 40) Sorbitan Monosteate; Tween 60 , Polyoxyethylene sorbitan monooleate (Polyoxyethylene Sorbitan Monooleate; Tween 80), polyoxyethylene polyoxypropylene glycol (Emulgen PP-290 (manufactured by Kao Corporation)), sucrose behenate, sucrose stearate, sucrose Palmitic acid ester, sucrose myristic acid ester, sucrose lauric acid ester, sucrose erucic acid ester and sucrose oleic acid ester (Surf Hope (registered trademark) (manufactured by Mitsubishi Chemical Foods)) are preferred. Among them, sucrose behenic acid ester, sucrose stearic acid ester, sucrose palmitic acid ester, sucrose myristic acid ester, sucrose laurin from the viewpoint of significantly preventing adsorption of the oxidoreductase of the present invention to the electrode surface Acid ester, sucrose erucic acid ester and sucrose oleate (Surf Hope (registered trademark) (manufactured by Mitsubishi Chemical Corporation)) are more preferable, and from the viewpoint of increasing the solubility of the oxidoreductase of the present invention, Polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-octylphenol; Triton (registered trademark) X-100)] is more preferable. These may be used alone or in the form of a mixture of two or more.

  The alkylglycoside nonionic surfactant is not particularly limited, but alkylglycoside and alkylthioglycoside having an alkyl group having 7 to 12 carbon atoms are preferable. The carbon number is more preferably 7 to 10, and particularly preferably 8 carbon atoms. The sugar moiety is preferably glucose or maltose, more preferably glucose. More specifically, n-octyl-β-D-glucoside and n-octyl-β-D-thioglucoside are preferable. Alkyl glycoside nonionic surfactants can be applied easily and uniformly during the manufacturing process when used in biosensors. In particular, when n-octyl-β-D-thioglucoside is contained in the reaction layer 10 (first reaction layer 8, second reaction layer 9), the spread when the sample solution is dropped is very good. Good wettability (makes surface tension less likely to occur). Therefore, alkylthioglycoside is very preferable to alkylglycoside from the viewpoint of spread and wettability. These may be used alone or in the form of a mixture.

  The amphoteric surfactant is not particularly limited, and examples thereof include 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS) and 3-[(3-cholamidopropyl) dimethylammoni. O) -2-hydroxypropanesulfonic acid (CHAPSO), n-alkyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid (Zwittergent (registered trademark)), and the like. These may be used alone or in the form of a mixture. Preferably, 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS) or 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonic acid (CHAPSO) ). CHAPS is particularly preferable. This is because CHAPS is a low hemolytic agent among the surfactants.

  The cationic surfactant is not particularly limited, and examples thereof include cetylpyridinium chloride and trimethylammonium bromide. These may be used alone or in the form of a mixture.

  The anionic surfactant is not particularly limited, and examples thereof include sodium cholate and sodium deoxycholate. These may be used alone or in the form of a mixture.

  Although it does not restrict | limit especially as a natural type surfactant, For example, phospholipid is mentioned, Preferably, lecithins, such as egg yolk lecithin, soybean lecithin, hydrogenated lecithin, high purity lecithin, etc. are mentioned. These may be used alone or in the form of a mixture.

  Among the above surfactants, from the viewpoint of further improving the accuracy of the biosensor, it is preferable to use a low hemolytic surfactant when using whole blood as a sample. As specific examples, the above-mentioned Surf Hope (registered trademark) (manufactured by Mitsubishi Chemical Corporation), CHAPS, Tween, Emulgen PP-290 (manufactured by Kao Corporation) (polyoxyethylene polyoxypropylene glycol) are preferable.

  There is no restriction | limiting in particular about content of surfactant, According to the addition amount etc. of a sample, it can adjust suitably.

  In the case of using amphoteric surfactants, it is preferable from the viewpoint that per one sensor, the solubility of the oxidoreductase of the present invention is increased, the enzyme activity is not deactivated, and it is easy to apply in the production process. 0.01 to 100 μg, more preferably 0.05 to 50 μg, and particularly preferably 0.1 to 10 μg may be contained. When a nonionic surfactant is used as the surfactant, the solubility of the oxidoreductase of the present invention is increased per sensor, the enzyme activity is not deactivated, and it is easy to apply in the production process. From the viewpoint, preferably 0.01 to 100 μg, more preferably 0.05 to 50 μg, and particularly preferably 0.1 to 10 μg is included. Such a surfactant is also preferably prepared in a buffer solution such as glycylglycine. In addition, when 2 or more types of surfactant is contained in 1 sensor, or when it contains in 2 or more layers, the total amount is meant.

  Further, in order to form the surfactant layer, it is preferable that the enzyme is prevented from adsorbing to the electrode surface, and that excessive addition may cause hemolysis, and preferably 0.001 to 100 μg, more preferably 0. 0.025 to 50 μg, more preferably 0.05 to 10 μg can be used.

  Further, when the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) contains a surfactant and when the upper surfactant layer 14 is provided, each surfactant contained in these layers. The types may be the same or different. At this time, the surfactant layer, the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9), and the interaction with other constituents contained in the upper surfactant layer 14 are selected. It is preferable to do. The surfactant layer 11 and the upper surfactant layer 14 may contain other components such as sugar, salt, and amino acid as necessary.

  In the biosensor of the present invention, it is preferable that the reaction layer 10 (first reaction layer 8) containing the oxidoreductase contains a surfactant. By including the surfactant, dissolution of the oxidoreductase can be promoted. For example, when PQQ-dependent glycerol dehydrogenase is included as an oxidoreductase, since they are highly hydrophobic, the addition of a surfactant is effective for dissolving the oxidoreductase.

  Further, the layer containing the electron carrier in the sample supply unit preferably contains a surfactant to promote dissolution of the electron carrier. Also in this case, it is preferable to use the above-mentioned low hemolytic surfactant from the viewpoint of spreading and wettability. By applying such a device, the accuracy as a biosensor is further improved.

(Hydrophilic polymer)
The intermediate layer 13 in the biosensor of the present invention includes a hydrophilic polymer layer 12 containing a hydrophilic polymer. In the case of the biosensor of the first embodiment, the reaction layer 10 may contain a hydrophilic polymer. In the case of the biosensors of the second and third embodiments, the hydrophilic polymer may be included in either the first reaction layer 8 or the second reaction layer 9, or may be included in both. . In the case of the biosensor of the third embodiment, a hydrophilic polymer may be included in the upper surfactant layer 14.

  Since the enzyme is composed of protein, there is a possibility that the electrode surface is passivated when it is attached to the electrode surface. In the present invention, by interposing the hydrophilic polymer layer 12 between the electrode and the reaction layer 10 as a part of the intermediate layer 13, it is possible to significantly suppress / prevent the enzyme from adhering to the electrode. It is possible to suppress / prevent the passivation of the electrode and the deactivation of the enzyme due to adhesion to the electrode. As a result, the storage stability is further improved. In addition, the conversion efficiency of the oxidized electron carrier by the enzyme to the reduced electron carrier in the vicinity of the electrode can be improved. In other words, the correlation with the substrate concentration in the sample solution can be further increased. . The hydrophilic polymer layer 12 also serves as an adhesive layer between the reaction layer 10 (first reaction layer 8 and second reaction layer 9) and the electrode. By providing, there also exists an advantage that the reaction layer 10 (the 1st reaction layer 8, the 2nd reaction layer 9) becomes difficult to peel from an electrode. This is effective in increasing the reliability of the biosensor. Further, when a hydrophilic polymer is contained in the reaction layer 10 (first reaction layer 8, second reaction layer 9), these layers are prevented from peeling off from other layers, and Surface cracks can be prevented.

  A conventionally well-known thing can be used as a hydrophilic polymer which can be used for this invention. More specifically, as the hydrophilic polymer, polyamino acids such as carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, and polylysine, Examples thereof include polystyrenesulfonic acid, gelatin and derivatives thereof, acrylic acid polymer or derivatives thereof, maleic anhydride polymer or salts thereof, starch and derivatives thereof, and the like. Of these, carboxymethyl cellulose, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol and derivatives thereof are particularly preferred from the viewpoint that the hydrophilic polymer does not inactivate the enzyme activity of the oxidoreductase of the present invention and has high solubility. . These may be used alone or in the form of a mixture.

  In addition, the blending amount of such a hydrophilic polymer is preferably 0.01 to 100 μg per sensor from the viewpoint of preventing adsorption of the enzyme to the electrode surface and fast solubility in blood per sensor. Preferably it is 0.05-50 micrograms, Most preferably, it is 0.1-10 micrograms. As will be described later, the hydrophilic polymer is preferably prepared in a buffer solution such as glycylglycine. Further, when the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) and the upper surfactant layer 14 contain a hydrophilic polymer, the kind of the hydrophilic polymer contained in these layers May be the same or different. At this time, the hydrophilic polymer is selected in consideration of the interaction with the constituent elements contained in the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) and the upper surfactant layer 14. It is preferable to do. When two or more types of hydrophilic polymers are contained in one sensor, or when contained in two or more layers, the above means the total amount.

  In order to form the hydrophilic polymer layer 12, the hydrophilic polymer is preferably 0.01 to 100 μg from the viewpoint of preventing the enzyme from adsorbing to the electrode surface and rapidly dissolving in blood. More preferably, it is 0.05-50 micrograms, More preferably, 0.1-10 micrograms can be used. The hydrophilic polymer layer 12 may contain other components such as sugar, salt, and amino acid as necessary.

(Oxidoreductase)
The first reaction layer 8 in the present invention comprises an oxidoreductase containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group (also referred to as “coenzyme”). including. In particular, a polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group is preferable. In the present invention, the oxidoreductase of the present invention may be used alone or in the form of a mixture.

  In the present invention, the oxidoreductase containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group is not particularly limited and depends on the type of sample. Examples of redox enzymes containing pyrroloquinoline quinone (PQQ) as a prosthetic group include glycerol dehydrogenase, sorbitol dehydrogenase, mannitol dehydrogenase, arabitol dehydrogenase, galactitol dehydrogenase, xylitol dehydrogenase, adonitol dehydrogenase, erythritol dehydrogenase, ribitol dehydrogenase , Propylene glycol dehydrogenase, fructose dehydrogenase, glucose dehydrogenase, gluconic acid Hydrogenase, 2-ketogluconic acid dehydrogenase, 5 ketogluconic acid dehydrogenase, 2,5 Jiketogurukon dehydrogenase, alcohol dehydrogenase, cyclic alcohol dehydrogenase, acetaldehyde dehydrogenase, amine dehydrogenase, shikimate dehydrogenase, galactose oxidase, and the like.

  Examples of oxidoreductases containing flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group include glucose oxidase, glucose dehydrogenase, D-amino acid oxidase, succinate dehydrogenase, monoamine oxidase, sarcosine dehydrogenase, glycerol dehydrogenase, Examples include sorbitol dehydrogenase, D-lactate dehydrogenase, and cholesterol oxidase.

  Among them, glycerol dehydrogenase containing at least one of pyrroloquinoline quinone (PQQ) or flavin adenine dinucleotide (FAD) is preferred as the prosthetic group, and glycerol dehydrogenase containing pyrroloquinoline quinone (PQQ) as the prosthetic group (hereinafter referred to as “PQQ-dependent”). Particularly preferred is also referred to as “synthetic glycerol dehydrogenase”.

  As the oxidoreductase of the present invention, a commercially available product may be purchased and used, or one prepared by itself may be used. As a method for preparing the oxidoreductase itself, for example, a known method of culturing bacteria producing the oxidoreductase in a nutrient medium and extracting the oxidoreductase from the culture (for example, JP, 2008-220367, A).

  Specifically, taking PQQ-dependent glycerol dehydrogenase as an example, examples of bacteria that produce glycerol dehydrogenase include bacteria belonging to various genera such as Gluconobacter and Pseudomonas. In particular, PQQ-dependent glycerol dehydrogenase present in the membrane fraction of bacteria belonging to the genus Gluconobacter can be preferably used. Among these, Gluconobacter albidas (Gluconobacter albidus) NBRC 3250, 3273, 103509, 103510, 103516, 103520, 103521, 103524; 3276; Gluconobacter frateurii NBRC 3171, 3251, 3253, 3262, 3264, 3265, 3268, 3270, 3285, 3286, 3290, 16669, 103413, 103342, 103427, 103428, 103429, 103437, 103438, 103439, 103440 103441, 103446, 103453, 103454, 103456, 103457, 103458, 103459, 103461, 103462, 103465, 103466, 103467, 103468, 103469, 103470, 103471, 103472, 103347, 103474, 103475, 103476, 103477, 103482, 103487, 103488, 103490, 103491, 103493, 103494, 103499, 103500, 103503, 103502, 103503, 103504, 103506, 103507, 103515, 103517, 103518, 103519, 103523; Gluconobacter japonica ponicus NBRC 3260, 3263, 3269, 3271, 3272; Gluconobacter kanchanaburiensis NBRC 1035887, 103588; Gluconobacter koncter NBRC 3130, 3189, 3244, 3287, 3292, 3293, 3294, 3462, 12528, 14819; Gluconobacter roseus NBRC 3990; Gluconobacter sp NBRC 3259 103508; Gluconobacter sphaericus NBRC 12467; Gluconobacter thylandicus NBRC 3172, 3254, 3255, 3256, 3257, 3258, 3289, 3291, 100600, 60 be able to.

  The medium for cultivating the PQQ-dependent glycerol dehydrogenase may be a synthetic medium or a natural medium as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance, and other necessary nutrients that can be assimilated by the strain used. There may be. As the carbon source, for example, glucose, glycerol, sorbitol and the like are used. Examples of the nitrogen source include nitrogen-containing natural products such as peptones, meat extracts and yeast extracts, and inorganic nitrogen-containing materials such as ammonium chloride and ammonium citrate. As the inorganic substance, potassium phosphate, sodium phosphate, magnesium sulfate or the like is used. In addition, specific vitamins are used as needed. The above carbon source, nitrogen source, inorganic substance, and other necessary nutrients may be used alone or in combination of two or more.

  The culture is preferably performed by shaking culture or aeration and agitation culture. The culture temperature is preferably 20 ° C to 50 ° C, more preferably 22 ° C to 40 ° C, and most preferably 25 ° C to 35 ° C. The culture pH is preferably 4-9, more preferably 5-8. Even under conditions other than these, it is carried out if the strain to be used grows. The culture period is usually preferably 0.5 to 5 days. By the above culture, oxidoreductase is accumulated in the cells. These oxidoreductases may be enzymes obtained by the above culture or recombinant enzymes obtained by transducing oxidoreductase genes into E. coli and the like.

  Next, the obtained PQQ-dependent glycerol dehydrogenase is extracted. As the extraction method, a commonly used extraction method can be used. For example, an ultrasonic crushing method, a French press method, an organic solvent method, a lysozyme method, or the like can be used. The method for purifying the extracted oxidoreductase is not particularly limited. For example, salting-out method such as ammonium sulfate or sodium nitrate, metal aggregation method using magnesium chloride or calcium chloride, denucleic acid using streptomycin or polyethyleneimine, or DEAE (diethylamino) An ion exchange chromatography method such as ethyl) -sepharose or CM (carboxymethyl) -sepharose can be used.

  The partially purified enzyme or purified enzyme solution obtained by these methods may be used as it is or in a chemically modified form. In the present invention, when a chemically modified form of the oxidoreductase is used, the oxidoreductase derived from the culture obtained by the above-described method is, for example, as described in JP-A-2006-271257. It can be used after being appropriately chemically modified using a method or the like. The chemical modification method is not limited to the method described in the above publication.

  There is no restriction | limiting in particular about content of the oxidoreductase of this invention, It can select suitably by the kind of sample to measure, the addition amount of a sample, the kind of electron carrier, the quantity of the hydrophilic polymer mentioned later, etc. . For example, when a PQQ-dependent glycerol dehydrogenase is used, for example, the amount of enzyme (enzyme activity amount) that rapidly decomposes glycerol and does not decrease the solubility of the reaction layer per sensor. From the viewpoint, it is preferably 0.01 to 100 U, more preferably 0.05 to 50 U, and particularly preferably 0.1 to 10 U. The definition and measurement method of the activity unit (U) of the PQQ-dependent glycerol dehydrogenase is based on the method described in JP-A-2006-271257. In addition, an oxidoreductase containing a PQQ-dependent glycerol dehydrogenase will be described later, but it is also preferable to prepare it in a buffer solution such as glycylglycine.

(Lipolytic enzyme)
The reaction layer 10 (second reaction layer 9) in the present invention includes a lipolytic enzyme that hydrolyzes an ester bond constituting the lipid. Therefore, the biosensor of the present invention can be used as a neutral fat sensor. Although it does not restrict | limit especially as such a lipolytic enzyme, Specifically, lipoprotein lipase (LPL), lipase, and esterase are mentioned suitably. In particular, lipoprotein lipase (LPL) is preferable from the viewpoint of reactivity.

  There is no restriction | limiting in particular about content of LPL, According to the kind of sample to measure, the addition amount of a sample, the quantity of the hydrophilic polymer to be used, the kind of electron carrier, etc., it can select suitably. For example, from the viewpoint of the amount of enzyme (enzyme activity amount) that rapidly decomposes neutral fat and does not reduce the solubility of the reaction layer, preferably 0.1 to 1000 activity units (U ), More preferably 1 to 500 U, particularly preferably 10 to 100 U. In addition, the definition and measuring method of the activity unit (U) of LPL are based on the method as described in international publication 2006/104077 pamphlet. In addition, as will be described later, LPL is preferably prepared with a buffer solution such as glycylglycine.

  In the biosensors of the second and third embodiments, the oxidative degradation enzyme and the lipolytic enzyme exist in separate layers, a first reaction layer and a second reaction layer, respectively. If it is such a form, the hydrolysis reaction by a lipolytic enzyme will advance efficiently.

(Electronic carrier)
The biosensor of the present invention preferably includes an electron carrier (sometimes referred to as “electron acceptor”). The electron carrier may be included in any of the layers constituting the sample supply unit. That is, in the biosensor of the first embodiment, a layer that is included in the reaction layer 10 or that is separated from the reaction layer 10 and includes an electron carrier on the cover 7 side may be provided. In the biosensor of the second embodiment, it can be included in at least one of the first reaction layer 8 and the second reaction layer 9, and is separated from the first and second reaction layers to transmit electrons to the cover 7 side. A layer including a body may be provided. In the biosensor of the third embodiment, a layer that is included in at least one of the first reaction layer 8, the second reaction layer 9, and the upper surfactant layer 14, or that includes an electron carrier on the cover 7 side is further provided. You can also. At this time, the electron carrier is more preferably separated from the electrode. Thereby, it is possible to suppress and prevent the electron carrier from being automatically reduced due to a phenomenon such as a local battery, and to provide a biosensor with improved accuracy. However, as described above, the electron carrier is preferably not included in the same layer as the oxidoreductase.

  When the biosensor is used, the electron carrier receives electrons generated by the action of the oxidoreductase, that is, is reduced. The reduced electron carrier is electrochemically oxidized by applying a potential to the electrode after completion of the enzyme reaction. The concentration of a desired component in the sample can be calculated from the magnitude of the current flowing at this time (hereinafter also referred to as “oxidation current”).

  As the electron carrier used in the present invention, a conventionally known electron carrier can be used, and can be appropriately determined according to the sample and the oxidoreductase used. In addition, an electron carrier may be used independently or may be used in combination of 2 or more type.

  More specifically, examples of the electron carrier include potassium ferricyanide, sodium ferricyanide, ferrocene and derivatives thereof, phenazine methosulfate and derivatives thereof, p-benzoquinone and derivatives thereof, 2,6-dichlorophenolindophenol, methylene blue, Nitrotetrazolium blue, osmium complexes, ruthenium complexes such as hexaammineruthenium (III) chloride, and the like can be suitably used. Of these, hexaammineruthenium (III) chloride and potassium ferricyanide are preferable, and hexaammineruthenium (III) chloride is more preferably used.

  There is no restriction | limiting in particular about content of an electron carrier, According to the addition amount etc. of a sample, it can adjust suitably. As an example, from the viewpoint of containing a sufficient amount per base mass per sensor, preferably 1 to 2000 μg, more preferably 5 to 1000 μg, and particularly preferably 10 to 500 μg of an electron carrier is included. Good. The electron carrier is also preferably prepared with a buffer solution such as glycylglycine.

(sugar)
The reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) of the biosensor of the present invention may further contain sugar. Saccharides can be used by appropriately selecting those that do not participate in the enzyme reaction involved in the measurement and that do not react by themselves. Sugar is replaced by hydration water around the enzyme or crystallized by covering the outside of the surrounding hydration water to prevent inactivation due to environmental changes of oxidoreductase or lipolytic enzyme. , Exerts the effect of stabilizing the enzyme. That is, the storage stability of the biosensor of the present invention is further improved by the addition of sugar.

  As the sugar that can be included in the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9), a non-reducing sugar that does not have a free aldehyde group or a ketone group and does not have a reducing property is preferable. Examples of such non-reducing sugars include trehalose-type microsaccharides in which reducing groups are bonded to each other, glycosides in which saccharide reducing groups and non-saccharides are bonded, sugar alcohols reduced by hydrogenation of saccharides, and the like. More specifically, trehalose-type small sugars such as sucrose, trehalose and raffinose; glycosides such as alkyl glycosides, phenol glycosides and mustard oil glycosides; and sugar alcohols such as arabitol, xylitol and sorbitol Is mentioned. These non-reducing sugars may be used alone or in the form of a mixture of two or more. Among these, trehalose, raffinose and sucrose are preferable, and trehalose and raffinose are particularly preferable because of the high effect of improving storage stability.

  The amount of sugar contained in the reaction layer 10 (first reaction layer 8, second reaction layer 9) is 0.1 to 500 μg, preferably 0.5 to 400 μg, more preferably 1 to 300 μg per sensor. is there. If the sugar is in the form of a mixture of two or more, the blending amount means the sum of all components. If it is said range, sufficient storage stability effect will be acquired.

(protein)
In the biosensor of the present invention, the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) may further contain a protein. A protein that does not participate in an enzyme reaction related to measurement, does not react with itself, and does not exhibit physiological activity can be appropriately selected and used. Since the oxidoreductase and the lipolytic enzyme are themselves proteins, the presence of proteins that do not contribute to such a reaction in the surroundings makes them more stabilized and maintained in the reaction layer. Therefore, the storage stability of the biosensor of the present invention can be further improved by adding protein to the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9).

  Examples of the protein that can be included in the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) include bovine serum albumin (BSA), casein, sericin, and hydrolysates thereof. These proteins may be used alone or in the form of a mixture of two or more. Among these, BSA which is easily available and inexpensive is preferable. The molecular weight of the preferred protein is 10 to 1000 kDa, more preferably 25 to 500 kDa, and still more preferably 50 to 100 kDa. The molecular weight at that time employs a value measured by gel filtration chromatography.

  The amount of protein contained in the reaction layer 10 (first reaction layer 8, second reaction layer 9) is 0.1 to 200 μg, preferably 0.5 to 100 μg, more preferably 1 to 50 μg per sensor. It is. If the protein is in the form of a mixture of two or more, the blending amount means the sum of all components. If it is said range, it can contribute to fixation and stabilization of each layer, without reducing the performance of a sensor.

<Manufacturing method of biosensor>
The intermediate layer 13 (hydrophilic polymer layer 12, surfactant layer 11), reaction layer 10 (first reaction layer 8, second reaction layer 9), and upper surfactant layer 14 in the biosensor of the present invention. There is no particular limitation on the method of forming. Below, description is abbreviate | omitted about the structure common to the biosensor of 1st-3rd embodiment, or the structure common to 2nd and 3rd embodiment.

  In the biosensor of the present invention, the intermediate layer 13 (hydrophilic polymer layer 12, surfactant layer 11), reaction layer 10 (first reaction layer 8, second reaction layer 9), and upper surfactant layer 14 are used. May be formed by any method, but the intermediate layer 13 (hydrophilic polymer layer 12, surfactant layer 11), reaction layer 10 (first reaction layer 8, second reaction layer 9) and It is preferably formed by applying a solution containing the constituent components of the upper surfactant layer 14. Here, the coating method is not particularly limited, and a solution containing the constituent components of each layer is dropped, or spraying device, bar coater, die coater, reverse coater, comma coater, gravure coater, spray coater, doctor knife, etc. The method of apply | coating using an applicator can be used.

  In the biosensor of the first embodiment shown in FIG. 1, first, a surfactant (for example, Surf Hope (registered trademark) (Mitsubishi Chemical Corporation)) and, if necessary, a salt and a saccharide are mixed to obtain a surfactant. An agent solution is prepared. Next, a predetermined amount of the surfactant solution is dropped on the electrode system (action part). After dripping, the surfactant layer 11 is formed on the electrode system (action part) by drying in a constant temperature bath or a hot plate maintained at a predetermined temperature.

  Next, the hydrophilic polymer layer 12 is formed next as follows. First, a hydrophilic polymer solution is prepared by mixing a hydrophilic polymer (for example, polyvinyl alcohol) and, if necessary, a salt or a saccharide. Next, a predetermined amount of the hydrophilic polymer solution is dropped on the surfactant layer 11. After dripping, the hydrophilic polymer layer 12 is formed on the surfactant layer 11 by drying in a constant temperature bath or a hot plate maintained at a predetermined temperature. The lamination order of the surfactant layer 11 and the hydrophilic polymer layer 12 may be reversed. Note that an adhesive may be installed on the substrate 1 in advance.

  The reaction layer 10 is formed on the intermediate layer 13 as follows. An oxidoreductase (eg, PQQ-dependent glycerol dehydrogenase), a lipolytic enzyme (eg, lipoprotein lipase (LPL)), and optionally a surfactant (eg, Emulgen PP-290 (manufactured by Kao Corporation)) ) And a desired component such as glycylglycine buffer are mixed to prepare a reaction layer solution. If necessary, a volatile organic solvent such as ethanol may be added. By adding a volatile organic solvent, it is easy to dry quickly and crystallization is small. A predetermined amount of this reaction layer solution is dropped on the intermediate layer 13 (the hydrophilic polymer layer 12 in the examples shown in FIGS. 1 and 2). After dropping, the reaction layer 10 is formed on the intermediate layer 13 by drying in a constant temperature bath or a hot plate maintained at a predetermined temperature.

  The method for forming the reaction layer 10 is not particularly limited, and a method equivalent to the method for forming the intermediate layer 13 can be used. At this time, the formation method of the reaction layer 10 may be the same as or different from that of the intermediate layer 13. However, in view of ease of production, production cost, and the like, it is preferable to use the same method, and a method of drying a coating film after applying a solution containing a predetermined component dropwise is particularly preferable. Such a method is preferable in that a biosensor can be easily produced and the manufacturing cost during mass production can be reduced.

  In the second embodiment, the intermediate layer 13 can be formed on the electrode system in the same manner as in the first embodiment. Therefore, description of the manufacturing method of the intermediate layer 13 is omitted.

  The first reaction layer 8 is formed on the intermediate layer 13 as follows. First, an oxidoreductase (for example, PQQ-dependent glycerol dehydrogenase) and, if necessary, a surfactant (for example, Emulgen PP-290 (manufactured by Kao Corporation)), saccharides (for example, trehalose and raffinose), hydrophilic A desired component such as a conductive polymer (for example, polyethylene glycol), protein (for example, BSA), and glycylglycine buffer is mixed to prepare an oxidoreductase solution. If necessary, a volatile organic solvent such as ethanol may be added. By adding a volatile organic solvent, it is easy to dry quickly and crystallization is small. A predetermined amount of this oxidoreductase solution is dropped on the intermediate layer 13 (the hydrophilic polymer layer 12 in the examples shown in FIGS. 3 and 4). After dripping, the 1st reaction layer 8 is formed on the intermediate | middle layer 13 by making it dry in the thermostat kept at predetermined temperature, or on a hotplate.

  Next, the second reaction layer 9 is formed as follows. That is, a lipolytic enzyme (eg, lipoprotein lipase (LPL)) is optionally added to a saccharide (eg, trehalose and raffinose), a hydrophilic polymer (eg, polyethylene glycol), a protein (eg, BSA) and a glycine. A lipolytic enzyme solution is prepared by mixing with a desired component such as sylglycine buffer. A predetermined amount of this lipolytic enzyme solution is dropped on the first reaction layer 8. After dropping the lipolytic enzyme solution, the second reaction layer 9 is formed on the first reaction layer 8 by drying in a constant temperature bath or a hot plate maintained at a predetermined temperature. In addition, you may add other components (for example, surfactant, hydrophilic polymer, etc.) mentioned above as needed. Moreover, you may add volatile organic solvents, such as ethanol, as needed. By adding a volatile organic solvent, it is easy to dry quickly and crystallization is small.

  The formation method of the 1st reaction layer 8 and the 2nd reaction layer 9 is not restrict | limited to the above, The method equivalent to the formation method of the said intermediate | middle layer 13 can be used. At this time, the formation method of the first reaction layer 8 and the second reaction layer 9 may be the same or different. However, in consideration of ease of production, production cost, etc., it is preferable to use the same method as that for the intermediate layer 13, and a method of drying the coating film after applying a solution containing a predetermined component by dropping is particularly preferable. . Such a method is preferable in that a biosensor can be easily produced and the manufacturing cost during mass production can be reduced.

  Finally, the substrate 1 on which the intermediate layer 13, the first reaction layer 8, and the second reaction layer 9 are formed and the cover 7 are bonded to each other via the adhesives 6 a and 6 b, thereby allowing the second embodiment. The biosensor can be manufactured.

  In the biosensor of the third embodiment, the intermediate layer 13, the first reaction layer 8, and the second reaction layer 9 can be formed in the same manner as in the second embodiment. Therefore, description of the manufacturing method of the intermediate layer 13, the first reaction layer 8, and the second reaction layer 9 is omitted.

  The upper surfactant layer 14 can be formed as follows. First, a surfactant (for example, Emulgen PP-290 (manufactured by Kao Corporation)) and components of a glycylglycine buffer solution are mixed as necessary to prepare an upper surfactant solution. Next, a predetermined amount of the upper surfactant solution is dropped on the inner surface of the cover 7. After dripping, the upper surfactant layer 14 is formed on the cover 7 side by drying in a constant temperature bath maintained at a predetermined temperature or on a hot plate. Note that an adhesive may be installed on the cover 7 in advance.

  Finally, the substrate 1 on which the intermediate layer 13, the first reaction layer 8 and the second reaction layer 9 are formed, and the cover 7 on which the upper surfactant layer 14 is formed are bonded to the adhesives 6a and 6b. The biosensor of the third embodiment can be manufactured by pasting together.

  The method for forming the first reaction layer 8, the second reaction layer 9 and the upper surfactant layer 14 is not particularly limited, and a method equivalent to the method for forming the intermediate layer 13 can be used. At this time, the formation method of the first reaction layer 8, the second reaction layer 9, and the upper surfactant layer 14 may be the same or different. However, in consideration of ease of production, production cost, etc., it is preferable to use the same method as that for the intermediate layer 13, and a method of drying the coating film after applying a solution containing a predetermined component by dropping is particularly preferable. . Such a method is preferable in that a biosensor can be easily produced and the manufacturing cost during mass production can be reduced.

<Application of biosensor>
The sample used in the present invention is preferably in the form of a solution. It does not restrict | limit especially as a solvent in a solution form, A conventionally well-known solvent can be referred suitably or can be applied in combination.

  The sample is not particularly limited, but for example, biological samples such as whole blood, plasma, serum, saliva, urine, bone marrow; drinking water such as juice, foods such as soy sauce, sauce; drainage, rain water, pool water, etc. Is mentioned. Preferred is whole blood, plasma, serum, saliva, bone marrow, and more preferred is whole blood.

  As the sample, the stock solution may be used as it is, or a solution diluted with an appropriate solvent may be used for the purpose of adjusting the viscosity or the like. The substrate contained in the sample is not particularly limited, and can react with each enzyme contained in the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) to generate a measurable current as described later. Any substance can be used.

  Examples of the desired component (substrate) in the sample include sugars such as glucose, polyhydric alcohols such as glycerol, sorbitol, and arabitol, lipids such as neutral fat and cholesterol, organic acids such as glutamic acid and lactic acid, creatine, and creatinine Etc. For the same reason as above, it is preferable to select a lipid such as neutral fat or cholesterol as the substrate.

  The form in particular of supplying a sample to a sample supply part is not restrict | limited, For example, you may supply a sample from the horizontal direction with respect to the reaction layer 10 using a capillary phenomenon.

  When the sample is supplied to the reaction layer 10, a desired component (substrate) in the sample is oxidized by the action of a lipolytic enzyme and an oxidoreductase, and electrons are released simultaneously with its own oxidation. The electrons released from the substrate are captured by the electron carrier contained in the sample supply unit, and the electron carrier changes from the oxidized type to the reduced type. After the sample is added, the biosensor is allowed to stand for a predetermined time, whereby the substrate is completely oxidized by the lipolytic enzyme and the oxidoreductase, and a certain amount of electron carrier is converted from the oxidized form to the reduced form.

  At this time, the reaction time (measurement time) for completing the reaction between the substrate and the enzyme is not particularly limited, but is usually 1 second to 120 seconds, preferably 1 second to 90 seconds, more preferably 1 to 90 seconds after the sample is added. 60 seconds, particularly preferably 1 to 45 seconds. In particular, according to the biosensors of the second and third embodiments, the reaction layer 10 is divided into a first reaction layer 8 containing an oxidoreductase and a second reaction layer 9 containing a lipolytic enzyme. The reaction time (that is, measurement time) for completing the reaction between the enzyme and the enzyme can be significantly shortened.

  Thereafter, a predetermined potential is applied between the working electrode 2 and the counter electrode 4 through the electrode for the purpose of oxidizing the reduced electron carrier. As a result, the reduced electron carrier is electrochemically oxidized and converted into an oxidized form. From the value of the current measured at this time (hereinafter, also referred to as “oxidation current”), the amount of the reduced electron carrier before applying the potential is calculated, and the amount of the substrate reacted with the enzyme can be quantified. . The value of the potential applied when flowing the oxidation current is not particularly limited, and can be appropriately adjusted with reference to known knowledge. For example, a potential of about −200 to +700 mV, preferably 0 to +500 mV may be applied between the counter electrode 4 and the working electrode 2. The potential applying means for applying the potential is not particularly limited, and a conventionally known potential applying means can be appropriately used.

  As a method for measuring the oxidation current value and converting the current value to the substrate concentration, a chronoamperometry method for measuring a current value after a predetermined time after applying a predetermined potential may be used. Alternatively, a chronocoulometry method may be used in which a charge amount obtained by integrating the current response by the chronoamperometry method with time is measured. The chronoamperometry method can be preferably used in that it is measured by a simple apparatus system.

  As described above, the mode of calculating the substrate concentration by measuring the current (oxidation current) when oxidizing the reduced electron carrier has been described as an example, but in some cases, it remains without being reduced. A form in which the substrate concentration is calculated by measuring a current (reduction current) when reducing the oxidized electron carrier may be employed.

  The biosensor of the present invention may be used in any form and is not particularly limited. For example, it can be used for various applications such as a disposable biosensor for disposable use, a biosensor for continuously measuring a predetermined value by embedding at least an electrode part in a human body.

  The biosensor of the present invention can be applied to conventionally known sensors such as a neutral fat sensor and a cholesterol sensor.

  The effects of the present invention are summarized below.

  In the biosensor of the present invention, an intermediate layer including a surfactant layer and a hydrophilic polymer layer is interposed between a reaction layer containing an oxidoreductase and a lipolytic enzyme and an electrode, thereby preserving the biosensor. Stability is improved. Therefore, even if the biosensor is stored for a long period of time, it is possible to prevent an increase in dispersion of measured values and a decrease in the system, and to measure accurately. Furthermore, by reducing the inactivation of the enzyme due to storage, the amount of the enzyme can be reduced, and the production cost can also be reduced.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples. Unless otherwise specified, “%” means “mass%”.

<Manufacture of biosensor for measuring neutral fat concentration>
Example 1
The electrode used was a three-electrode system designed and manufactured independently. In this electrode, a working electrode 2 made of carbon, a reference electrode 3 and a counter electrode 4 are formed on an insulating substrate 1, and a working electrode working part 2-1 made of carbon, silver chloride is sandwiched between insulating layers 5. A reference electrode working part 3-1 made of silver and a counter electrode working part 4-1 made of carbon are formed.

  A surfactant layer constituting the intermediate layer was formed on the electrode system by the following procedure.

  Dissolve Surf Hope (Mitsubishi Chemical Foods Co., Ltd.) in distilled water to a final concentration of 0.01% (0.1 μg) per sensor (1 μl of sample “whole blood” supplied). After dropping so as to cover the electrode surface, the electrode was dried at 40 ° C. for 10 minutes to form a first surfactant layer.

  On the surfactant layer, a hydrophilic polymer layer constituting an intermediate layer together with the surfactant layer was formed by the following procedure.

  Polyvinyl alcohol 500 completely saponified type (manufactured by Wako Pure Chemical Industries, Ltd.) is 0.25% (2.5 μg) at a final concentration per sensor (amount 1 μl of sample “whole blood” to be supplied). After dissolving in distilled water and dropping so as to cover the first surfactant layer, it was dried at 40 ° C. for 10 minutes to form a hydrophilic polymer layer.

  A first reaction layer (GLDH layer) was formed on the molecular layer with high hydrophilicity by the following procedure.

  Per sensor (volume 1 μl of sample “whole blood” to be supplied) at a final concentration, 1.0 U of PQQ-dependent glycerol dehydrogenase and 5 mM glycylglycine (Wako Pure Chemical Industries, Ltd.) 65 μg), Emulgen PP-290 (manufactured by Kao Corporation) 0.025% (0.25 μg), trehalose dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.) 5% (50 μg), BSA pH 7.0 (Japanese) 0.5% (5 μg), manufactured by Kojun Pharmaceutical Co., Ltd., polyethylene glycol 6,000 (manufactured by Wako Pure Chemical Industries, Ltd.) 0.345% (3.45 μg), raffinose (manufactured by Wako Pure Chemical Industries, Ltd.) Was mixed to 1% (10 μg) to obtain a solution (GLDH solution). The obtained GLDH solution was dropped so as to cover the hydrophilic polymer layer, and dried at 40 ° C. for 5 minutes to obtain a first reaction layer (GLDH layer).

  The second reaction layer (electron carrier-containing LPL layer) was formed by the following procedure.

  Per one sensor (1 μl of sample “whole blood” to be supplied) at a final concentration, lipoprotein lipase (LPL, manufactured by Asahi Kasei Co., Ltd.) is 80 U, glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) is 5 mM ( 0.65 μg), hexaammineruthenium (III) chloride (Wako Pure Chemical Industries, Ltd.) 100 mM (65 μg), trehalose dihydrate (Wako Pure Chemical Industries, Ltd.) 5% (50 μg), BSA pH 7 0.0 (manufactured by Wako Pure Chemical Industries, Ltd.) 0.5% (5 μg), polyethylene glycol 6,000 (manufactured by Wako Pure Chemical Industries, Ltd.) 0.345% (3.45 μg), raffinose (Wako Pure Chemical Industries, Ltd.) Kogyo Co., Ltd.) was mixed at 1% (10 μg) to obtain a solution (electron carrier-containing LPL solution). The obtained electron carrier-containing LPL solution was dropped on the formed GLDH layer so as to be layered (coated), dried at 40 ° C. for 5 minutes, and then the second reaction layer (electron carrier-containing LPL layer) Got.

  In this way, an electron carrier-containing LPL layer as a second reaction layer was formed (multilayered) on the GLDH layer as the first reaction layer.

  The upper surfactant layer was formed by the following procedure.

  50 mM (6.5 μg) of glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) and Emulgen PP-290 (manufactured by Kao Corporation) at a final concentration per sensor (amount of 1 μl of sample “whole blood” to be supplied) Was mixed so as to be 0.1% (1 μg) to obtain a surfactant solution. The obtained surfactant solution was dropped into a gap formed by bonding an adhesive (double-sided tape) to a cover made of PET, and then dried at 50 ° C. for 5 minutes to form an upper surfactant layer.

  A cover on which the upper surfactant layer is formed, and an adhesive (double-sided tape) adhered to the substrate on which the surfactant layer, the hydrophilic polymer layer, the first reaction layer and the second reaction layer are formed Were attached to each other to produce a neutral fat sensor. At this time, the thicknesses of the first surfactant layer, the hydrophilic polymer layer, the first reaction layer, the second reaction layer, and the second surfactant layer are each 5 μm, and the second reaction The separation distance between the layer and the surfactant layer was 0.075 mm.

  With respect to the biosensor for measuring neutral fat concentration thus produced, storage stability was evaluated according to the following procedure.

  The biosensor was enclosed in a lami zip together with silica gel and kept at 40 ° C. Enzymes in which GLDH and LPL are dissolved by immersing the biosensor that has been kept warm after a certain number of days in a 10 mM phosphate buffer (pH 7.0) containing 0.1% Triton (registered trademark) X-100 A lysis solution was prepared.

  The activity of GLDH in the obtained enzyme solution was measured by measuring DCIP (2,6-dichlorophenolindophenol) at 50 μM, PMS (5-methylphenazinium methyl sulfate) at 0.2 mM, glycerol at 450 mM, and Triton. (Registered trademark) 10 mM phosphate buffer (pH 7.0) containing 0.1% of X-100 was used as a substrate solution, and a certain amount of enzyme solution was added thereto, followed by reaction at 37 ° C. to reduce absorbance at 600 nm. It was measured. The enzyme activity unit was the amount of enzyme that reduced 1 μmol of DCIP per minute under the above conditions.

  LPL activity in the obtained enzyme solution was measured by 10% olive oil (manufactured by Wako Pure Chemical Industries, Ltd.), 0.5% Triton (registered trademark) X-100, and BSA pH 7.0 (Japanese A certain amount of enzyme solution was added to an olive oil emulsion solution adjusted to 2% (manufactured by Kojun Pharmaceutical Co., Ltd.) and phosphate buffer (pH 7.0) to 30 mM, and the reaction was performed at 37 ° C. After the time, the reaction was stopped by adding 0.2 M trichloroacetic acid solution. Thereafter, the amount of glycerol produced by the reaction between the enzyme and the substrate was measured using Free Glycerol Reagent (manufactured by SIGMA ALDRICH). The enzyme activity unit was defined as the amount of enzyme that produces 1 μmol of glycerol per minute under the above conditions. The measurement results are shown in the following Table 1 and FIG. 5 with the activity of the enzyme used when producing the biosensor as 100%. In FIG. 5 and FIG. 6, the results of the examples are represented by white circles (◯).

(Comparative Example 1)
The same electrode as used in Example 1 was used.

  The first reaction layer (GLDH layer) was formed by the following procedure.

  Per sensor (volume 1 μl of sample “whole blood” to be supplied) at a final concentration, 1.0 U of PQQ-dependent glycerol dehydrogenase and 5 mM glycylglycine (Wako Pure Chemical Industries, Ltd.) 65 μg) and Emulgen PP-290 (manufactured by Kao Corporation) were mixed so as to be 0.025% (0.5 μg) to obtain a solution (GLDH solution). The obtained GLDH solution was dropped so as to cover the working electrode working part, reference electrode working part and counter electrode working part of the electrode, and dried at 40 ° C. for 5 minutes to obtain a first reaction layer (GLDH layer). .

  The second reaction layer (electron carrier-containing LPL layer) was formed by the following procedure.

  Per sensor (sample volume 1 μl), lipoprotein lipase (LPL, manufactured by Asahi Kasei Corporation) is 80 U, glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) is 5 mM (0.65 μg), hexaammineruthenium (III) Chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was mixed to 200 mM (65 μg) to obtain a solution (electron carrier-containing LPL solution). The obtained electron carrier-containing LPL solution was dropped on the formed GLDH layer so as to be layered (coated), dried at 40 ° C. for 5 minutes, and then the second reaction layer (electron carrier-containing LPL layer) Got.

  In this way, an electron carrier-containing LPL layer as a second reaction layer was formed (multilayered) on the GLDH layer as the first reaction layer.

  The upper surfactant layer was formed in the same procedure as in Example 1.

  By attaching the cover on which the upper surfactant layer is formed and the adhesive (double-sided tape) adhered to the substrate on which the first reaction layer and the second reaction layer are formed to each other, the neutral fat A sensor was fabricated. At this time, the thicknesses of the first reaction layer, the second reaction layer, and the upper surfactant layer are each 5 μm, and the separation distance between the second reaction layer and the upper surfactant layer is 0.085 mm. there were.

  The biosensor manufactured as described above was evaluated for storage stability in the same manner as in Example 1. The evaluation results are shown in the following Table 1, FIG. 7 and FIG. 7 and 8, the result of Comparative Example 1 is represented by a black square (♦).

(Comparative Example 2)
The same electrode as used in Example 1 was used.

  The first reaction layer (GLDH layer) was formed by the following procedure.

  Per sensor (volume 1 μl of sample “whole blood” to be supplied) at a final concentration, 1.0 U of PQQ-dependent glycerol dehydrogenase and 5 mM glycylglycine (Wako Pure Chemical Industries, Ltd.) 65 μg), Emulgen PP-290 (manufactured by Kao Corporation) 0.025% (0.25 μg), and trehalose dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.) 5% (50 μg) To obtain a solution (GLDH solution). The obtained GLDH solution was dropped so as to cover the working electrode working part, reference electrode working part and counter electrode working part of EP-N, and dried at 40 ° C. for 5 minutes, and the first reaction layer (GLDH layer) was formed. Obtained.

  The second reaction layer (electron carrier-containing LPL layer) was formed by the following procedure.

  For each sensor (amount of 1 μl of sample “whole blood” to be supplied), lipoprotein lipase (LPL, manufactured by Asahi Kasei Co., Ltd.) is 80 U and glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) is 5 mM (final concentration). 0.65 μg) and hexaammineruthenium (III) chloride (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed so as to be 100 mM (65 μg) to obtain a solution (electron carrier-containing LPL solution). The obtained electron carrier-containing LPL solution was dropped on the formed GLDH layer so as to be layered (coated), dried at 40 ° C. for 5 minutes, and then the second reaction layer (electron carrier-containing LPL layer) Got.

  In this way, an electron carrier-containing LPL layer as a second reaction layer was formed (multilayered) on the GLDH layer as the first reaction layer.

  The upper surfactant layer was formed in the same procedure as in Example 1.

  By attaching the cover on which the upper surfactant layer is formed and the adhesive (double-sided tape) adhered to the substrate on which the first reaction layer and the second reaction layer are formed to each other, the neutral fat A sensor was fabricated. At this time, the thicknesses of the first reaction layer, the second reaction layer, and the upper surfactant layer are each 5 μm, and the separation distance between the second reaction layer and the upper surfactant layer is 0.085 mm. there were.

  The biosensor manufactured as described above was evaluated for storage stability in the same manner as in Example 1. The evaluation results are shown in the following Table 1, FIG. 7 and FIG. 7 and 8, the result of Comparative Example 2 is represented by black triangles (▲).

  As is apparent from Table 1, FIG. 7 and FIG. 8, the biosensor of the present invention maintains a high enzyme activity exceeding 90% even after 30 days of storage and is excellent in storage stability. I understand that. In particular, with respect to lipolytic enzymes, the biosensor of the present invention is hardly inactivated, and very excellent storage stability is realized. On the other hand, in Comparative Examples 1 and 2, which do not have an intermediate layer, it can be seen that, in both oxidoreductase and lipolytic enzyme, the enzyme begins to be deactivated immediately after storage, and the storage stability is not sufficient. . In particular, the inactivation of the oxidoreductase is large. In Comparative Example 1, the residual activity of the enzyme is less than 50% after storage for 30 days. In comparison with Comparative Example 1, it can be seen that the biosensor of Example 1 has greatly improved storage stability. This result shows the effect of improving the storage stability by providing the intermediate layer including the surfactant layer and the hydrophilic polymer layer in the biosensor of the present invention. Furthermore, it is considered that the storage stability is further improved by adding hydrophilic polymers, sugars and proteins to the first and second reaction layers.

  Further, when Comparative Example 1 is compared with Comparative Example 2 in which trehalose is added to the first reaction layer, the residual activity of the enzyme of Comparative Example 2 is improved. Therefore, the addition of sugar is effective for storage stability. However, the storage stability improvement effect is not sufficient by itself, and storage stability is achieved by providing an intermediate layer between the reaction layer and the electrode, and further adding a hydrophilic polymer, sugar and protein in the reaction layer. Is further improved.

1 Insulating substrate,
2 working electrode,
2-1 Working electrode working part,
3 Reference electrode,
3-1 Reference electrode working part,
4 counter electrode,
4-1 Counter electrode action part,
5 Insulating layer,
6 (6a, 6b) adhesive,
7 Cover,
8 First reaction layer,
9 Second reaction layer,
10 reaction layer,
11 Surfactant layer,
12 hydrophilic polymer layer,
13 Middle layer,
14 Upper surfactant layer,
S space part.

Claims (12)

A biosensor having an insulating substrate, an electrode system including at least a working electrode and a counter electrode formed on the insulating substrate, and a sample supply unit formed on the electrode system,
The sample supply unit is
An intermediate layer formed on the electrode system, and a reaction layer formed on the intermediate layer,
The intermediate layer includes a surfactant layer containing a surfactant and a hydrophilic polymer layer containing a hydrophilic polymer,
A biosensor in which the reaction layer includes at least an oxidoreductase containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group, and a lipolytic enzyme . The reaction layer includes at least a first reaction layer including an oxidoreductase containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group,
The biosensor according to claim 1, further comprising: a second reaction layer formed by applying a solution containing a lipolytic enzyme on the first reaction layer.   The biosensor according to claim 1 or 2, wherein the sample supply unit further includes an electron carrier.   The biosensor according to any one of claims 1 to 3, wherein the oxidoreductase is a polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group.   The biosensor according to any one of claims 1 to 4, wherein the hydrophilic polymer is at least one of carboxymethyl cellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and derivatives thereof.   The biosensor according to any one of claims 1 to 5, wherein the surfactant is a nonionic surfactant.   The nonionic surfactant is sucrose behenate, sucrose stearate, sucrose palmitate, sucrose myristic ester, sucrose laurate, sucrose erucate and sucrose oleate The biosensor according to claim 6, which is at least one of the following.   The biosensor according to any one of claims 1 to 7, wherein the reaction layer contains a non-reducing sugar.   The biosensor according to claim 8, wherein the non-reducing sugar is at least one of trehalose, raffinose, and sucrose.   The biosensor according to claim 1, wherein the reaction layer contains a protein.   The biosensor according to claim 10, wherein the protein is at least one of BSA, casein, and sericin.   The biosensor according to any one of claims 1 to 11, wherein the sample supply unit further includes an upper surfactant layer containing a surfactant separated from the intermediate layer and the reaction layer.