JP2010233545A - Method for measuring polyol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a prompt method for determining a specific component having improved accuracy, and a determination kit. <P>SOLUTION: In determining a polyol by reacting the polyol with a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group, the reaction is performed in the presence of a specific surfactant. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method capable of quickly and accurately quantifying the concentration of a specific component in a sample, particularly a specific component (polyol) contained in a biological sample, using an enzyme reaction.

  As a method for quantifying a specific component in a sample, a colorimetric method or an electrochemical quantification method is known. For example, regarding a biosensor that is an electrochemical quantification method, as a typical example, an electrode system including at least a working electrode and a counter electrode is formed on an insulating substrate, and an electrode system is formed on the electrode system. A reaction layer containing a hydrophilic polymer, an oxidoreductase and an electron carrier is formed in contact therewith.

  When a sample solution containing a substrate is supplied to the reaction layer of the biosensor manufactured in this way, the reaction layer dissolves in the sample solution, whereby the enzyme and the substrate react, and the electron carrier is reduced accordingly. The reduced electron carrier is electrochemically oxidized, and the substrate concentration in the sample solution can be quantified from the obtained oxidation current value (for example, Patent Document 1).

  For example, when glycerol is used as a sample as a polyol, the following method is known. Glycerol is obtained by using glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) or glycerol-3-phosphate dehydrogenase (GPDH) as shown in the following formulas (1) and (2). It can be quantified. That is, in the following formula, glycerol can be quantified by electrochemically measuring the increased amount of reduced electron mediator.

However, this method requires the use of two kinds of expensive enzymes and has a problem that the reaction is complicated. Further, when glycerol-3-phosphate oxidase is used, there is a problem that it is affected by dissolved oxygen. Further, when glycerol-3-phosphate dehydrogenase is used, it is necessary to add expensive NAD ⁺ or NADP.

As a method using one type of enzyme without being affected by dissolved oxygen, a method using NAD ⁺ dependent glycerol dehydrogenase (NAD-GDH) is known as shown in the following formula (3).

However, like this glycerol-3-phosphate dehydrogenase, this reaction also requires the addition of expensive NAD ⁺ .

As a method for quantifying glycerol more easily and cheaply, there is a method using a polyol dehydrogenase (hereinafter also referred to as “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, there is no need to use a plurality of enzymes, and there is no need to add expensive NAD ^+. There is.

  As a characteristic of PQQ-PDH, a hydrophilic part is known, but since it is generally a membrane-bound enzyme, it is known that its solubility in water is low. Therefore, when PQQ-PDH is used as a quantitative reagent, it is preferable to add a surfactant. In Patent Document 2, Triton (registered trademark) X-100 is used as a surfactant.

Japanese Patent No. 2517153 JP 2006-271257 A

  However, further improvements were considered necessary in order to provide a method for quantifying specific components that is faster and more accurate.

  Therefore, an object of the present invention is to provide a method for quantifying a specific component and a quantification kit that are faster and more accurate.

  The present inventors have intensively studied to solve the above problems. As a result, the above problem is solved by performing the reaction in the presence of a specific surfactant when the polyol is quantified by reacting with a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group. The headline, the present invention has been completed.

  According to the present invention, it is possible to provide a quantification method and a quantification kit for a specific component that are more rapid and accurate.

It is a disassembled perspective view which shows one form of the biosensor which is 2nd Embodiment of this invention. It is sectional drawing of the biosensor shown in FIG. It is a disassembled perspective view which shows one form of the biosensor which is 2nd Embodiment of this invention. It is sectional drawing of the biosensor shown in FIG.

<First of the present invention>
The first of the present invention is 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid, n-dodecyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid, polyoxy In the presence of at least one surfactant selected from the group consisting of ethylene polyoxypropylene glycol, n-octyl-β-D-glucoside and n-octyl-β-D-thioglucoside and an electron carrier. A method for quantifying a polyol, comprising reacting with a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group.

<Second of the present invention>
The second of the present invention relates to 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS), n-dodecyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid (Zwittergent 3-12), polyoxyethylene polyoxypropylene glycol (registered trademark Emulgen PP290), at least one selected from the group consisting of n-octyl-β-D-glucoside and n-octyl-β-D-thioglucoside A polyol quantification kit comprising a surfactant of a kind, an electron carrier, and a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group.

  As described above, the present inventors have conducted intensive research in order to provide a method for quantifying a specific component more quickly and with improved accuracy. In the process, attention was paid to the surfactant used for the determination of polyol, and detailed investigations were made.

  As described above, a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group is a membrane-bound enzyme, has a strong hydrophobic property, and in order to maintain the enzyme activity of the solubilized enzyme, an interface in solution is used. Preferably an activator is present.

  Therefore, Patent Document 2 discloses that a surfactant is preferably used. However, in the conventional literature, it has not been known that the combination of a specific enzyme and a specific surfactant affects the rate of the enzyme reaction.

  Therefore, in the present invention, by using a specific surfactant at the time of quantification of a polyol (for example, glycerol), a conventionally commonly used surfactant as disclosed in Patent Document 2 (for example, for example, It was found that the reaction rate was significantly improved as compared with using a registered trademark Triton X-100 or the like, and the present invention was completed.

  The first polyol quantification method of the present invention and the second quantification kit of the present invention are applicable to any quantification method and quantification kit such as quantification by electrochemical reaction (biosensor) and quantification by colorimetric method. Can do.

  Hereinafter, several embodiments of the present invention will be described with reference to the drawings. In addition, the dimension ratio of drawing is exaggerated on account of description, and may differ from an actual ratio.

[Quantification by electrochemical reaction (biosensor)]
First, the first polyol quantification method of the present invention and the second polyol quantification kit of the present invention will be described by taking a quantification (biosensor) by an electrochemical reaction as a specific example.

  FIG. 1 is an exploded perspective view showing one embodiment of a biosensor which is an embodiment of the second (quantitative kit) of the present invention. FIG. 2 is a cross-sectional view of the biosensor of FIG.

  As shown in FIGS. 1 and 2, a working electrode 2, a reference electrode 3, and a counter electrode 4 are formed on an insulating substrate 1 (also simply referred to as “substrate” in the present specification). Further, an 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 outside of 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 electrodes. 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. The reaction layer 10 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. In FIG. 1, the reaction layer 10 and the space S located between the reaction layer 10 and the cover 7 form a sample supply unit. This reaction layer 10 comprises an electron carrier, a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group (hereinafter also referred to as “polyol dehydrogenase of the present invention”), 3-[(3-cholamidopropyl). ) Dimethylammonio] -1-propanesulfonic acid (CHAPS), n-dodecyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid (registered trademark zwittergent 3-12), polyoxyethylene polyoxypropylene glycol (Registered trademark Emulgen PP290), at least one surfactant selected from the group consisting of n-octyl-β-D-glucoside and n-octyl-β-D-thioglucoside (hereinafter referred to as “surface activity of the present invention”). And also referred to as “agent”). The reaction layer 10 may contain at least one of cyclodextrin and a cyclodextrin derivative.

  This action part (2-1, 3-1, 4-1) detects a potential application means for applying a potential to the sample in the reaction layer 10 and a current flowing in the sample when the biosensor is used. Functions as a 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 this embodiment is configured by adhering 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.

  Subsequently, FIG. 3 is an exploded perspective view showing an embodiment of a biosensor which is an embodiment of the second (quantitative kit) of the present invention. 4 is a cross-sectional view of the biosensor of FIG.

  As shown in FIGS. 3 and 4, the basic structure is the same as that of the biosensor shown in FIGS. 1 and 2, but the difference from such a biosensor is that there are two reaction layers (first reaction layer). 8 and the second reaction layer 9). At this time, the first reaction layer 8, the second reaction layer 9, and the space S disposed between the first reaction layer 8 and the second reaction layer 9 include a sample supply unit. Form. The first reaction layer 8 may include one of the polyol dehydrogenase of the present invention and the electron carrier, and the second reaction layer 9 may include the other. For convenience, the one formed on the cover 7 side is referred to as the first reaction layer 8 and the one formed on the electrode side is referred to as the second reaction layer 9. In addition, the 1st reaction layer 8 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.

  In the biosensor of this embodiment, the adhesive (double-sided tape) 6b adhered to the substrate 1 on which the second reaction layer 9 is formed and the adhesion adhered to the cover 7 on which the first reaction layer 8 is formed. The agent (double-sided tape) 6a is formed by sticking together.

  Hereinafter, each component will be described in detail. As described above, the difference between the structure of the biosensor shown in FIGS. 1 and 2 and the structure of the biosensor shown in FIGS. 3 and 4 is that there is only one reaction layer or two reaction layers. (First reaction layer 8 and second reaction layer 9). Since the other points are the same, unless otherwise specified, the specific description of the constituent elements described below also applies to these two forms of biosensors. In addition, the term “1 sensor” may be used when describing the content of each constituent element in the biosensor of the embodiment of the present invention. “1 sensor” refers to a sample supplied to the sample supply unit. Is assumed to be “0.1 to 20 μL (preferably about 2 μl)”. Therefore, a biosensor that is smaller or larger than that can be controlled by appropriately adjusting the content of each constituent element.

(Insulating substrate)
The insulating substrate 1 used in the present embodiment is not particularly limited, and a conventionally known substrate can be used. For example, plastic, paper, glass, ceramic and the like can be mentioned. 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, and the like can be given.

(electrode)
The electrode used in this embodiment includes at least a working electrode 2 and a counter electrode 4.

  The electrode of the present embodiment is not particularly limited as long as the reaction between the sample (substrate) and the polyol dehydrogenase of the present invention can be detected electrochemically. 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 embodiment, 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 chloride / silver.

(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 biosensor shown in FIGS. 1 and 2, the sample supply unit has the reaction layer 10 including the electron carrier, the polyol dehydrogenase of the present invention, and the surfactant of the present invention. . The reaction layer 10 may include at least one of cyclodextrin and a cyclodextrin derivative.

  1 and 2 is preferable in that a biosensor can be easily produced. Moreover, it is preferable at the point that the manufacturing cost at the time of mass production becomes cheap.

  Although there is no restriction | limiting in particular also in the thickness of the reaction layer 10, Preferably it is 0.01-50 micrometers, More preferably, it is 0.05-40 micrometers, More preferably, it is good to set it as 0.1-25 micrometers. 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.

  On the other hand, in the biosensor shown in FIGS. 3 and 4, the sample supply unit has a reaction layer containing an electron carrier, the polyol dehydrogenase of the present invention, and the surfactant of the present invention. The reaction layer has a first reaction layer 8 formed on the electrode and a second reaction layer 9 formed separately from the first reaction layer 8. Here, the first reaction layer 8 preferably includes one of the polyol dehydrogenase and the electron carrier of the present invention, and the second reaction layer preferably includes the other. Further, it is preferable that at least one of the first reaction layer 8 and the second reaction layer 9 includes at least one of cyclodextrin and a cyclodextrin derivative. When the first reaction layer 8 includes one of the polyol dehydrogenase and the electron carrier of the present invention, and the second reaction layer includes the other, the polyol dehydrogenase and the electron carrier of the present invention are included. Since it can be made to contain in a separate reaction layer, it is preferable at the point which can prevent the deterioration during a preservation | save by an electron carrier and this oxidoreductase contacting.

  The thickness of each of the first reaction layer 8 and the second reaction layer 9 is not particularly limited, but is preferably 0.01 to 10 μm, more preferably 0.025 to 10 μm, and still more preferably 0.05. It should be ˜8 μm. Here, the thickness of the first reaction layer 8 and the second reaction layer 9 may be the same or different. 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 addition, although there is no restriction | limiting in particular in the separation distance of the 1st reaction layer 8 and the 2nd reaction layer 9, Preferably it is 0.05-1.5 mm, More preferably, it is 0.075-1.25 mm, More preferably, it is 0.1-1 mm. It is less than 0.05 mm. If it exceeds 1.5 mm, capillary action is unlikely to occur, and the sample may not be sucked into the 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 first reaction layer 8 and the second reaction layer 9.

<Redox enzyme>
The biosensor reaction layer 10 (first reaction layer 8, second reaction layer 9) in one embodiment of the present invention contains pyrroloquinoline quinone (PQQ) as a prosthetic group (also referred to as “coenzyme”). Contains polyol dehydrogenase.

  In the present invention, the polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group may be any polyol as a substrate, and if it is an alcohol having two or more hydroxy groups (including a sugar alcohol), Although not particularly limited, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, disaccharide-derived alcohols such as lactitol, triols such as glycerol, tetritols such as erythritol, and arabitol Pentitol such as xylitol and ribitol, hexitol such as mannitol and sorbitol, and cyclitol such as inositol. Among them, glycerol (pyrroloquinoline quinone dependent glycerol dehydrogenase), arabitol (pyrroloquinoline quinone dependent arabitol dehydrogenase), sorbitol (pyrroloquinoline quinone dependent sorbitol dehydrogenase), and mannitol (pyrroloquinoline quinone dependent) are preferable. Sex mannitol dehydrogenase) as a substrate, more preferably glycerol as a substrate (hereinafter also referred to as “PQQ-dependent glycerol dehydrogenase”).

  More specifically, the polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group includes 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, gluconate dehydrogenase, 2-ketogluconate dehydrogenase, 5 keto-gluconate dehydrogenase, 2,5-dikegluconate dehydrogenase, alcohol dehydrogenase, cyclic alcohol dehydrogenase, Acetaldehyde dehydrogenase, amine dehydrogenase, shikimate dehydrogenase, and galactose oxidase and the like.

  PQQ-dependent glycerol dehydrogenase is a coenzyme-linked oxidoreductase having a function to oxidize glycerol in a sample to reduce an electron carrier. Here, the PQQ-dependent glycerol dehydrogenase uses only the electron carrier in the solution for the reaction and is not affected by dissolved oxygen. Therefore, by measuring the oxidation current of the reduced electron carrier using PQQ-dependent glycerol dehydrogenase, the neutral fat concentration in the sample can be measured more accurately.

  As the polyol dehydrogenase of the present invention, commercially available products 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 oxydans NBRC 3130, 3171, 3172, 3189, 3244, 3250, 3253, 3255, 3256, 3257, 3258, 3285, 3287, 3289, 3290, 3291 are easy to obtain. , 3292, 3293, 3294, 3462, 3990, 12467, 14819; Gluconobacter frateurii NBRC 3251, 3254, 3260, 3264, 3265, 3268, 3270, 3271, 3272, 3273, 3274, 3286, 16669 Gluconobacter cerinus NBRC 3262, 3263, 3266, 3267, 3269, 3275, 3276, etc. can be used. A representative strain of such a microorganism is Gluconobacter oxydans NBRC 3291.

  The 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 There 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 inorganic nitrogen-containing materials 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 necessary. 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 20 ° C to 50 ° C, preferably 22 ° C to 40 ° C, and most preferably 25 ° C to 35 ° C. The culture pH is 4-9, 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.

  Moreover, there is no restriction | limiting in particular about content of the polyol dehydrogenase of this invention, It selects suitably 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. Can do. 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, when 0.01 to 100 U, preferably 0.05 to 50 U, more preferably 0.1 to 10 U of enzyme is included in the reaction layer 10 (first reaction layer 8, second reaction layer 9). Good. The PQQ-dependent glycerol dehydrogenase is preferably prepared in a buffer solution such as glycylglycine.

  In the biosensor shown in FIGS. 3 and 4, the polyol dehydrogenase of the present invention may be contained in any one of the first reaction layer 8 and the second reaction layer 9, but preferably Is contained in the second reaction layer 9. Here, a general biosensor adopts a configuration in which an enzyme does not directly contact an electrode. This is because the enzyme is composed of protein, and if it is attached to the electrode surface, the electrode surface may be passivated. Since there is such general common sense, the present invention is applied to the first reaction layer 8 that is not in direct contact with the electrode in the case of two reaction layers such as the biosensor shown in FIGS. It is generally considered that the polyol dehydrogenase is contained. However, in the biosensor shown in FIGS. 3 and 4, the polyol dehydrogenase of the present invention is contained in the second reaction layer 9 on the side in contact with the electrode. This is because in the present invention, the oxidoreductase of the present invention is significantly prevented from sticking to the electrode. By containing the polyol dehydrogenase of the present invention in the second reaction layer 9, the conversion efficiency of the oxidized electron carrier to the reduced electron carrier in the vicinity of the electrode is increased. There is an advantage that the correlation with the neutral fat concentration in the medium becomes high.

<Electron carrier>
The reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) in the present embodiment includes an electron carrier (sometimes referred to as “electron acceptor”).

  The electron carrier receives the electrons generated by the action of the polyol dehydrogenase of the present invention at the time of quantification of the polyol, 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”). In addition, since the peak of the absorption band varies depending on the oxidized electron carrier and the reduced electron carrier, the quantification by the colorimetric method described later is performed by appropriately combining the types of the electron carrier and measuring the absorbance. Can do. In the quantification by the colorimetric method, a coloring reagent or the like may be combined as necessary.

  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, phenazinium methyl sulfate and derivatives thereof, p-benzoquinone and derivatives thereof, 2,6-dichlorophenolindophenol, Methylene blue, nitrotetrazolium blue, osmium complexes, ruthenium complexes, aromatic nitroso compounds, and equivalent oximes of tautomers can be preferably used. Particularly preferably, at least one selected from the group consisting of phenazinium methyl sulfate, phenazinium methyl sulfate derivatives, potassium ferricyanide, 2,6-dichlorophenolindophenol, aromatic nitroso compounds, and equivalent oximes of tautomers. It is a seed.

  The phenazinium methyl sulfate derivative is not particularly limited, but 5-methylphenazinium methyl sulfate and 1-methoxy-5-methylphenazinium methyl sulfate are preferable.

  The equivalent oxime of the aromatic nitroso compound and tautomer is not particularly limited, but N, N-bis- (2-hydroxyethyl) -4-nitrosoaniline, p-nitrosoaniline, p-benzoquinone-dioxime, etc. Is mentioned.

  There is no restriction | limiting in particular about content of an electron carrier, According to the addition amount of a sample, etc., it can adjust suitably. For example, 1 to 2000 μg, preferably 5 to 1000 μg, more preferably 10 to 500 μg of an electron carrier may be included from the viewpoint of containing a sufficient amount of the polyol serving as a substrate per sensor. . The electron carrier is also preferably prepared in a buffer solution such as glycylglycine, as will be described later.

  In the biosensor shown in FIGS. 3 and 4, the electron carrier may be included in any reaction layer of the first reaction layer 8 and the second reaction layer 9. Included in the reaction layer 8. The reason is that when an electron carrier exists in the second reaction layer 9 in contact with the electrode, that is, when an electron carrier exists on the electrode, a phenomenon like a local battery occurs, and the electron carrier is automatically reduced. This is because there is a risk of being done. Therefore, in view of providing a biosensor with improved accuracy, it is included in the first reaction layer 8 (that is, the one not in contact with the electrode).

<Surfactant>
The reaction layer 10 (first reaction layer 8 and second reaction layer 9) in the present embodiment includes the surfactant of the present invention.

  In the present invention, a specific surfactant is used when quantifying a polyol (for example, glycerol). More specifically, a specific nonionic surfactant or amphoteric surfactant is used. As a result, the reaction rate is significantly improved as compared with a conventionally commonly used surfactant as disclosed in Patent Document 2 (for example, Triton X-100).

  As the surfactant of the present invention, 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -1-propane Sulfonic acid (CHAPS), n-dodecyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid (registered trademark zwittergent 3-12), polyoxyethylene polyoxypropylene glycol (registered trademark Emulgen PP290), n- At least one selected from the group consisting of octyl-β-D-glucoside and n-octyl-β-D-thioglucoside is used. Among them, polyoxyethylene polyoxypropylene glycol (registered trademark Emulgen PP290) is preferable from the viewpoint of particularly improving the reaction rate.

  These surfactants can be obtained by purchasing commercially available products or synthesizing them with appropriate reference to known knowledge.

  In the biosensor shown in FIG. 3 and FIG. 4, the surfactant of the present invention may be contained in any reaction layer of the first reaction layer 8 and the second reaction layer 9, or both reactions. Although it may be included in the layer, it is preferably included in both reaction layers. The type of each surfactant included in both reaction layers 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 and the second reaction layer 9.

  There is no restriction | limiting in particular about content of the surfactant of this invention, According to the addition amount of a sample, etc., it can adjust suitably. From the viewpoint of significantly improving the reactivity of PQQ-dependent PDH per sensor, 0.01 to 100 μg, preferably 0.05 to 50 μg, and more 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.

  In the biosensor according to the embodiment of the present invention, other components (cyclodextrin, cyclodextrin derivative, hydrophilic polymer, swellable lamellar viscous mineral particles, fine particles, etc.) are added to the reaction layer 10 (first reaction layer). 8, preferably included in the second reaction layer 9). This will be described below.

<Cyclodextrin / cyclodextrin derivative>
The reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) in the biosensor of the embodiment of the present invention preferably contains at least one of cyclodextrin and a cyclodextrin derivative.

  There are no particular limitations on the type of cyclodextrin and cyclodextrin derivative used in the present invention. The cyclodextrin may be any of α type, β type, γ type, δ type, and ε type. However, γ-cyclodextrin is particularly preferable from the viewpoint of high solubility.

  The cyclodextrin derivative may be any of α-type, β-type, γ-type, δ-type, and ε-type. However, from the viewpoint of high solubility, β-cyclodextrin derivatives or γ-cyclodextrin derivatives are preferable, and γ-cyclodextrin derivatives are more preferable. The cyclodextrin derivative is intended to be a chemical modification such as amino, tosyl, methyl, propyl, monoacetyl, triacetyl, benzoyl, sulfonyl, and monochlorotriazinyl. (Of course, not limited to these).

For example, as cyclodextrin derivatives, 2-hydroxypropyl-α-cyclodextrin, 2,6-di-O-methyl-α-cyclodextrin, 6-O-α-maltosyl-α-cyclodextrin, 6-O- α-D-glucosyl-α-cyclodextrin mono, hexakis (2,3,6-tri-O-acetyl) -α-cyclodextrin, hexakis (2,3,6-tri-O-methyl) -α-cyclo Dextrin, hexakis (6-O-tosyl) -α-cyclodextrin, hexakis (6-amino-6-deoxy) -α-cyclodextrin, hexakis (2,3-acetyl-6-bromo-6-deoxy) -α -Cyclodextrin, hexakis (2,3,6-tri-O-octyl) -α-cyclodextrin, mono (2-O-phosphoryl) -α-cyclodex Phosphorus, mono [2, (3) -O- (carboxymethyl)] - alpha-cyclodextrin, octakis (6-O-t- butyldimethylsilyl)-.alpha.-cyclodextrin, succinyl-.alpha.-cyclodextrin,
Glucuronylglucosyl-β-cyclodextrin, heptakis (2,6-di-O-methyl) -β-cyclodextrin, heptakis (2,6-di-O-ethyl) -β-cyclodextrin, heptakis (6- O-sulfo) -β-cyclodextrin, heptakis (2,3-di-O-acetyl-6-O-sulfo) -β-cyclodextrin, heptakis (2,3-di-O-methyl-6-O- Sulfo) -β-cyclodextrin, heptakis (2,3,6-tri-O-acetyl) -β-cyclodextrin, heptakis (2,3,6-tri-O-benzoyl) -β-cyclodextrin, heptakis ( 2,3,6-tri-O-methyl) -β-cyclodextrin, heptakis (3-O-acetyl-2,6-di-O-methyl) -β-cyclodextrin, heptakis (2,3 O-acetyl-6-bromo-6-deoxy) -β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, (2-hydroxy -3-N, N, N-trimethylamino) propyl-β-cyclodextrin, 6-O-α-maltosyl-β-cyclodextrin, methyl-β-cyclodextrin, hexakis (6-amino-6-deoxy)- β-cyclodextrin, bis (6-azido-6-deoxy) -β-cyclodextrin, mono (2-O-phosphoryl) -β-cyclodextrin, hexakis [6-deoxy-6- (1-imidazolyl)]- β-cyclodextrin, monoacetyl-β-cyclodextrin, triacetyl-β-cyclodextrin Thrin, monochlorotriazinyl-β-cyclodextrin, 6-O-α-D-glucosyl-β-cyclodextrin, 6-O-α-D-maltosyl-β-cyclodextrin, succinyl-β-cyclodextrin, succinyl -(2-hydroxypropyl) -β-cyclodextrin, 2-carboxymethyl-β-cyclodextrin, 2-carboxyethyl-β-cyclodextrin, butyl-β-cyclodextrin, sulfopropyl-β-cyclodextrin, 6- Monodeoxy-6-monoamino-β-cyclodextrin, silyl [(6-Ot-butyldimethyl) 2,3-di-O-acetyl] -β-cyclodextrin, 2-hydroxyethyl-γ-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, butyl-γ- Cyclodextrin, 3A-amino-3A-deoxy- (2AS, 3AS) -γ-cyclodextrin, mono-2-O- (p-toluenesulfonyl) -γ-cyclodextrin, mono-6-O- (p-toluene) Sulfonyl) -γ-cyclodextrin, mono-6-O-mesitylenesulfonyl-γ-cyclodextrin, octakis (2,3,6-tri-O-methyl) -γ-cyclodextrin, octakis (2,6-di-) O-phenyl) -γ-cyclodextrin, octakis (6-Ot-butyldimethylsilyl) -γ-cyclodextrin, octakis (2,3,6-tri-O-acetyl) -γ-cyclodextrin, etc. Can be mentioned. Among them, from the viewpoint of availability and cost, 2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2-hydroxyethyl-γ-cyclodextrin, 2-hydroxypropyl-γ-cyclo Dextrin, 3A-amino-3A-deoxy- (2AS, 3AS) -γ-cyclodextrin, mono-2-O- (p-toluenesulfonyl) -γ-cyclodextrin, mono-6-O- (p-toluenesulfonyl) ) -Γ-cyclodextrin and butyl-γ-cyclodextrin are preferable, and 2-hydroxyethyl-γ-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, 3A-amino-3A-deoxy- (2AS, 3AS) -γ-cyclodextrin, mono-2-O- (P-toluenesulfonyl) -γ-cyclodextrin, mono-6-O- (p-toluenesulfonyl) -γ-cyclodextrin, butyl-γ-cyclodextrin.

  Moreover, you may contain the cyclodextrin and cyclodextrin derivative used for this invention with the form of a salt. Such salts are not particularly limited, but phosphates, sodium salts, potassium salts, sulfates and the like are preferable.

  There is no restriction | limiting in particular about content of a cyclodextrin and a cyclodextrin derivative, According to the addition amount of a sample, etc., it can adjust suitably. As an example, 1 to 1000 μg, preferably 5 to 500 μg, more preferably 1 to 1000 μg per sensor from the viewpoint of significantly suppressing hemolysis, significantly suppressing background current, and not lowering the solubility of the reaction layer. Is preferably contained in an amount of 10 to 100 μg.

  In addition, it is also preferable to prepare at least one of cyclodextrin and a cyclodextrin derivative with a buffer solution such as glycylglycine.

  In the biosensor shown in FIGS. 3 and 4, the cyclodextrin and the cyclodextrin derivative may be included in any one of the first reaction layer 8 and the second reaction layer 9, or both. It may be included in the layer.

<Hydrophilic polymer, swellable layered viscosity mineral particles>
It is preferable that the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) in the biosensor of the embodiment of the present invention further includes at least one of a hydrophilic polymer and a swellable layered viscosity mineral particle.

  The hydrophilic polymer has a function of immobilizing the polyol dehydrogenase or the electron carrier of the present invention on the electrode. Further, since the reaction layer 10 (first reaction layer 8 and second reaction layer 9) contains a hydrophilic polymer, the reaction layer 10 (first reaction layer 8 and second reaction layer 8 from the substrate 1 and the electrode surface). The reaction layer 9) can be prevented from peeling off. The hydrophilic polymer also has an effect of preventing the surface of the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) from cracking, and improves the reliability of the biosensor according to the embodiment of the present invention. It is effective to raise. Furthermore, adsorption of adsorptive components such as proteins to the electrode can also be suppressed. In addition, when the reaction layer 10 (the 1st reaction layer 8, the 2nd reaction layer 9) contains a hydrophilic polymer, it may have the form in which a hydrophilic polymer is contained in the reaction layer, or It may have a form in which a hydrophilic polymer layer containing a hydrophilic polymer is formed so as to cover the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9).

  A conventionally well-known thing can be used as a hydrophilic polymer which can be used for this invention. More specifically, examples of the hydrophilic polymer include carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polylysine and other polyamino acids, polystyrene sulfonic acid , Gelatin and derivatives thereof, polymers of acrylic acid or derivatives thereof, polymers of maleic anhydride or salts thereof, starch and derivatives thereof, and the like. Of these, carboxymethylcellulose is preferred from the viewpoint of not deactivating the enzyme activity of the polyol dehydrogenase (PQQ-dependent PDH) of the present invention and having high solubility. These may be used alone or in the form of a mixture.

  The blending amount of such a hydrophilic polymer is preferably from the viewpoint of immobilizing the polyol dehydrogenase or electron carrier of the present invention and not reducing the solubility of the reaction layer per sensor. It is 01-100 micrograms, More preferably, it is 0.05-50 micrograms, Most preferably, it is 0.1-10 micrograms. It is also preferable to prepare the hydrophilic polymer in a buffer solution such as glycylglycine.

  The swellable laminar viscosity mineral particles also have a function of immobilizing the polyol dehydrogenase of the present invention, the electron carrier, etc. on the electrode in the same manner as the hydrophilic polymer. The electron carrier can be fixed more uniformly by fixing with swellable layered viscosity mineral particles than by fixing with a conventional hydrophilic polymer. If immobilization is insufficient, the solubility in the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) becomes insufficient even after the sample is supplied, and there is a possibility that the measurement may vary. . Therefore, by fixing the electron carrier with the swellable lamellar viscous mineral particles, it is possible to provide a biosensor with uniform immobilization, suppressing variation in current value, and thus improved accuracy.

As the swellable lamellar viscosity mineral particles that can be used in the present invention, conventionally known particles can be used. More specifically, smectite, bentonite, synthetic fluorine mica, vermiculite and the like can be mentioned. Among these, smectite is preferable from the viewpoint of not deactivating the enzyme activity of the polyol dehydrogenase (GLDH) of the present invention and having high solubility.
These may be used alone or in the form of a mixture. These can be prepared by purchasing commercially available products. For example, as the smectite, Lucentite (trade name) and the like are preferable.

  In addition, the blending amount of such swellable layered viscosity mineral particles is preferably 0.01 to 100 μg from the viewpoint that the solubility of the reaction layer is not lowered per sensor and the electron carrier can be held uniformly. Yes, more preferably 0.05 to 50 μg, and particularly preferably 0.1 to 10 μg. It is also preferable to prepare the swellable layered viscosity mineral particles with a buffer solution such as glycylglycine.

  In the biosensor shown in FIGS. 3 and 4, the hydrophilic polymer / swellable layered viscosity mineral particles may be contained in either the first reaction layer 8 or the second reaction layer 9, but preferably Since the first reaction layer 8 contains an electron carrier, it is preferable that the swellable layered viscosity mineral particles are also contained in the first reaction layer 8 containing the electron carrier. On the other hand, preferably, since the polyol dehydrogenase of the present invention is contained in the second reaction layer 9, it is preferable that the second reaction layer 9 containing the oxidoreductase contains a hydrophilic polymer. In addition, when the polyol dehydrogenase of this invention is contained in the 2nd reaction layer 9, when the effect of fixation is considered, a hydrophilic polymer is more preferable than a swellable layered viscosity mineral particle. In this case, considering the solubility of the hydrophilic polymer, it is preferable to contain fine particles described later.

<Fine particles>
The reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) in the biosensor according to the embodiment of the present invention preferably further includes fine particles. The fine particles have a Brownian motion, and also have a role of diffusion and stirring.

  Thus, by making the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) contain fine particles, the reaction layer becomes porous and the sample solution penetrates faster. As a result, the layer containing the electron carrier and the polyol dehydrogenase of the present invention dissolves rapidly, and the entire reaction layer becomes uniform. As a result, highly accurate measurement can be performed in a short time. More specifically, when a hydrophilic polymer is contained in the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9), the hydrophilic polymer has a property of being hardly soluble. However, the problem can be solved by adding fine particles.

  The fine particles of the present invention are not particularly limited, and conventionally known fine particles can be used regardless of whether they are high polymers or low molecules. However, the fine particles used in the present invention preferably do not contain impurities that cause electrolysis and are electrochemically inactive. In addition, the fine particles used in the present invention are preferably insoluble or hardly soluble in water.

  More specifically, examples of the fine particles include polymer compounds, inorganic compounds, metal oxides, carbonates, and the like. These may be used alone or in the form of a mixture.

  More specifically, the polymer compound may be a polymer or copolymer containing at least one of acrylic acid, methacrylic acid, maleic acid, acrylic acid ester, methacrylic acid ester, maleic acid ester and styrene derivative monomer. Can be mentioned. These may be used alone or in the form of a mixture.

  Examples of the polymer containing the styrene derivative include polystyrene, styrene, and alkylstyrene.

  In addition, polyamide is also exemplified. Examples thereof include urethane compounds such as polyurethane and polyurea, and polyolefin compounds such as polyethylene and polypropylene.

  Examples of the inorganic compound include silica gel, alumina, zeolite, apatite, ceramics typified by glass and alite. These may be used alone or in the form of a mixture.

  Examples of the metal oxide include diamond powder and titanium oxide. These may be used alone or in the form of a mixture.

  Examples of the carbonate include calcium carbonate.

  In addition, as the fine particles of the present invention, latex spheres, gold coated with a polymer, fine cellulose powder, fine crystalline cellulose powder, and the like can also be used.

  The fine particles of the present invention may change the surface characteristics. Specifically, a carboxyl group or an amino group may be introduced on the surface of the fine particles. The method for introducing a carboxyl group or an amino group can be carried out by referring to or appropriately combining conventionally known knowledge, or a commercially available product may be purchased. In this way, by introducing a carboxyl group or an amino group, since it has an electric charge, it may be adsorbed with impurities (such as blood cells) contained in the sample, and may have an effect of suppressing a decrease in sensitivity due to the impurities.

  The average particle size of the fine particles is not particularly limited, but is preferably 0.1 to 20 μm. If it is less than 0.1 μm, the fine particles are too small, and the effects of the present invention may not be obtained. Moreover, when larger than 20 micrometers, microparticles | fine-particles may precipitate and it may adhere to the electrode surface. More preferably, it is 0.1-15 micrometers, and it is further more preferable that it is 0.1-10 micrometers.

  In addition, an average particle diameter can be measured by SEM observation and TEM observation, for example. The average particle diameter mentioned above is expressed by an absolute maximum length because the shape of the particles may not be uniform. Here, the absolute maximum length is an average of the maximum length L among the distances between any two points on the outline of the single bond. The value is an average value obtained from 10 pieces.

  The amount of such fine particles is preferably 0.01 to 1000 μg, more preferably 0.1 to 500 μg, and particularly preferably from the viewpoint of improving the solubility of the reaction layer per sensor. Is 1-50 μg.

  In the biosensor shown in FIGS. 3 and 4, the fine particles may be contained in any one of the first reaction layer 8 and the second reaction layer 9 or in both layers. Good. For example, if the first reaction layer 8 does not contain swellable lamellar viscosity mineral particles but contains a hydrophilic polymer, and the second reaction layer 9 also contains a hydrophilic polymer, it may be contained in both. preferable. In this way, by adding fine particles to both reaction layers (the first reaction layer 8 and the second reaction layer 9), the reaction layer becomes porous and the sample penetrates faster. As a result, the layer containing the electron carrier and the enzyme dissolves quickly, and the entire reaction layer becomes uniform. As a result, highly accurate measurement can be performed in a short time. In addition, when the first reaction layer 8 contains swellable layered viscosity mineral particles, does not contain a hydrophilic polymer, and the second reaction layer 9 also contains a hydrophilic polymer, the second reaction is preferably performed. Only included in layer 9. This is because the swellable lamellar viscosity mineral particles can firmly fix the electron carrier even in the absence of fine particles.

[Manufacturing method of biosensor]
(Biosensor shown in FIGS. 1 and 2)
The method for forming the reaction layer 10 in the biosensor shown in FIGS. 1 and 2 is not particularly limited. For example, the following method can be considered.

  The raw material for forming the reaction layer 10 containing the polyol dehydrogenase of the present invention, the electron carrier, and the surfactant of the present invention is prepared with a glycylglycine buffer solution, and the prepared raw material is Then, a predetermined amount is dropped on the electrode (working part) or the cover 7. After the prepared sample is dropped, it is dried in a constant temperature bath or a hot plate maintained at a predetermined temperature. Note that the surfactant may be simply contained in the reaction layer, or a surfactant-containing layer containing a surfactant may be formed so as to cover the reaction layer. Moreover, you may add other components (for example, cyclodextrin or its derivative (s), microparticles | fine-particles, 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. Finally, the biosensor shown in FIG. 1 and FIG. 2 can be manufactured by pasting the reaction layer 10 on the reaction layer 10 through an adhesive.

(Biosensor shown in FIGS. 3 and 4)
The method for forming the first reaction layer 8 and the second reaction layer 9 in the biosensor shown in FIGS. 3 and 4 is not particularly limited. For example, the following method can be considered.

  For the first reaction layer 8, an electron carrier (eg, 5-methylphenazinium methyl sulfate, 2,6-dichlorophenolindophenol), swellable lamellar viscous mineral particles (eg, smectite), and A raw material for forming the first reaction layer 8 containing a surfactant is prepared with a glycylglycine buffer solution or the like, and a predetermined amount of the prepared raw material is dropped onto the cover 7. At this time, the surfactant of the present invention may be simply contained in the reaction layer, or a surfactant-containing layer containing the surfactant of the present invention may be formed so as to cover the reaction layer. . Note that an adhesive may be previously installed on the cover 7. After dripping the prepared raw material, it is dried in a constant temperature bath or a hot plate maintained at a predetermined temperature. In this way, the first reaction layer 8 can be produced.

  On the other hand, for the second reaction layer 9, the polyol dehydrogenase of the present invention, a hydrophilic polymer (for example, carboxymethylcellulose), the surfactant of the present invention, fine particles (for example, polystyrene beads), The raw material for forming the 2nd reaction layer 9 containing is prepared with a glycylglycine buffer etc., and the prepared raw material is dripped at a predetermined amount to an electrode. Moreover, you may add other components (for example, cyclodextrin or its derivative (s)) mentioned above as needed. At this time, the surfactant may be simply contained in the reaction layer, or a surfactant-containing layer containing a surfactant may be formed so as to cover the reaction layer. Note that an adhesive may be installed on the substrate 1 in advance. After dripping the prepared raw material, it is dried in a constant temperature bath or a hot plate maintained at a predetermined temperature. In this way, the second reaction layer 9 can be produced.

  Finally, the substrate 1 on which the second reaction layer 9 is formed and the cover 7 on which the first reaction layer 8 is formed are bonded to each other with an adhesive, as shown in FIGS. Biosensors can be manufactured.

[sample]
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, food such as soy sauce, sauce; drainage, rain water, pool water, etc. Is mentioned.

  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. The substrate contained in the sample is not particularly limited as long as it can react with the polyol dehydrogenase of the present invention.

  Examples of the desired component (substrate) in the sample include glycerol, sorbitol, mannitol, arabitol, and the like.

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

  When the sample is supplied to the reaction layer 10 (first reaction layer 8, second reaction layer 9), the desired component (substrate) in the sample is the polyol dehydrogenase of the present invention contained in the reaction layer. It is oxidized by the action of and releases electrons simultaneously with its own oxidation. Electrons released from the substrate (polyol) are captured by the electron carrier, and the electron carrier changes from an oxidized form to a reduced form. After the sample is added, the biosensor is allowed to stand for a predetermined time, whereby the substrate is completely oxidized by the polyol dehydrogenase of the present invention, and a certain amount of electron carrier is converted from the oxidized form to the reduced form.

  There is no particular limitation on the standing time for completing the reaction between the substrate and the polyol dehydrogenase of the present invention, but after adding the sample, the biosensor is usually attached for 1 second to 5 minutes, preferably 3 seconds to 3 minutes. Just leave it alone.

  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 measured at this time (hereinafter also referred to as “oxidation current”), the amount of the reduced electron carrier before application of the potential is calculated, and further, the amount of the substrate reacted with the polyol dehydrogenase of the present invention is calculated. The amount 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 according to the embodiment 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, and a biosensor for continuously measuring a predetermined value by embedding at least an electrode part in a human body.

  In the present embodiment, since the polyol in the sample is quantified by reacting with a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group in the presence of a specific surfactant, speed and accuracy are increased. It is possible to provide a method and a kit for quantitatively determining a polyol by an electrochemical reaction, which are improved.

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

[Quantification by colorimetric method]
Next, the first polyol quantification method of the present invention and the second polyol quantification kit of the present invention will be described with specific examples of quantification by a colorimetric method. Needless to say, the present invention is not limited to the following embodiments.

  A first aspect of the present invention is a method for quantifying a polyol in a sample, wherein the polyol dehydrogenase of the present invention is reacted with the polyol in the presence of the surfactant and the electron carrier of the present invention. It is.

  The quantification by the first colorimetric method of the present invention can be performed by appropriately referring to known knowledge or combining them.

  Examples of the sample containing a polyol include food, serum, and blood clot as described above.

  The surfactant of the present invention is as described above.

  In addition, the decrease in absorbance is measured using potassium ferricyanide as described above as an electron carrier, or the decrease in absorbance is measured in the presence of 2,6-dichlorophenolindophenol and phenazinium methyl sulfate. Can be done. Alternatively, it can be quantified by measuring the formazan color produced in the presence of phenazinium methyl sulfate and nitrotetrazolium blue.

  For the content of each component such as an electron carrier, refer to the examples, refer to conventionally known knowledge (for example, Japanese Patent Publication No. 06-087793, Japanese Patent No. 2825754, etc. as appropriate, or combine them). This can be adjusted.

  Regarding the concentration of the surfactant, although depending on the surfactant to be used, the surfactant is 0.005 to 10 g, more preferably 0.0075 to 7.5 g, and most preferably 0.000 to 100 mL of the reaction solution. 01 to 5 g. If it is less than 0.005 g, PQQ-dependent PDH having strong hydrophobicity may aggregate to cause the enzyme reaction not to proceed, and if it is 10 g or more, the viscosity of the reaction solution may become too high and the reaction rate of the enzyme may decrease. Because there is.

  Subsequently, a quantitative kit (polyol quantitative kit according to the second colorimetric method of the present invention) held in another substance will be described.

  When each component used in the first quantification method of the present invention is held in a liquid to form the second quantification kit of the present invention, the color change is visually determined after mixing and reacting each component. Even if it is a thing to measure, a transmission light absorbency may be measured with a spectrophotometer.

  Moreover, when each component used for the 1st fixed_quantity | assay method of this invention is used as the 2nd fixed_quantity | assay kit of this invention, it is good also as a test device hold | maintained at a support | carrier used by dry chemistry. Here, when used in a state of being held on a carrier, a measurement layer, a spreading layer, a filtration layer, a holding layer, and the like may be included. Thus, when using it by hold | maintaining to a support | carrier, after giving a test substance, besides changing a color change visually, you may measure a reflected light absorbency with a spectrophotometer. The measured value can be converted into the amount of substrate using a calibration curve prepared in advance.

  As the carrier material, porous materials such as paper, fabric, and polymer membrane can be used. In particular, a polymer membrane is preferable in terms of coloring performance. The polymer film is a water-insoluble porous body made of a polymer. Polymers include polysulfone, polyethersulfone, cellulose, cellulose acetate, cellulose nitrate, polyacrylonitrile, polyamide, aromatic polyamide, polycarbonate, polyethylene terephthalate, polyimide, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, polychlorinated Examples include vinyl and polyvinyl alcohol. These polymers can form a film by using a generally known film forming method. Among these polymer membranes, polysulfone or polyethersulfone is particularly preferable from the viewpoint of coloring performance.

  There is no particular limitation on the method of holding the carrier, but the reagent composition (the surfactant of the present invention, the present invention) dissolved in a suitable solvent (for example, phosphate buffer, glycylglycine buffer, etc.) in the carrier. In addition to coating a carrier with a solution of (polyol dehydrogenase, electron carrier, etc.), a known method such as forming a matrix precursor containing a reagent composition to form a test layer can be used. For coating, a general coating method used for industrial use can be used. However, when the carrier is porous, uneven coating due to liquid movement immediately after coating or uneven drying is a problem. Become. The physical properties of the carrier and the coating liquid, and the coating drying method, equipment, and conditions can be important factors governing these. For this reason, the precision printing method may be effective.

  In the present embodiment, since the polyol in the sample is quantified by reacting with a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group in the presence of a specific surfactant, speed and accuracy are increased. Can provide a method and a kit for quantifying a polyol by a colorimetric method.

  The test device of the embodiment of the present invention can be applied to conventionally known test devices such as a neutral fat test device and a glucose test device.

  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>
50 μM DCIP (2,6-dichlorophenolindophenol), 0.2 mM PMS (5-methylphenazinium methyl sulfate), 450 mM glycerol and n-octyl-β-D-glucoside (Dojindo) 0.1% 20 μl of PQQ-dependent glycerol dehydrogenase solution was added to 10 mM phosphate buffer pH 7.0 containing, and the reaction between the PQQ-dependent glycerol dehydrogenase and the substrate (glycerol) was followed by the change in absorbance of DCIP at 600 nm, The rate of decrease in absorbance was taken as the enzyme reaction rate. Enzyme activity that reduces 1 μmol of DCIP per minute was defined as 1 unit (U). The molar extinction coefficient of DCIP at pH 7.0 was 16.3 mM- ¹ . The results are shown in Table 1.

<Example 2>
The enzyme activity and reaction rate were measured in the same manner as in Example 1 except that n-octyl-β-D-glucoside was changed to n-octyl-β-D-thioglucoside (manufactured by Dojindo).

<Example 3>
The enzyme activity and reaction rate were measured in the same manner as in Example 1 except that n-octyl-β-D-glucoside was changed to Emulgen PP290 (manufactured by Kao Chemical Co., Ltd.).

<Example 4>
The enzyme activity and reaction rate were measured in the same manner as in Example 1 except that n-octyl-β-D-glucoside was changed to CHAPS (manufactured by Dojindo).

<Example 5>
The enzyme activity and reaction rate were measured in the same manner as in Example 1 except that n-octyl-β-D-glucoside was changed to Zwittergent 3-12 (Calbiochem).

<Comparative Example 1>
The enzyme activity and reaction rate were measured in the same manner as in Example 1 except that n-octyl-β-D-glucoside was changed to Triton X-100 (manufactured by MP Bio Inc.).

  From Table 1, it can be seen that the reaction rate is improved by 1.5 times or more by using the surfactant of the present invention instead of the conventionally used Triton X-100.

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,
S space part.

Claims (11)

3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid, n-dodecyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid, polyoxyethylene polyoxypropylene glycol, n In the presence of at least one surfactant and electron carrier selected from the group consisting of -octyl-β-D-glucoside and n-octyl-β-D-thioglucoside,
A method for quantifying a polyol, comprising reacting a polyol with a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group.   The quantification according to claim 1, further comprising reacting the polyol with a polyol dehydrogenase containing pyrroloquinoline quinone as the prosthetic group in the presence of at least one of γ-cyclodextrin and a derivative thereof. Method.   The electron carrier is selected from the group consisting of phenazinium methyl sulfate, phenazinium methyl sulfate derivatives, potassium ferricyanide, 2,6-dichlorophenolindophenol, aromatic nitroso compounds, and equivalent oximes of tautomers. The quantification method according to claim 1 or 2 which is at least one sort.   The quantification method according to any one of claims 1 to 3, wherein the polyol is glycerol, sorbitol, mannitol or arabitol.   The quantification method according to any one of claims 1 to 4, wherein the polyol dehydrogenase containing pyrroloquinoline quinone as the prosthetic group is obtained from a bacterium belonging to the genus Gluconobacter.   The quantification method according to any one of claims 1 to 5, wherein the polyol is quantified by a colorimetric method.   The quantification method according to claim 1, wherein the polyol is quantified by an electrochemical reaction. 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid, n-dodecyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid, polyoxyethylene polyoxypropylene glycol, n -At least one surfactant selected from the group consisting of -octyl-β-D-glucoside and n-octyl-β-D-thioglucoside;
An electron carrier;
A polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group,
A polyol quantification kit comprising:   Furthermore, the quantification kit of Claim 8 containing at least 1 sort (s) of (gamma) -cyclodextrin and its derivative (s).   The quantification kit according to claim 8 or 9, wherein the polyol is quantified by a colorimetric method.   The quantification kit according to claim 8 or 9, wherein the polyol is quantified by an electrochemical reaction.

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