JP5979937B2 - Biosensor containing trisboric acid - Google Patents

Description

  The present invention relates to a biosensor, particularly a biosensor for measuring neutral fat concentration. In particular, the present invention relates to a biosensor capable of quickly and accurately quantifying the concentration of a specific component in a specific sample such as a biological sample using an enzyme reaction, and more particularly to a biosensor for measuring neutral fat concentration.

  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 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 (also referred to as an “electron carrier”) are immobilized on the electrode system, such as a hydrophilic polymer. A biosensor has been proposed in which an enzyme reaction layer immobilized with an agent is provided, and then a filtration layer (blood cell removal layer) is provided on the enzyme reaction layer, and a cover is placed over the filtration layer and integrated. Yes.

  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, for example, in Patent Document 1, the enzyme cost is reduced by using two types of enzymes, ie, neutral lipolytic enzyme and glycerol dehydrogenase (GLDH), as enzymes used for the neutral lipolytic reaction. A reduced biosensor is disclosed. 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, as a method using one kind of enzyme without being affected by dissolved oxygen, a method using NAD ⁺ dependent glycerol dehydrogenase (NAD-GLDH) is known 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), 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 ⁺ There are merits such as.

  In view of the above, for the purpose of a biosensor capable of measuring neutral fat in a short time and with high accuracy, for example, Patent Documents 2 and 3 disclose a biosensor in which neutral lipolytic enzyme and glycerol dehydrogenase are arranged in different layers. It has been reported. The biosensor disclosed in 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. It is a feature. 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 on a nonwoven fabric are sequentially laminated on the electrode. It is characterized by that.

  However, even with the biosensors described in Patent Documents 2 and 3, the measurement time is still not sufficiently short, and a biosensor capable of measuring in a shorter time has been desired.

  Therefore, in order to measure the target component concentration in a shorter time, for example, in Patent Document 4, a reaction layer is coated with a layer containing an oxidoreductase and a solution containing a lipolytic enzyme directly on the layer. A biosensor having a two-layer structure with a layer containing a lipolytic enzyme formed by the above is disclosed. According to Patent Document 4, such a two-layer structure improves the reaction rate by the two enzymes and can measure the concentration of a specific component such as neutral fat in a sample in a short time.

International Publication No. 2006/104077 Pamphlet JP 2009-244013 A JP 2009-244014 A International Publication No. 2011-125750 Pamphlet

  However, the biosensor described in Patent Document 4 has insufficient measurement accuracy and has been desired to be improved.

  Therefore, an object of the present invention is to provide a biosensor having sufficient accuracy.

  The present inventors have intensively studied to solve the above problems. As a result, in a specific oxidoreductase, a reduced electron carrier is generated by reacting with an electron carrier despite the absence of such a substrate for the oxidoreductase, resulting in a reaction other than the reaction with the substrate. It has been found that the response current value may occur (hereinafter also referred to as “background current”). This means that the following problems occur.

  In other words, a biosensor has a storage period from when it is manufactured to storage in a warehouse, transportation to a wholesaler or a retail store, and use in a hospital or home. However, since the background current is generated during the storage period, the background current value is added to the response current value for the substance to be measured, and a value different from the actual value is displayed. The problem may become more pronounced as the biosensor storage period increases. The problem may become more pronounced as the biosensor storage period increases.

  In view of the above research results, as a result of intensive studies to solve the above problems, it was learned that the above problems can be solved by adding trisboric acid to the reaction layer of the biosensor, and the present invention was completed. did.

  That is, the above-described problems are 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 including a reaction layer formed on the electrode system. Wherein 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 second reaction layer formed by applying a solution containing a lipolytic enzyme on the first reaction layer, and the sample supply unit contains trisboric acid. Achieved by a biosensor.

  According to the biosensor of the present invention, a biosensor having sufficient accuracy can be provided. More specifically, a biosensor having sufficient accuracy can be provided by suppressing the background current.

It is a disassembled perspective view which shows one Embodiment of the biosensor of this invention. It is sectional drawing of the biosensor of FIG. It is a disassembled perspective view which shows other embodiment of the biosensor of this invention. FIG. 4 is a cross-sectional view of the biosensor of FIG. 3. It is a graph which shows the response current value of the biosensor of the Example of this invention, and a comparative example.

  A first aspect of the present invention is a sample supply unit including an insulating substrate, an electrode system including at least a working electrode and a counter electrode formed on the insulating substrate, and a reaction layer formed on the electrode system. The reaction layer includes at least an oxidoreductase containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group. A reaction layer including a first reaction layer and a second reaction layer formed by applying a solution containing a lipolytic enzyme on the first reaction layer; Is a biosensor characterized by containing trisboric acid.

  Conventionally, as described above, in a biosensor including a specific oxidoreductase as described in Patent Document 4, there is no substrate for such oxidoreductase when not used (before supplying a sample). Nevertheless, the phenomenon of reacting with the electron carrier occurred. When the electron carrier is reduced by a reaction other than the reaction between the substrate and the oxidoreductase, resulting in a background current, the response current resulting from the reaction between the substrate and the oxidoreductase, the original measurement target It becomes difficult to measure accurately.

  Furthermore, as described above, the background current may become more prominent as the storage period of the biosensor becomes longer.

  In contrast, the present invention is characterized in that trisboric acid is contained in at least a part of the sample supply unit. When trisboric acid is contained in at least a part of the sample supply unit, trisboric acid is oxidized with the substrate. It is thought that the electrons generated by reactions other than the reaction with the reductase are chelated, and the electron transfer to the electron carrier is reduced. As a result, the generation of background current (particularly, the generation of background current associated with a prolonged storage period) is suppressed, so that a highly accurate biosensor can be obtained. In addition, the said mechanism is an inference to the last, and this invention is not limited by the said mechanism.

  Hereinafter, embodiments of the biosensor of the present invention will be specifically described with reference to the drawings. It should be noted that the present invention is not limited to the following embodiments unless departing from the concept defined by the claims. 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 an embodiment of the biosensor of the present invention. FIG. 2 is a cross-sectional view of the biosensor of FIG. In the present specification, the biosensor shown in FIGS. 1 and 2 is also referred to as a “first biosensor”.

  As shown in FIGS. 1 and 2, the first biosensor includes an electrode system including a working electrode 2, a reference electrode 3, and a counter electrode 4 on an insulating substrate 1 (also simply referred to as “substrate” in the present specification). Is formed. The electrode system only needs to include at least the working electrode 2 and the counter electrode 4. 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 known knowledge such as a screen printing method, an ink jet method, and a sputtering method.

  And it coat | covers with the insulating layer 5 so that a part of electrode system (The working electrode 2, the reference electrode 3, and the counter electrode 4) formed on the insulating board | substrate 1 may be exposed. The insulating layer 5 functions as an insulating means for preventing a short circuit between the electrodes. The method for forming the insulating layer is not particularly limited, and the insulating layer can be formed by a conventionally known method such as a screen printing method, an ink jet method, or an adhesion method.

  Each part of the electrode system (working electrode 2, reference electrode 3 and counter electrode 4) exposed from the insulating layer 5 is in contact with the sample supply unit (more specifically, the first reaction layer 8). doing. In this specification, the part of the electrode system (working electrode 2, reference electrode 3 and counter electrode 4) in contact with the first reaction layer 8 is referred to as “working part (working electrode working part 2-1, see FIG. These are also referred to as “polar action part 3-1 and counter electrode action part 4-1)”, and the shape of these action parts is not particularly limited. Further, as shown in FIGS. 1 and 2, on the surfaces of the working electrode working part 2-1, the reference electrode working part 3-1, and the counter electrode working part 4-1, the first reaction layer 8 and the second reaction layer 8 are formed. The reaction layer 9 is sequentially laminated. A space S (not shown in FIG. 1) formed between the second reaction layer 9 and the cover 7, the first reaction layer 8, and the second reaction layer 9 are supplied as a sample. Forming part.

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

  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.

  The biosensor according to the present embodiment contains trisboric acid in at least a part of the sample supply unit, whereby generation of background current is suppressed and a highly accurate biosensor can be obtained. Trisboric acid may be contained in any of the sample supply units. In the first biosensor shown in FIGS. 1 and 2, the form contained in the first reaction layer 8 and the second reaction layer 9 are included. Examples include a form included, a form included in the space portion S located between the second reaction layer 9 and the cover 7, and a combination of these.

  As the form in which the trisboric acid is contained in the first reaction layer 8, even if trisboric acid is contained in the reaction layer together with the oxidoreductase, the surface of the first reaction layer is coated or immobilized. May be. Similarly, the trisboric acid is contained in the second reaction layer 9 even if trisboric acid is contained in the reaction layer together with the lipolytic enzyme (trisboric acid is added to the solution containing the lipolytic enzyme. Mixing), or may be coated or immobilized on the surface of the second reaction layer. Further, as a form in which trisboric acid is included in the space S located between the second reaction layer 9 and the cover 7, the trisboric acid covers or fixes at least a part of the surface of the second reaction layer. When trisboric acid is coated or immobilized on a part of the surface such as a wall (cover, third reaction layer, or surfactant layer) that forms the space portion S as well as the formed shape Including.

  Here, the action part (2-1, 3-1, 4-1) includes a potential applying means for applying a potential to the sample in the first reaction layer 8 and the sample when the biosensor is used. It functions as a current detection means for detecting the flowing current. 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 serve as current measuring means for measuring an oxidation current (response current) that flows when a potential is applied to the sample in the first reaction layer 8. Function. In the biosensor according to the present invention, when the reference electrode 3 is provided, a predetermined potential is applied between the counter electrode 4 and the working electrode 2 with reference to the reference electrode 3.

  The biosensor of this embodiment is configured by bonding a cover 7 so as to cover the first reaction layer 8 and the second reaction layer 9 via an adhesive (double-sided tape) 6 installed on the substrate 1. Is done. 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. In FIG. 1, the adhesive 6 is provided only on the cover 7 side.

  In the biosensor of the present invention, it is preferable that the sample supply unit further includes an electron carrier. The electron carrier in such a form may be present in the sample supply unit in any form. Specifically, (a) the first reaction layer 8 includes an electron carrier (an electron carrier is disposed in the first reaction layer 8), and (b) the second reaction layer 9 is an electron carrier. (The electron carrier is arranged in the second reaction layer 9), (c) a third reaction layer containing the electron carrier is further arranged, and the like. 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. That is, in the biosensor of the present invention, the second reaction layer has an electron carrier (the electron carrier is disposed in the second reaction layer), or a third reaction layer including the electron carrier is further provided. More preferably.

  In the case of (a) (the second reaction layer has an electron carrier), a layer containing a surfactant (hereinafter also referred to as a surfactant layer) is further added to the first reaction layer 8 and the second reaction. It is preferable to be provided in the sample supply unit so as to be separated from the layer 9. At this time, the arrangement of the surfactant layer is not particularly limited. For example, the surfactant layer is preferably formed on the cover side so as to be separated from the first reaction layer 8 and the second reaction layer 9. At that time, if the surfactant layer is formed on the cover side, the sample spreads to the cover side and wettability is better than when the cover 7 directly touches the sample (blood, etc.), and the sample is supplied to the sample. There is also an advantage that can be quickly introduced into the department.

  In the case of (c) (the third reaction layer has an electron carrier), a third reaction layer having an electron carrier is further added to the first reaction layer 8 and the second reaction layer 9. It is preferable to provide the sample supply unit so as to be separated. At this time, the arrangement of the third reaction layer is not particularly limited. For example, the third reaction layer is formed on the cover side apart from the first reaction layer 8 and the second reaction layer 9. Is preferred. By having the electron carrier in such an arrangement, the effect of improving the stability of the electron carrier itself or of the oxidoreductase during storage can be obtained. This is due to the fact that the electron carrier and the oxidoreductase do not contact each other.

  That is, according to another embodiment of the biosensor of the present invention including the third reaction layer, an electrode including at least a working electrode and a counter electrode formed on the insulating substrate and the insulating substrate. And a sample supply unit formed on the electrode system, wherein the sample supply unit is formed on the electrode system, and pyrroloquinoline quinone (PQQ) as a prosthetic group, It is formed by applying a first reaction layer containing an oxidoreductase containing flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), and a solution containing a lipolytic enzyme on the first reaction layer. And a second reaction layer, and a third reaction layer including an electron carrier is disposed in a form separated from the first reaction layer and the second reaction layer. Hereinafter, a biosensor according to another embodiment of the present invention will be described with reference to FIGS.

  FIG. 3 is an exploded perspective view showing another embodiment of the biosensor of the present invention including the third reaction layer. 4 is a cross-sectional view of the biosensor of FIG. In the present specification, the biosensor shown in FIGS. 3 and 4 is also referred to as a “second biosensor”.

  As shown in FIGS. 3 and 4, the basic structure of the second biosensor further includes a third reaction layer 10 containing an electron carrier (ie, the first reaction layer 8 and the second reaction layer). 9 is the same as the biosensor shown in FIGS. 1 and 2 except that a third reaction layer 10 is disposed. 3 and 4, the side formed on the cover 7 side is the third reaction layer 10, and the side formed on the electrode side is the first reaction layer 8 and the second reaction layer sequentially from the electrode side. Nine. At this time, the first reaction layer 8, the second reaction layer 9, the third reaction layer 10, and the space portion disposed between the second reaction layer 9 and the third reaction layer 10. S forms a sample supply section. The first reaction layer 8 contains an oxidoreductase, the second reaction layer 9 contains a lipolytic enzyme, and the third reaction layer 10 contains an electron carrier. In other words, in the second biosensor according to the other embodiment, the oxidoreductase, the lipolytic enzyme and the electron carrier are not simultaneously contained in the same reaction layer. In addition, the 3rd reaction layer 10 is formed in the clearance gap between the both ends on the cover 7 in which the adhesive agent (double-sided tape) 6a was installed in both ends.

  The trisboric acid according to the present invention is contained in at least one reaction layer selected from the group consisting of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10, as will be described later. Alternatively, it is preferably present on the reaction layer surface.

  In the second biosensor, an adhesive (double-sided tape) 6b adhered to the substrate 1 on which the first reaction layer 8 and the second reaction layer 9 are formed, and a third reaction layer 10 are formed. The adhesive (double-sided tape) 6a adhered to the cover 7 is bonded to each other to constitute a structure. 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.

  Also in the second biosensor of this embodiment, trisboric acid is included in at least a part of the sample supply unit. Thereby, generation | occurrence | production of background current is suppressed and it can be set as a highly accurate biosensor. Trisboric acid may be contained in any of the sample supply units. In the second biosensor shown in FIGS. 3 and 4, the form contained in the first reaction layer 8 and the second reaction layer 9 are included. Forms included, forms included in the third reaction layer 10, forms included in the space S disposed between the second reaction layer 9 and the third reaction layer 10, and combinations thereof Etc.

  In addition, since the said form in a 2nd biosensor is the same as that of a 1st biosensor, it abbreviate | omits here.

  Hereinafter, each component will be described in detail. Note that, as described above, the structure of the first biosensor and the structure of the second biosensor are the same except that the second biosensor further has the third reaction layer 10, and thus is clearly specified. Unless otherwise specified, the specific description of the constituent elements described below applies to the first biosensor and the second biosensor of the present invention. 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, in the reference electrode 3, the reference electrode working part 3-1 may be made of silver / silver chloride, and the other part may be made of carbon. 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>
As described above, in the first biosensor of the present invention, the sample supply unit includes the first reaction layer 8 including the oxidoreductase, the second reaction layer 9 including the lipolytic enzyme, and trisboric acid. Have. In addition, if necessary, the sample supply section is separated from the space section, the first reaction layer, and the second reaction layer, and when the surfactant layer is provided on the cover 7 side, the sample supply section It is also preferred to have an activator layer.

  On the other hand, in the second biosensor of the present invention, the sample supply unit includes a first reaction layer 8 containing an oxidoreductase, a second reaction layer 9 containing a lipolytic enzyme, and the first reaction layer 8. And a third reaction layer 10 formed separately from the reaction layer 8 and the second reaction layer 9, and the third reaction layer 9 preferably contains an electron carrier. By providing the third reaction layer as in the second biosensor of the present invention, the oxidoreductase and the electron carrier can be contained in separate reaction layers. Deterioration during storage due to contact with the enzyme can be prevented.

  In the present invention, the average thickness of the first reaction layer 8 and the second reaction layer 9 is not particularly limited, and can be appropriately selected so as to be an average thickness of a 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 average 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 average thickness of the average thicknesses 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. Good. 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 first biosensor according to the present invention, when a surfactant layer is formed on the cover 7 side by being separated from the first reaction layer 8 and the second reaction layer 9, the average thickness is 0.01-25 micrometers is preferable, More preferably, it is 0.025-10 micrometers, More preferably, it is 0.05-8 micrometers. The average separation distance between the second reaction layer 9 and the surfactant layer is preferably 0.05 to 1.5 mm, more preferably 0.07 to 1.25 mm, and still more preferably 0.09 to 1 mm. 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. Here, the average thickness of the first reaction layer 8, the second reaction layer 9, and the surfactant layer formed as necessary may be the same or different.

  In addition, the average thickness of the first reaction layer 8, the second reaction layer 9, and the third reaction layer in the case of the second biosensor according to the present invention is not particularly limited, and the average thickness of the normal reaction layer It can select suitably so that it may become. 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 average 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 average thickness of the average thicknesses 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. Good.

  The average thickness of the third reaction layer 10 is preferably 0.01 to 10 μm, more preferably 0.025 to 10 μm, and particularly preferably 0.05 to 8 μm. Here, the average thickness of the 1st reaction layer 8, the 2nd reaction layer 9, and the 3rd reaction layer 10 may be the same, or may differ. The method for controlling the average thickness at this time is not particularly limited. For example, the thickness can be controlled by appropriately adjusting the coating amount (for example, the amount to be dropped) of a solution containing a predetermined component. In addition, although there is no restriction | limiting in particular in the separation distance of the 2nd reaction layer 9 and the 3rd reaction layer 10, Preferably it is 0.05-1.5 mm, More preferably, it is 0.075-1.25 mm, Especially preferably, it is 0.1-1 mm. If it is the said range, an oxidation reductase and an electron carrier will not contact during a preservation | save, a capillary phenomenon will occur easily, and a sample will be attracted | sucked easily to a reaction layer. 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 third reaction layer 10.

  As described above, the sample supply unit of the biosensor according to the present invention has the space S and the reaction layer (concept including the first reaction layer, the second reaction layer, and the third reaction layer). And the reaction layer contains oxidoreductase, lipolytic enzyme, and trisboric acid as essential components, and if necessary, consists of an electron carrier, a surfactant, a hydrophilic polymer, a sugar, and a protein. At least one component selected from the group. Hereinafter, each component will be described.

(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 culture medium for cultivating the PQQ-dependent glycerol dehydrogenase is a natural medium, even if it is a synthetic medium, as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients that can be assimilated by the strain used. It may be. As the carbon source, for example, glucose, glycerol, sorbitol and the like are used. As the nitrogen source, for example, nitrogen-containing natural products such as peptones, meat extracts and yeast extracts, and nitrogen-containing compounds such as ammonium chloride and ammonium citrate are used. As the inorganic substance, potassium phosphate, sodium phosphate, magnesium sulfate or the like is used. In addition, specific vitamins are used as needed. The carbon source, nitrogen source, inorganic substance, and other necessary nutrients described above 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 other conditions, culture is possible if the strain 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 are originally obtained by transducing oxidoreductase genes into E. coli or the like, even if they are enzymes obtained by culturing bacteria having oxidoreductase production ability. It may be a replacement enzyme.

  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)
Moreover, the 2nd reaction layer 9 in this invention contains the lipolytic enzyme which hydrolyzes the ester bond which comprises 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 present invention, the oxidative degradation enzyme and the lipolytic enzyme are separately present in separate layers, a first reaction layer and a second reaction layer. If it is such a form, the hydrolysis reaction by a lipolytic enzyme will advance efficiently.

(Trisboric acid)
The trisboric acid according to the present invention is used in a mixture of an equimolar concentration of tris (hydroxymethyl) aminomethane solution (solvent is water) and boric acid solution (solvent is water), and the mixing ratio is changed. The pH can be adjusted appropriately. Preferably it is pH 5-9, More preferably, it is pH 6-8.

  There is no restriction | limiting in particular about content of the said trisboric acid, According to the addition amount etc. of a sample, it can adjust suitably. For example, it is preferable that 10 to 500 mM, more preferably 50 to 400 mM, and particularly preferably 100 to 300 mM are contained from the viewpoint of containing a sufficient amount per base mass per sensor.

(Electronic carrier)
The biosensor according to the present invention preferably includes an electron carrier. Here, the electron carrier may be included in the first reaction layer 8 or the second reaction layer 9, but is preferably included in the third reaction layer 10 separately from these reaction layers. In this case, the reaction layer containing the electron carrier is more preferably separated from the electrode, and is separated from the first reaction layer 8 or the second reaction layer 9, particularly the second reaction layer 9. It is particularly preferable to make it exist. That is, the present invention is a biotechnology 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 sensor, wherein the sample supply unit is formed on the electrode system and includes pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group And a second reaction layer formed by applying a solution containing a lipolytic enzyme on the first reaction layer, and further, electron transfer A biosensor (second biosensor) is provided in which a third reaction layer including a body is formed apart from the first and second reaction layers.

  In the second biosensor of the present invention, the third reaction layer 10 includes an electron carrier (sometimes referred to as “electron acceptor”). In this way, by allowing the electron carrier to be separated from the electrode, it is possible to suppress / prevent the electron carrier from being automatically reduced due to a phenomenon such as a local battery. Thereby, a biosensor with improved accuracy can be provided.

  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. In addition, as will be described later, the electron carrier is preferably prepared with a buffer solution using the above-described salt component.

(Surfactant)
In the biosensor according to the present invention, the first reaction layer 8, the second reaction layer 9, or the third reaction layer 10 has a surfactant as necessary. In the first biosensor according to the present invention, a surfactant layer may be formed on the cover 7 side so as to be separated from the first reaction layer 8 and the second reaction layer 9.

  Since the oxidoreductase is composed of protein, if the electrode surface adheres to the electrode surface, the electrode surface may be passivated. Therefore, in conventional general biosensors, the enzyme is not in direct contact with the electrode. The structure was taken. However, inclusion of a surfactant in the first reaction layer 8 significantly suppresses and prevents the redox enzyme from adhering to the electrode, and as a result, oxidized electrons by the redox enzyme in the vicinity of the electrode. The conversion efficiency of the transmitter to the reduced electron carrier can be improved. In other words, the correlation with the substrate concentration in the sample solution can be further increased. In addition, when the surfactant is formed on the cover 7 side, the spread and wettability of the sample such as whole blood to the cover side is improved compared to the case where the cover 7 directly touches the sample, and the sample is supplied to the sample supply unit. It is preferable because it can be introduced quickly.

  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 nonionic surfactant is not particularly limited, but polyalkylene oxide, polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-t- octylphenol; Triton (registered trademark) X-100)], polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalitanate, polyoxyethylene sorbite monopolymitolate Monostearate (Polyoxyethylene Sorbitan Monostea rate; Tween 60), polyoxyethylene sorbitan monooleate (Polyoxyethylene Sorbitan Monooleate; Tween 80), polyoxyethylene polyoxypropylene glycol (Emulgen PP-290 (manufactured by Kao Corporation)) and the like are preferable. Among them, 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)], polyoxyethylene polyoxypropylene glycol (Emulgen PP-290 (manufactured by Kao Corporation)) is preferred.

  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 (the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10), the sample solution is dropped. Spreads very well and has 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. When a specific example is given, said CHAPS, Tween, and Emulgen PP-290 (made by Kao Corporation) (polyoxyethylene polyoxypropylene glycol) are preferable.

  In the second biosensor of the present invention, the surfactant may be contained in any reaction layer of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10. However, preferably the first reaction layer 8 or the third reaction layer 10, more preferably the first reaction layer 8 and the third reaction layer 10 contain a surfactant. Thereby, dissolution of an oxidoreductase or an electron carrier can be promoted. In addition, when a surfactant is present in two or more reaction layers of the above three reaction layers, the types and blending amounts of the surfactants included in these reaction layers are the same or different. Also good. At this time, it is preferable to select in consideration of the interaction with each constituent element contained in the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10.

  More specifically, first, as described above, the first reaction layer 8 contains an oxidoreductase. For example, when a PQQ-dependent glycerol dehydrogenase is contained as the oxidoreductase, they are hydrophobic. Therefore, it is preferable that at least the first reaction layer 8 contains a surfactant. In this case, as the type of surfactant, from the viewpoint of further improving the accuracy of the biosensor, when whole blood is used as a sample, a low hemolytic surfactant (for example, CHAPS, Tween, Emulgen PP-290, etc.) ) Is preferably used. On the other hand, as described above, in the second biosensor, the third reaction layer 10 contains an electron carrier (for example, hexaammon ruthenium (III) chloride). From the viewpoint of improving the accuracy of the biosensor by improving, it is more preferable that the third reaction layer 10 also contains a surfactant. Also in this case, it is preferable to use a low hemolytic surfactant (for example, CHAPS, Tween, Emulgen PP-290, etc.) from the viewpoint of spreading and wettability. By applying such a device, the accuracy as a biosensor is further improved.

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

  When using amphoteric surfactants, it is preferable from the viewpoint of increasing the solubility of the oxidoreductase of the present invention per sensor, not deactivating the enzyme activity, and being 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. In addition, as described later, such a surfactant is preferably prepared in a buffer solution such as glycylglycine. When two or more kinds of surfactants are contained in one sensor, the content means the total amount.

  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.

(Hydrophilic polymer)
The first reaction layer 8, the second reaction layer 9, or the third reaction layer 10 in the present invention may further contain a hydrophilic polymer.

  As described above, since the oxidoreductase is composed of a protein, if the oxidoreductase is attached to the electrode surface, there is a possibility that the electrode surface may be passivated. Had a configuration that would not be in direct contact. However, the inclusion of a hydrophilic polymer in the first reaction layer 8 significantly suppresses and prevents the redox enzyme from adhering to the electrode, and as a result, the oxidized form of the redox enzyme in the vicinity of the electrode. The conversion efficiency of the electron carrier to the reduced electron carrier can be improved. In other words, the correlation between the substrate concentration in the sample solution and the oxidation current can be further increased.

  Further, the hydrophilic polymer may be the first reaction layer 8 or the second reaction layer 9 in the case of the first biosensor, or the first reaction layer 8 in the case of the second biosensor. It may be contained in either the second reaction layer 9 or the third reaction layer 10. The hydrophilic polymer has a function of immobilizing the oxidoreductase or electron carrier of the present invention on the electrode. For this reason, when the first reaction layer 8, the second reaction layer 9 or the third reaction layer 10, particularly the first reaction layer 8 or the third reaction layer 10 contains a hydrophilic polymer, the substrate 1 and Peeling of these reaction layers from the electrode surface can be prevented. The hydrophilic polymer also has an effect of preventing cracks on the surfaces of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10, and increases the reliability of the biosensor. It is effective. Furthermore, adsorption of adsorptive components such as proteins to the electrode can also be suppressed. In addition, when the 1st reaction layer 8, the 2nd reaction layer 9, or the 3rd reaction layer 10 contains a hydrophilic polymer, it may have a form in which a hydrophilic polymer is contained in the reaction layer. Alternatively, a hydrophilic polymer layer containing a hydrophilic polymer may be formed so as to cover the first reaction layer 8, the second reaction layer 9, or the third reaction layer 10.

  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 polyethylene glycol, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, 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, polyvinyl pyrrolidone, polyethylene glycol, and polyvinyl alcohol are preferred from the viewpoint of not deactivating the enzyme activity of the oxidoreductase of the present invention and having 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 from the viewpoint that an enzyme or an electron carrier can be immobilized per sensor and the solubility of the reaction layer is not lowered. More 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 a hydrophilic polymer is present in two or more reaction layers of the first reaction layer 8, the second reaction layer 9, or the third reaction layer 10, each hydrophilic layer included in these reaction layers The type and blending amount of the polymer may be the same or different. At this time, it is preferable to select in consideration of the interaction with each constituent element contained in the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10.

(sugar)
In the first biosensor and the second biosensor of the present invention, at least one of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10 may further contain sugar. Sugars do not participate in enzyme reactions related to measurement, and can be appropriately selected and used without reacting themselves, and can contribute to immobilization and stabilization of each layer. Sugar may be contained in any of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10, but is preferably contained in at least the first reaction layer 8.

  The sugar that at least one of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10 may contain is a non-reducing non-reducing aldehyde group or ketone group that has no free aldehyde group or ketone group. Reducing sugar is preferred. 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, examples include 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 and xylitol. It is done. 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 is particularly preferable.

  The amount of sugar contained in at least one of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10 is preferably 0.1 to 500 μg, more preferably 0.5 per sensor. ˜400 μg, more preferably 1 to 300 μg. If the sugar is in the form of a mixture, 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.

(protein)
In the first and second biosensors of the present invention, at least one of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10 may further contain a protein. Proteins do not participate in enzyme reactions related to measurement, and do not react with themselves, and those that do not exhibit biological activity against samples can be selected and used as appropriate, contributing to the immobilization and stabilization of each layer Can do. The protein may be contained in any of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10, but is preferably contained in at least the first reaction layer 8.

  Examples of proteins that can be included in at least one of the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10 include bovine serum albumin (BSA), casein, sericin, and hydrolysates thereof. It is done. These proteins may be used alone or in the form of a mixture of two or more. Of these, BSA is preferred because it is readily available and inexpensive. 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. At this time, the molecular weight employs a value measured using a gel filtration chromatography method.

  The amount of protein contained in the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10 is preferably 0.1 to 200 μg, more preferably 0.5 to 100 μg, more preferably 1 sensor. Preferably it is 1-50 micrograms. 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 method for forming the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10 in the first and second biosensors of the present invention is not particularly limited. Below, preferable embodiment of the production method of the 2nd biosensor which concerns on this invention is described. The present invention is not limited to the following method. Also, the production of the first biosensor according to the present invention is not particularly limited. For example, it can be carried out in the same manner except that the third reaction layer is not formed in the following.

  The first reaction layer 8 and the third reaction layer 10 may be formed by any method, but the second reaction layer 9 can be formed by applying a solution containing a lipolytic enzyme. Formed on layer 8. Here, the application method is not particularly limited, and a solution containing a lipolytic enzyme is dropped or applied to a spray device, a bar coater, a die coater, a reverse coater, a comma coater, a gravure coater, a spray coater, a doctor knife, or the like. A method of applying using an instrument can be used.

  Moreover, the formation method in particular of said 1st reaction layer 8 and the 3rd reaction layer 10 is not restrict | limited, The method equivalent to the formation method of said 2nd reaction layer can be used. At this time, the formation method of the second reaction layer 9 and the formation method of the first reaction layer 8 and the third reaction layer 10 may be the same or different. In consideration of easiness and production cost, it is preferable to use the same method, and a method of drying the 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.

  More specifically, first, the first reaction layer is formed as follows. That is, a redox enzyme (for example, PQQ-dependent glycerol dehydrogenase) and, if necessary, a surfactant (for example, emulgen) and a desired component such as a buffer solution such as glycylglycine are mixed to obtain a redox Prepare enzyme solution. A predetermined amount of this oxidoreductase solution is dropped onto the electrode (working part) (in this case, it is preferable to apply the oxidoreductase solution to all parts not covered with the insulating film including the working part of each electrode). .) After dropping the prepared oxidoreductase solution, the first reaction layer is formed on the electrode (action part) by drying it in a thermostat kept at a predetermined temperature or on a hot plate. The surfactant may be simply contained in the first reaction layer, or a layer containing a surfactant may be formed so as to cover the first reaction layer, or on the electrode. A surfactant layer may be formed, and the first reaction layer 8 may be formed thereon. Moreover, you may add other components (for example, hydrophilic polymer etc.) mentioned above to the oxidoreductase solution as needed. If necessary, a volatile organic solvent such as ethanol may be added to the oxidoreductase solution. By adding a volatile organic solvent, it is easy to dry quickly and crystallization is small. Note that an adhesive may be installed on the substrate 1 in advance.

  In the case of forming a surfactant layer, a surfactant (for example, Emulgen PP-290 (manufactured by Kao Corporation) is mixed with a desired component such as a buffer solution such as glycylglycine, a volatile organic solvent, etc. Prepare an activator solution on the electrode or first reaction layer, or as described below, on a second reaction layer, third reaction layer or cover 7 in a predetermined amount. After the surfactant solution is dropped, the surfactant layer is formed by drying in a constant temperature bath or a hot plate maintained at a predetermined temperature.

  Next, the second reaction layer is formed as follows. That is, a lipolytic enzyme (for example, lipoprotein lipase (LPL)) is mixed with a desired component such as a buffer solution such as glycylglycine to prepare a lipolytic enzyme solution. A predetermined amount of this lipolytic enzyme solution is dropped onto the oxidoreductase layer prepared above. After dropping the prepared lipolytic enzyme solution, the second reaction layer is formed on the first reaction layer by drying in a thermostat or a hot plate maintained at a predetermined temperature. A surfactant may be further added to the second reaction layer. The surfactant may be simply contained in the second reaction layer, or a layer containing the surfactant may be formed as described above so as to cover the second reaction layer. Moreover, you may add other components (for example, hydrophilic polymer etc.) mentioned above to the lipolytic enzyme solution as needed. If necessary, a volatile organic solvent such as ethanol may be added to the lipolytic enzyme solution. By adding a volatile organic solvent, it is easy to dry quickly and crystallization is small.

  The third reaction layer is formed as follows. That is, an electron carrier (for example, hexaammineruthenium (III) chloride) and, if necessary, a surfactant (for example, emulgen) and a desired component such as a buffer solution such as glycylglycine are mixed to form an electron. Prepare a transmitter solution. A predetermined amount of this electron carrier solution is dropped on the cover 7. After the prepared electron carrier solution is dropped, the third reaction layer is formed on the cover by drying in a constant temperature bath maintained at a predetermined temperature or on a hot plate. At this time, the surfactant may be simply contained in the reaction layer, or a layer containing the surfactant may be formed as described above so as to cover the reaction layer, or the surfactant may be formed on the cover. A layer may be formed, and the third reaction layer 10 may be formed thereon. Note that an adhesive may be installed on the cover 7 in advance.

  The biosensor of the present invention preferably contains trisboric acid in the reaction layer that is the configuration of the sample supply unit, but the method for containing trisboric acid in that case is not particularly limited. For example, when the first reaction layer contains trisboric acid, trisboric acid is added when the above-mentioned oxidoreductase solution is prepared. And as above-mentioned, the 1st reaction layer is formed in an electrode (action part) by dripping the oxidoreductase solution containing the said trisboric acid to the electrode (action part) predetermined amount, and making it dry. Similarly, when trisboric acid is added to the surfactant layer, the second reaction layer, and the third reaction layer, similarly, when preparing the surfactant solution, the lipolytic enzyme solution, and the electron carrier solution, respectively. Each layer can be formed by adding trisboric acid and dropping the obtained solution onto a predetermined portion and drying.

  Finally, the substrate 1 on which the first reaction layer 8 and the second reaction layer 9 are formed and the cover 7 on which the third reaction layer 10 is formed are bonded together with adhesives 6a and 6b. Thus, the second biosensor can be manufactured.

<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. There are no particular restrictions on the substrate contained in the sample as long as it is a substance that can react with each enzyme contained in the first reaction layer and the second reaction layer of the present invention and generate a measurable current as described later. Good.

  As the desired component (substrate) in the sample, for the same reason as described above, lipids such as neutral fat and cholesterol are preferably selected as the substrate.

  The form in particular of supplying a sample to a sample supply part is not restrict | limited, For example, using a capillary phenomenon, with respect to the reaction layer (the 1st reaction layer 8, the 2nd reaction layer 9, the 3rd reaction layer 10) The sample may be supplied from the horizontal direction.

  When the sample is supplied to the reaction layers (the first reaction layer 8, the second reaction layer 9, and the third reaction layer 10), the desired component (substrate) in the sample is converted into the second reaction layer 9. And oxidoreductase contained in the first reaction layer 8 are oxidized and release electrons simultaneously with their own oxidation. The electrons released from the substrate are captured by the electron carrier dissolved from the third reaction layer 10, 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.

  According to the biosensor of the present invention, the reaction time (ie, measurement time) for completing the reaction between the substrate and the enzyme can be significantly shortened. 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.

  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 can be summarized as follows. Since the biosensor of the present invention contains trisboric acid in at least a part of the sample supply unit, generation of background current (particularly generation of background current associated with prolonged storage period) ) Is suppressed. Therefore, the biosensor of the present invention can exhibit excellent accuracy.

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%”.
Example 1
"Manufacture of neutral fat sensor"
The electrode used was a three-electrode system designed and manufactured independently. The electrode has a working electrode 2 made of carbon, a reference electrode 3 and a counter electrode 4 formed on an insulating substrate 1, and a working electrode working part 2-1 made of carbon, silver / A reference electrode working part 3-1 made of silver chloride and a counter electrode working part 4-1 made of carbon are formed.

(Formation of first reaction layer (GLDH layer))
Per sensor (volume of 1 μl of sample “whole blood” to be supplied) at a final concentration, 0.5 U of PQQ-dependent glycerol dehydrogenase and 10 mM of glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) .3 μg), Emulgen PP-290 (manufactured by Kao Corporation), 0.05% (0.5 μg), trisboric acid (tris (hydroxymethyl) aminomethane and boric acid are mixed in equimolar concentrations to adjust to pH 7.5. ) Was mixed to 150 mM 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 average thickness of the first reaction layer was 5 μm.

  For each sensor (sample volume 1 μl), lipoprotein lipase (LPL, manufactured by Asahi Kasei Co., Ltd.) 80 U, trisboric acid (mixed with equimolar concentrations of tris (hydroxymethyl) aminomethane and boric acid to adjust to pH 7.5) 150 mM and hexaammineruthenium (III) chloride (Wako Pure Chemical Industries, Ltd.) were mixed so as to be 200 mM (124 μg) to obtain a solution (LPL solution). The obtained LPL solution was dropped so as to be layered (coated) on the formed GLDH layer and dried at 40 ° C. for 5 minutes to obtain a second reaction layer (LPL layer). The average thickness of the second reaction layer was 5 μm.

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

(Formation of third reaction layer (surfactant layer))
Glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) 50 mM (7.5 μg) and Emulgen PP-290 (Kao Corporation) at a final concentration per sensor (amount 1 μl of sample “whole blood” to be supplied) Manufactured to a concentration of 0.1% (4 μg) to obtain a mediator solution. The obtained mediator 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 a third reaction layer (surfactant layer). The average thickness of the third reaction layer was 5 μm.

By attaching the cover in which the third reaction layer is formed and the adhesive (double-sided tape) adhered to the substrate in which the first reaction layer and the second reaction layer are formed to each other, the present invention Such a neutral fat sensor was produced. At this time, the separation distance between the second reaction layer and the third reaction layer was 0.1 mm.
(Comparative example)
A neutral fat sensor was produced by the same method except that trisboric acid was not added to the first and second reaction layers of the above Examples.
"Evaluation of background current"
About the neutral fat sensor of an Example and a comparative example, it enclosed with the silica gel in the laminate film packaging which can be sealed light-shielding, and preserve | saved for 48 hours in a 50 degreeC thermostat. Then, it took out from the thermostat and left it to reach room temperature to dissipate heat. 45 seconds after inhaling 1 μl of the sample solution (PBS pH 7.4), a potential of +200 mV is applied between the working electrode and the counter electrode with reference to the reference electrode, and one second later, the electric current flows between the working electrode and the counter electrode. The current value was measured. This current value indicates the concentration of the reduced electron carrier, that is, the background current value, and the difference between the value immediately after production and the value after 72 hours means an increase during the storage period. The results are shown in Table 1 and FIG.

  In FIG. 5, the values immediately after production (after 0 hours) of each of the examples and comparative examples are filled (■ in FIG. 5), and the values after storage for 72 hours in a constant temperature bath at 50 ° C. are outlined (FIG. 5). □) in each.

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 Third reaction layer,
S space part.

Claims (6)

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 including a reaction layer formed on the electrode system. There,
The reaction layer is
A first reaction layer comprising at least an oxidoreductase containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group , and trisboric acid ;
Wherein formed on the first reaction layer is perforated with lipolytic enzyme, the electron mediator, and a Tris-borate, and a second coating reaction layer, a biosensor. The biosensor according to claim 1, wherein the trisboric acid is contained only in the first reaction layer and the second coating reaction layer. The biosensor according to claim 1 or 2 , wherein the oxidoreductase is a polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group. The sample supply unit further includes a third reaction layer, and the third reaction layer is provided in the sample supply unit apart from the first reaction layer and the second coating reaction layer. Item 4. The biosensor according to any one of Items 1 to 3 . The biosensor according to claim 4 , wherein the third reaction layer further includes an electron carrier . At least one layer selected from the group consisting of the first reaction layer, the second coating reaction layer , and the third reaction layer is selected from the group consisting of a surfactant, a hydrophilic polymer, a sugar, and a protein. The biosensor according to claim 4 or 5 , further comprising at least one selected.