JP2010237140A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor more enhanced in precision. <P>SOLUTION: The biosensor includes an insulating base plate, the electrode, which includes at least an acting electrode and a counter electrode, formed on the insulating base plate and the sample supply part formed on the electrode, wherein the sample supply part includes a reaction layer containing oxidoreductase, an electron conductor and at least one of cyclodextrin and a cyclodextrin derivative. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

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

  Conventionally, the following biosensor is known as a method for easily quantifying a biological component or a specific component contained in food without diluting or stirring. That is, an electrode composed of a working electrode and a counter electrode is formed on an insulating substrate by a method such as screen printing, and after further forming an insulating layer, a hydrophilic polymer, an oxidoreductase, and an electron carrier are formed on the electrode. An enzyme reaction layer made of is formed.

  When a sample containing a substrate is dropped onto the enzyme reaction layer, the enzyme reaction layer is dissolved, the substrate reacts with the enzyme, the substrate is oxidized, and the electron carrier is reduced accordingly. After the completion of the enzyme reaction, the reduced electron carrier is electrochemically oxidized, and the substrate concentration in the sample is obtained from the oxidation current value obtained at this time (Patent Document 1).

Japanese Patent No. 2517153

  However, the accuracy of conventional biosensors is not sufficient.

  Therefore, an object of the present invention is to provide a biosensor with improved accuracy.

  The inventors of the present invention conducted intensive research to solve the above problems as follows. As a result, particularly in the case of a specific oxidoreductase, a reduced electron carrier is generated by reacting with the electron carrier even though the substrate of the oxidoreductase is not present. It was found that a response current value other than the reaction may occur (hereinafter also referred to as “background current”). This means that the following problems occur.

  In other words, in general, in biosensors, an excessive amount of enzyme is used in consideration of inactivation of the enzyme, and even if a part of the enzyme is inactivated during storage, the response current value corresponding to the measurement target substance is not obtained. It is designed not to change. However, with regard to the background current, the amount of enzyme (enzyme activity) itself affects the response current value. Therefore, the lower the inactivation, the higher the background current value, and the greater the inactivation, the smaller the background current value. There is a problem that the response current value fluctuates. The problem may become prominent depending on the storage period of the biosensor.

  In view of the above research results, as a result of intensive studies to solve the above problems, it has been found that the above problems can be solved by adding cyclodextrin or a cyclodextrin derivative to the reaction layer of the biosensor.

  That is, an object of the present invention is to provide a biomaterial having an insulating substrate, an electrode including at least a working electrode and a counter electrode formed on the insulating substrate, and a sample supply unit formed on the electrode. Solved by providing a biosensor in which the sample supply unit has a reaction layer including an oxidoreductase, an electron carrier, and at least one of cyclodextrin and a cyclodextrin derivative. As a result, the present invention has been completed.

  According to the present invention, a biosensor with improved accuracy can be provided. More specifically, a biosensor with improved accuracy can be provided by suppressing the background current.

It is a disassembled perspective view which shows one Embodiment of the 1st biosensor of this invention. It is sectional drawing of the biosensor of FIG. It is a disassembled perspective view which shows one Embodiment of the 2nd biosensor of this invention. FIG. 4 is a cross-sectional view of the biosensor of FIG. 3. 6 is a graph showing measurement results of neutral fat concentration in whole blood of Example 1 and Comparative Example 1. It is a graph showing the measurement result of the background current of Examples 2-11 and Comparative Example 2. It is a graph showing the measurement result of the background current of Example 12 and Comparative Example 3.

  Hereinafter, embodiments of the biosensor 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.

1. First of the present invention
A first aspect of the present invention is a biosensor having an insulating substrate, an electrode including at least a working electrode and a counter electrode formed on the insulating substrate, and a sample supply unit formed on the electrode. The sample supply unit is a biosensor having a reaction layer including an oxidoreductase, an electron carrier, and at least one of cyclodextrin and a cyclodextrin derivative.

2. Second of the present invention
A second aspect of the present invention is a biosensor having an insulating substrate, an electrode including at least a working electrode and a counter electrode formed on the insulating substrate, and a sample supply unit formed on the electrode. The sample supply unit includes a reaction layer including an oxidoreductase, an electron carrier, and at least one of cyclodextrin and a cyclodextrin derivative, and the reaction layer is formed on the electrode. A first reaction layer and a second reaction layer formed separately from the first reaction layer, wherein the first reaction layer includes one of the oxidoreductase and the electron carrier. And a second biosensor comprising the other reaction layer.

  FIG. 1 is an exploded perspective view showing an embodiment of the first biosensor 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. The reaction layer 10 includes an oxidoreductase, an electron carrier, and 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 first biosensor is configured by bonding a cover 7 so as to cover the reaction layer 10 via an adhesive (double-sided tape) 6 installed on the substrate 1. The adhesive (double-sided tape) 6 may be installed on the electrode side, may be installed only on the cover 7 side, or may be installed on both.

  Subsequently, FIG. 3 is an exploded perspective view showing an embodiment of the second biosensor 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 first biosensor shown in FIGS. 1 and 2, but the difference from the first biosensor is that there are two reaction layers (first The reaction layer 8 and the second reaction layer 9) are provided. 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 includes one of an oxidoreductase and an electron carrier, and the second reaction layer 9 includes the other. In other words, in the second biosensor of the present invention, the oxidoreductase and the electron carrier are not simultaneously contained in the same reaction layer. Specifically, if the first reaction layer 8 includes an electron carrier, no oxidoreductase is included, and if the second reaction layer 9 includes an oxidoreductase, an electron carrier is included. Absent. 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.

  The second biosensor has an adhesive (double-sided tape) 6b bonded to the substrate 1 on which the second reaction layer 9 is formed and an adhesive bonded to the cover 7 on which the first reaction layer 8 is formed. The agent (double-sided tape) 6a is configured to be bonded to each other. The adhesive (double-sided tape) 6 may be installed only on the substrate 1 side, or may be installed only on the cover 7 side.

  Hereinafter, each component will be described in detail. As described above, the difference between the structure of the first biosensor of the present invention and the structure of the second biosensor of the present invention is that there is only one reaction layer or two reaction layers (first The first reaction layer 8 and the second reaction layer 9). Since the other points are the same, unless otherwise specified, the specific description of the constituent elements described below applies to the first biosensor of the present invention and the second biosensor of the present invention. Is done. In the present specification, the term “1 sensor” may be used to describe the content of each constituent element. The term “1 sensor” in this specification refers to the size of a general biosensor. The 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 invention is not particularly limited, and a conventionally known substrate can be used. For example, plastic, paper, glass, ceramics 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 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 (measuring object) and the oxidoreductase, 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 using only the working electrode 2 and the counter electrode 4 or a three-electrode system further including the reference electrode 3 may be used. Note that the three-electrode method can be preferably used rather than the two-electrode method from the viewpoint that the potential is controlled with higher sensitivity. In addition, a sensing electrode for sensing the amount of liquid may be included.

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

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

<Sample supply unit>
As described above, in the first biosensor of the present invention, the sample supply unit has the reaction layer 10 including an oxidoreductase, an electron carrier, and at least one of cyclodextrin and a cyclodextrin derivative.

  The form having one reaction layer like the first biosensor of the present invention 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 second biosensor of the present invention, the sample supply unit has a reaction layer containing an oxidoreductase, an electron carrier, and at least one of cyclodextrin and a cyclodextrin derivative, 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, and the first reaction layer 8 is , One of the oxidoreductase and the electron carrier, and the second reaction layer 9 includes the other.

  In the embodiment having two reaction layers like the second biosensor of the present invention, the oxidoreductase and the electron carrier can be contained in separate reaction layers. It is preferable in that it can prevent deterioration during storage due to contact with.

  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. If the thickness is less than 0.05 mm, the oxidoreductase may contact the electron carrier during storage. On the other hand, when the thickness exceeds 1.5 mm, the capillary phenomenon hardly occurs 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.

(Oxidoreductase)
The reaction layer 10 (first reaction layer 8 and second reaction layer 9) in the present invention contains an oxidoreductase. The oxidoreductase may be used alone or in the form of a mixture.

  There is no particular limitation on the oxidoreductase, but glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, ascorbate oxidase, alcohol oxidase, alcohol dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, lactate oxidase, lactate dehydrogenase, fructose oxidase, fructose dehydrogenase, glycerol oxidase Glycerol dehydrogenase, glycerol-3-phosphate oxidase, glycerol-3-phosphate dehydrogenase, uricase and the like. These may be used alone or in the form of a mixture.

  Alternatively, as an oxidoreductase, as a prosthetic group, pyrroloquinoline quinone (PQQ), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine nucleotide phosphate ( It may be at least one selected from the group consisting of NADP). These 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, and 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.

  Examples of the oxidoreductase containing nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine nucleotide phosphate (NADP) as prosthetic groups include alcohol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase, glycerol dehydrogenase and the like.

  Among these, glycerol dehydrogenase containing at least one of pyrroloquinoline quinone (PQQ) or flavin adenine dinucleotide (FAD) is preferable as the prosthetic group, and glycerol dehydrogenase containing pyrroloquinoline quinone (PQQ) as the prosthetic group is particularly preferable (hereinafter referred to as “prosthetic group”). , Also referred to as “PQQ-dependent glycerol dehydrogenase”).

  The inventors have identified a specific oxidoreductase, in particular an oxidoreductase comprising at least one of pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group (in particular, PQQ-dependent glycerol dehydrogenase) reacts with an electron carrier in spite of the absence of the enzyme's substrate to produce a reduced electron carrier, resulting in a response current other than the reaction with the substrate. In some cases, the current value is not negligible, and it was considered that drastic contrivance was required to provide a biosensor with improved accuracy.

  Therefore, in the present invention, at least one of cyclodextrin and a cyclodextrin derivative is contained in the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9). As a result, although the mechanism is unknown, an increase in background current is suppressed. That is, in the present invention, at least one of cyclodextrin and a cyclodextrin derivative is contained in the reaction layer 10 (the first reaction layer 8 or the second reaction layer 9), thereby suppressing the background current in the biosensor. A method is also provided. Regarding the degree of suppression, it is preferable that no background current is observed (below the detection limit). However, when a biosensor is used, it is preferably 0 to 1 μA per minute, more preferably 0 to 0. It is suppressed to an increase of 0.5 μA, more preferably 0 to 0.2 μA.

  As the oxidoreductase used in 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 a PQQ-dependent glycerol dehydrogenase as an example, examples of bacteria that produce the 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, when using the biosensor of this invention as a neutral fat sensor, you may further add the enzyme which hydrolyzes the ester bond which comprises lipid. Specific examples of such an enzyme include lipoprotein lipase (LPL), lipase, and esterase. 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. As an example, from the viewpoint of the amount of enzyme (enzyme activity amount) that quickly decomposes neutral fat and does not decrease the solubility of the reaction layer, 0.1 to 1000 activity units (U) per sensor, Preferably it is 1-500U, More preferably, it is 10-100U. The definition and measurement method of LPL activity unit (U) are based on the method described in WO2006 / 104077. In addition, as will be described later, LPL is preferably prepared with a buffer solution such as glycylglycine.

  Moreover, there is no restriction | limiting in particular about content of an oxidoreductase, 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, 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 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. The oxidoreductase is also described later, but it is also preferable to prepare it with a buffer solution such as glycylglycine.

  In the second biosensor of the present invention, if the oxidoreductase used in the present invention is not included in the same layer as the electron carrier, either the first reaction layer 8 or the second reaction layer 9 is used. Although it may be contained in the reaction layer, it is preferably 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, for example, in the case where there are two reaction layers as in the second biosensor of the present invention, the oxidoreductase is present in the first reaction layer 8 which is not in direct contact with the electrode. It is generally considered to contain. However, in the second biosensor of the present invention, the second reaction layer 9 on the side in contact with the electrode contains oxidoreductase. This is because in the present invention, the oxidoreductase is significantly prevented from sticking to the electrode. On the other hand, when the second reaction layer 9 contains an oxidoreductase, the conversion efficiency of the oxidized electron mediator to the reduced electron mediator near the electrode is increased, in other words, in the sample solution. There is an advantage that the correlation with the substrate concentration is high.

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

  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 complex, ruthenium complex and the like can be preferably used.

  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. As an example, from the viewpoint of containing a sufficient amount with respect to the base mass per sensor, 1 to 2000 μg, preferably 5 to 1000 μg, more preferably 10 to 500 μg of an electron carrier may be included. The electron carrier is also preferably prepared in a buffer solution such as glycylglycine, as will be described later.

  In the second biosensor of the present invention, the electron carrier is included in any of the first reaction layer 8 and the second reaction layer 9 unless it is included in the same layer as the oxidoreductase. However, it is preferably included in the first reaction layer 8. The reason is that, as described above, it is preferable to include the oxidoreductase in the second reaction layer 9. Further, if an electron carrier exists in the second reaction layer 9 in contact with the electrode, that is, if an electron carrier exists on the electrode, a phenomenon like a local battery occurs, and the electron carrier is automatically reduced. There is a risk of it. 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).

(Cyclodextrin and cyclodextrin derivatives)
The reaction layer 10 (first reaction layer 8, second reaction layer 9) in the present invention includes at least one of cyclodextrin and cyclodextrin derivatives.

  As described above, in the present invention, by containing at least one of cyclodextrin and a cyclodextrin derivative in the reaction layer 10 (first reaction layer 8 and second reaction layer 9), an increase in background current is suppressed. It is done.

  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 preferred from the viewpoint of significantly suppressing / preventing background current and high solubility.

  The cyclodextrin derivative may be any of α-type, β-type, γ-type, δ-type, and ε-type. However, a β-cyclodextrin derivative or a γ-cyclodextrin derivative is preferable, and a γ-cyclodextrin derivative is more preferable from the viewpoint of significantly suppressing / preventing background current and high solubility. 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 10 to 100 μg is contained from the viewpoint of significantly suppressing the background current per sensor and not lowering the solubility of the reaction layer. Good. Further, when considered in comparison with the electron carrier, at least one of the cyclodextrin and the cyclodextrin derivative is preferably 0.01 to 0.8 mol, more preferably 0.01 to 0.8 mol per mol of the electron carrier. 0.5 mol, more preferably 0.01 to 0.2 mol. If the content exceeds 0.8 mol with respect to 1 mol of the electron carrier, the familiarity with the sample supplied to the biosensor may be deteriorated. In addition, when the amount of at least one of cyclodextrin and cyclodextrin derivative contained is significantly small in this way, it is considered that they do not include an electron carrier (and, for example, potassium ferricyanide is used as the electron carrier). In this case, there is no meaning of inclusion because there is no problem of solubility).

  In addition, although content of cyclodextrin and a cyclodextrin derivative was described above, when using several types like the combination of a cyclodextrin and a cyclodextrin derivative, the total amount is meant.

  The intention of containing at least one of cyclodextrin and a cyclodextrin derivative in the present invention is to improve the accuracy of the biosensor by suppressing the background current in the absence of a substrate. At this time, the present inventors believe that the problem of the suppression of the background current is solved by including a substrate that induces an enzyme reaction and a part of the oxidoreductase.

  At least one of the cyclodextrin and the cyclodextrin derivative will be described later, but it is also preferable to prepare with a buffer solution such as glycylglycine, for example.

  In the second biosensor of the present invention, the cyclodextrin and the cyclodextrin derivative may be contained in any one of the first reaction layer 8 and the second reaction layer 9, or both layers. It may be contained in, but preferably in both layers.

  In the biosensor of the present invention, the other components (surfactant, hydrophilic polymer, swellable lamellar viscosity mineral particles, fine particles, etc.) are added to the reaction layer 10 (first reaction layer 8, second reaction layer 8). In the reaction layer 9). This will be described below.

(Surfactant)
In the biosensor of the present invention, the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) may have a surfactant. By adding the surfactant to the reaction layer 10 (first reaction layer 8, second reaction layer 9), the reaction layer 10 (first reaction layer 8, second reaction layer 9) is dissolved. Can be promoted.

  The surfactant used in the present invention is not particularly limited as long as the enzyme activity of the oxidoreductase to be used does not decrease. For example, nonionic surfactants, amphoteric surfactants, cationic interfaces are used. An activator, 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.

  Although it does not restrict | limit especially as a nonionic surfactant, From a viewpoint that it does not affect the enzyme activity of an oxidoreductase, 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 polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-octylphenol; TritonX-100)] Polyoxyethylene sorbitan monolaurate (Polyoxyethylene Sorbitan Monolaurate; Tween 20), polyoxyethylene sorbitan monopalmitate (Polyoxyethylene Sorbitan Monopalmitate; Tween 40), polyoxyethylene sorbitan monostearate (Polyoxyethylene Sorbitan Monostearate; Tween 60), Polyoxyethylene sorbitan monooleate (Tween 80) is preferred. Among them, polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-octylphenol; TritonX-100)] is preferable from the viewpoint of increasing the solubility of oxidoreductase.

  Although there is no restriction | limiting in particular as an alkylglycoside type nonionic surfactant, The alkylglycoside, alkylthioglycoside, etc. which have a C7-C12 alkyl group are preferable. The carbon number is more preferably 7 to 10, and particularly preferably 8 carbon atoms. The sugar moiety is preferably glucose or maltose, more preferably glucose. More specifically, n-octyl-β-D-glucoside and n-octyl-β-D-thioglucoside are preferable. Alkyl glycoside nonionic surfactants can be applied easily and uniformly during the manufacturing process when used in biosensors. In particular, when n-octyl-β-D-thioglucoside is contained in the reaction layer 10 (first reaction layer 8, second reaction layer 9), the spread when the sample solution is dropped is very good. Good wettability (makes surface tension less likely to occur). Therefore, from the viewpoint of spread and wettability, “alkylthioglycoside” is very preferable to “alkylglycoside”. 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), 3-[(3-cholamidopropyl) dimethylammoni. O] -2-hydroxypropanesulfonic acid (CHAPSO), n-alkyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid (Zwittergent) 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.

  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.

  Of the above surfactants, CHAPS, Tween, and Emulgen PP290 (manufactured by Kao) (polyoxyethylene polyoxypropylene glycol) are preferred. In particular, CHAPS and Tween are preferable from the viewpoint of solubility.

  Among the above surfactants, in the first biosensor of the present invention, as the surfactant, CHAPS, Tween, and Emulgen PP290 are preferable, and CHAPS and Tween are particularly preferable from the viewpoint of solubility.

  In the second biosensor of the present invention, the surfactant may be contained in any reaction layer of the first reaction layer 8 and the second reaction layer 9 or in both reaction layers. Preferably, it is 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.

  More specifically, first, as described above, the second reaction layer 9 preferably contains an oxidoreductase. For example, when a PQQ-dependent glycerol dehydrogenase is contained as the oxidoreductase, Since they are highly hydrophobic, it is preferable that at least the second reaction layer 9 contains a surfactant. In this case, as the type of surfactant, CHAPS, Tween, or the like is preferably used from the viewpoint of further improving the accuracy of the biosensor. On the other hand, as described above, the first reaction layer 8 preferably contains an electron carrier (for example, potassium ferricyanide), but improves the spread and wettability, thereby improving the accuracy of the biosensor. From the viewpoint of further improving the viscosity, it is preferable that the first reaction layer 8 also contains a surfactant. In this case, from the viewpoint of spreading and wettability, it is preferable to use the alkylthioglycoside (such as n-octyl-β-D-thioglucoside) as the surfactant.

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

  When using amphoteric surfactants, 0.01-100 μg per sensor from the viewpoint of increasing the solubility of oxidoreductase, not deactivating the enzyme activity, and easy to apply in the production process. , Preferably 0.05 to 50 μg, more preferably 0.1 to 10 μg. 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.

  In the case of using a nonionic surfactant as the surfactant, from the viewpoint of increasing the solubility of the oxidoreductase per sensor, not deactivating the enzyme activity, and easy to apply in the production process, 0.01 to 100 μg, preferably 0.05 to 50 μg, 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.

(Hydrophilic polymer, swellable layered viscosity mineral particles)
The reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) in the present invention may further have at least one of a hydrophilic polymer and swellable layered viscosity mineral particles.

  The hydrophilic polymer has a function of immobilizing an oxidoreductase or electron carrier 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 (first reaction layer 8 and second reaction layer 9) from cracking, and is effective in increasing the reliability of the biosensor. is there. 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. Among these, carboxymethylcellulose is preferable from the viewpoint of not deactivating the enzyme activity of the oxidoreductase 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.

  The swellable laminar viscosity mineral particles also have a function of immobilizing an oxidoreductase or an electron carrier on the electrode in the same manner as the hydrophilic polymer.

  The inventors of the present invention have been diligently studying to provide a biosensor with improved accuracy, and the electron carrier is swelled by a swellable layered viscosity mineral particle rather than fixing with a conventional hydrophilic polymer. It has been found that the fixation can be carried out more uniformly. 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 that the enzyme activity of the oxidoreductase is not inactivated and is highly soluble. 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) is 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. Although the swellable lamellar viscosity mineral particles will be described later, it is also preferable to prepare them with a buffer solution such as glycylglycine.

  In the second biosensor of the present invention, the hydrophilic polymer / swellable laminar viscosity mineral particles may be contained in either the first reaction layer 8 or the second reaction layer 9, but as described above. Preferably, since the first reaction layer 8 contains an electron carrier, the swellable lamellar viscosity mineral particles are also preferably contained in the first reaction layer 8 containing the electron carrier. On the other hand, preferably, since the second reaction layer 9 contains oxidoreductase, the second reaction layer 9 containing oxidoreductase preferably contains a hydrophilic polymer. In the case where the second reaction layer 9 contains an oxidoreductase, a hydrophilic polymer is preferable to the swellable layered viscosity mineral particles in view of the effect of immobilization. In this case, considering the solubility of the hydrophilic polymer, it is preferable to contain fine particles described later.

  As described above, in the form having two reaction layers as in the second biosensor of the present invention, depending on the respective components to be contained in each reaction layer (first reaction layer 8 and second reaction layer 9). It can be said that using a different fixing agent is a major feature.

(Fine particles)
The reaction layer 10 (first reaction layer 8 and second reaction layer 9) in the present invention may further have 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 oxidoreductase 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 latex spheres, 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, 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 second biosensor of the present invention, 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. For example, when the first reaction layer 8 does not contain swellable lamellar viscosity mineral particles but contains a hydrophilic polymer, and the second reaction layer 9 contains a hydrophilic polymer, it is preferably contained in both. . 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 oxidoreductase dissolves rapidly, and the entire reaction layer becomes uniform. As a result, highly accurate measurement can be performed in a short time. The first reaction layer 8 contains an electron carrier and swellable layered viscosity mineral particles, does not contain a hydrophilic polymer, and the second reaction layer 9 contains an oxidoreductase and a hydrophilic polymer. , Preferably contained only in the second reaction layer 9. This is because, even in the absence of fine particles, the swellable lamellar viscosity mineral particles can fix the electron carrier uniformly and dissolve it uniformly.

<Manufacturing method of biosensor>
(First biosensor of the present invention)
Although there is no restriction | limiting in particular also in the method of forming the reaction layer 10 in the 1st biosensor of this invention, For example, the following method can be considered.

  A raw material for forming the reaction layer 10 containing an oxidoreductase, an electron carrier, and at least one of cyclodextrin and a cyclodextrin derivative is prepared with a glycylglycine buffer solution, and the prepared raw material is A predetermined amount is dropped on the electrode (action part). After the prepared sample is dropped, it is dried in a constant temperature bath or a hot plate maintained at a predetermined temperature. A surfactant may be contained. 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, 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 first biosensor can be manufactured by covering the reaction layer 10 with the cover 7 and bonding them with an adhesive.

(Second biosensor of the present invention)
Although there is no restriction | limiting in particular also in the method of forming the 1st reaction layer 8 in the 2nd biosensor of this invention, and the 2nd reaction layer 9, For example, the following method can be considered.

  For the first reaction layer 8, an electron carrier (for example, potassium ferricyanide), a swellable layered viscosity mineral particle (for example, smectite), and at least one of cyclodextrin and a cyclodextrin derivative (for example, γ-cyclodextrin) And a raw material for forming the first reaction layer 8 containing glycylglycine buffer solution and the like, and a predetermined amount of the prepared raw material is dropped on the cover 7. A surfactant may be contained. 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 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, for example, an oxidoreductase (for example, PQQ-dependent glycerol dehydrogenase), lipoprotein lipase (LPL), a hydrophilic polymer (for example, carboxymethylcellulose), and fine particles A raw material for forming the second reaction layer 9 containing (for example, polystyrene beads) and at least one of cyclodextrin and a cyclodextrin derivative (for example, γ-cyclodextrin) is obtained using a glycylglycine buffer solution or the like. A predetermined amount of the prepared raw material is dropped onto the electrode. A surfactant may be included. 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 via the adhesives 6a and 6b. A sensor 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, food 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. The substrate contained in the sample is not particularly limited as long as it can react with the oxidoreductase.

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

  The form in particular of supplying a sample to a sample supply part is not restrict | limited, For example, 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 (the first reaction layer 8 and the second reaction layer 9), the desired component (substrate) in the sample is oxidized by the action of the oxidoreductase contained in the reaction layer. It emits electrons simultaneously with its own oxidation. The electrons released from the substrate are captured by the electron carrier, and the electron carrier changes from the oxidized form to the 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 oxidoreductase, 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 enzyme, but after adding the sample, the biosensor is usually left for 1 second to 5 minutes, preferably 3 seconds to 3 minutes.

  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 as a disposable application, and 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 glucose sensor.

  The effects of the present invention are summarized below.

  In the biosensor of the present invention, since at least one of cyclodextrin and a cyclodextrin derivative is contained in the reaction layer containing the oxidoreductase, generation of background current is significantly suppressed / prevented. The accuracy of is improved.

  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%”.

<Measurement of response current value 1 (measurement of neutral fat concentration in whole blood)>
Response current value measurement 1 is the production of the second biosensor of the present invention (that is, two reaction layers), and the accuracy as a biosensor for measuring neutral fat is observed depending on whether cyclodextrin is added. did.

Example 1
As an electrode, DEP Chip ER-N (manufactured by Biodevice Technology Co., Ltd.) was used. In the DEP Chip ER-N, a working electrode 2 made of carbon, a reference electrode 3 and a counter electrode 4 are formed on an insulating substrate 1, respectively, and a working electrode working part 2-1 made of gold is sandwiched between insulating layers 5. Then, a reference electrode working part 3-1 made of silver and silver chloride and a counter electrode working part 4-1 made of carbon were formed.

  First, the second reaction layer 9 (enzyme layer) was formed by the following procedure.

  Per sensor (volume of 2 μl of sample “whole blood” supplied), final concentration of 2.5 U of PQQ-dependent glycerol dehydrogenase, 80 U of lipoprotein lipase (manufactured by Amano Enzyme), particle size of 0.5 μm Polystyrene beads (manufactured by Polysciences) 0.625% (12.5 μg), γ-cyclodextrin (manufactured by Wako Pure Chemical Industries) 1.5% (30 μg), CHAPS (manufactured by DOJINDO) 0.05% (1 μg), Carboxymethylcellulose was mixed at 0.1% (2 μg) and glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) so as to be 50 mM (6.5 μg) to obtain an enzyme solution. The obtained enzyme solution was dropped so as to cover the working electrode working part 2-1, the reference working part 3-1 and the counter working part 4-1 of ER-N, and dried at 40 ° C. for 10 minutes, A first reaction layer (enzyme layer) was obtained.

  The first reaction layer 8 (electron carrier layer) was formed by the following procedure.

  200 mM (132 μg) potassium ferricyanide (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.1% n-octyl-β-D-thioglucoside at a final concentration per sensor (amount 2 μl of sample “whole blood” to be supplied) (2 μg), γ-cyclodextrin (manufactured by Wako Pure Chemical Industries), 0.1% (2 μg), swellable layered viscosity mineral particles Lucentite SWN (manufactured by Corp Chemical), 0.05% (1 μg), glycylglycine ( Wako Pure Chemical Industries, Ltd.) was mixed to 50 mM (6.5 μg) and ethanol to 50% (1 mg) to obtain an electron carrier solution. The obtained electron carrier solution was dropped into a gap formed by adhering an adhesive (double-sided tape) 6a to a cover 7 made of PET, and then dried at 50 ° C. for 5 minutes to obtain a first reaction layer 8 (electron carrier layer). ) Was formed.

  By sticking together the substrate on which the second reaction layer 9 is formed and the adhesives (double-sided tape) 6a, 6b adhered to the cover 7 on which the first reaction layer 8 is formed, A fat sensor was fabricated and characterized. At this time, the thicknesses of the first reaction layer 8 and the second reaction layer 9 were 5 μm, respectively, and the separation distance was 0.15 mm.

  55 seconds after inhaling 2 μl of the sample liquid (whole blood), a potential of 500 mV is applied between the working electrode 2 and the counter electrode 4 with reference to the reference electrode 3, and 5 seconds later, the working electrode 2 and the counter electrode 4 The current value flowing between them was measured. This current value is proportional to the concentration of the reduced electron carrier, that is, the neutral fat concentration in the sample solution, and the neutral fat concentration in whole blood can be obtained from this current value.

  Four measurements were performed using whole blood (121, 131, 204, 336 mg / dl) from four individuals with different triglyceride concentrations. The results are shown in Table 1 and FIG.

(Comparative Example 1)
Measurement was performed in the same manner as in Example 1 except that γ-cyclodextrin was changed to distilled water. The results are shown in Table 1 and FIG.

  As can be seen from Table 1 and FIG. 5, the example containing at least one of cyclodextrin and cyclodextrin derivative (γ-cyclodextrin) has a lower overall current value and variation in any whole blood. It can be seen that there are few. This is considered to be due to the suppression of an increase in background current by adding at least one of cyclodextrin and a cyclodextrin derivative (γ-cyclodextrin).

<Measurement of response current value 2 (measurement of background current)>
Response current value measurement 2 was evaluated by adding at least one of cyclodextrin and a cyclodextrin derivative to a solution containing a PQQ-dependent polyol dehydrogenase, a surfactant, and an electron carrier. This measurement system can also be applied to an actual electrode system.

(Example 2)
PQQ-dependent polyol dehydrogenase was dissolved in 50 mM glycylglycine-NaOH buffer (pH 7.5) containing 0.2% CHAPS, and adjusted to a concentration of 1.25 U / μl.

  After adding 1.25 U / μl PQQ-dependent polyol dehydrogenase 10 μl (12.5 U) and 6% (w / v) α-cyclodextrin 5 μl to a 1.5 ml plastic tube, the mixture is drawn with a vacuum pump for 1 hour. It was made to dry. After drying, 10 μl (658 μg) of 200 mM potassium ferricyanide (manufactured by Wako Pure Chemical Industries) was added and stirred. 2 μl is sampled after elapse of a predetermined time, dropped so as to cover each working part of the electrode, a potential of +500 mV is applied between the working electrode and the counter electrode with reference to the reference electrode, and after 5 seconds, the working electrode and the counter electrode The reaction between the PQQ-dependent PDH and the electron carrier in the absence of a substrate (polyol) was evaluated by measuring the current value flowing between them. The electrode used was DEP Chip ER-N (manufactured by Biodevice Technology Co., Ltd.). The results are shown in Table 2 and FIG. In Example 2, white turbidity was observed in the reaction layer 10.

Example 3
Evaluation was performed in the same manner as in Example 2 except that 6% of α-cyclodextrin was changed to 1.8% of β-cyclodextrin (90 μg). In Example 3, white turbidity was observed in the reaction layer 10.

Example 4
Evaluation was carried out in the same manner as in Example 2 except that α-cyclodextrin was changed to 2-HE-β-cyclodextrin (2-hydroxyethyl-β-cyclodextrin) (300 μg). In Example 4, no white turbidity was observed in the reaction layer 10.

(Example 5)
Evaluation was carried out in the same manner as in Example 2 except that α-cyclodextrin was changed to 2-HP-β-cyclodextrin (2-hydroxypropyl-β-cyclodextrin) (300 μg). In Example 5, no white turbidity was observed in the reaction layer 10.

(Example 6)
Evaluation was performed in the same manner as in Example 2 except that α-cyclodextrin was changed to γ-cyclodextrin (300 μg). In Example 6, white turbidity was observed in the reaction layer 10.

(Example 7)
Evaluation was performed in the same manner as in Example 2 except that α-cyclodextrin was changed to 2-HP-γ-cyclodextrin (2-hydroxypropyl-γ-cyclodextrin) (300 μg). In Example 7, no white turbidity was observed in the reaction layer 10.

(Example 8)
Evaluation was performed in the same manner as in Example 2 except that α-cyclodextrin was changed to γ-cyclodextrin phosphate (γ-cyclodextrin sodium phosphate) (300 μg). In Example 8, no white turbidity was observed in the reaction layer 10.

Example 9
The same as Example 2 except that α-cyclodextrin was changed to aminated γ-cyclodextrin (3A-amino-3A-deoxy- (2AS, 3AS) -γ-cyclodextrin hydrate) (300 μg). Evaluation was performed. In Example 9, no white turbidity was observed in the reaction layer 10.

(Example 10)
Evaluation was performed in the same manner as in Example 2 except that α-cyclodextrin was changed to tosylated γ-cyclodextrin [mono 2-O- (p-toluenesulfonyl) -γ-cyclodextrin]] (300 μg). . In Example 10, no white turbidity was observed in the reaction layer 10.

(Example 11)
Evaluation was performed in the same manner as in Example 2 except that 6% of α-cyclodextrin was changed to 3% of γ-cyclodextrin (150 μg). In Example 11, white turbidity was observed in the reaction layer 10.

(Comparative Example 2)
Evaluation was performed in the same manner as in Example 2 except that α-cyclodextrin was not added. In Comparative Example 2, no white turbidity was observed in the reaction layer 10.

  From Table 2 and FIG. 6, β-cyclodextrin derivative, γ-cyclodextrin and derivatives thereof have an effect of suppressing the reaction between PQQ-dependent glycerol dehydrogenase and electron transporter in the absence of a substrate (polyol). It was recognized that In particular, it can be seen that γ-cyclodextrin and its derivatives have a high inhibitory effect.

<Measurement of response current value 3 (measurement of background current)>
The measurement 3 of the response current value was evaluated using a simple measurement system (reaction in the electrode reaction layer was carried out in a tube) in order to measure the background current value in the addition of γ-cyclodextrin. This measurement system can also be applied to an actual electrode system.

(Example 12)
In a 1.5 ml plastic tube, 2 μl (528 μg) of an 800 mM potassium ferricyanide (Wako Pure Chemical Industries) solution using PBS (phosphate buffered saline) as a solvent and 2 μl of a 2.5 U PQQ-dependent glycerol dehydrogenase solution A reaction solution (6 μl) was obtained by adding 2 μl (80 μg) of 4% γ-cyclodextrin solution and mixing.

  To the obtained reaction solution, 2 μl of a 12 mM glycerol solution serving as a substrate was dropped and mixed, and reacted for a predetermined time. Thereafter, using an electrode (ER-N, manufactured by Biodevice Technology), 3 μl of the reaction solution is dropped and applied so as to cover each working part of the electrode, and between the working electrode 2 and the counter electrode 4 based on the reference electrode 3. A voltage of +500 mV was applied to the electrode, and the value of a current flowing between the working electrode 2 and the counter electrode 4 was measured after 5 seconds. This current value corresponds to the concentration of the reduced electron carrier, that is, the glycerol concentration in the sample solution.

  The result of glycerol measurement is shown in FIG.

(Comparative Example 3)
Measurement was performed in the same manner as in Example 12 except that 4% γ-cyclodextrin was changed to PBS (phosphate buffered saline). The result is shown in FIG.

  As can be seen from FIG. 7, in Comparative Example 3 in which γ-cyclodextrin was not added, the current value continued to increase with time. This is thought to be because the background current continues to rise. On the other hand, in Example 12 to which γ-cyclodextrin is added, the current value after 45 seconds is set to the maximum value, and a constant current value is shown over time. That is, it can be said that the glycerol decomposition reaction is completed in 45 seconds.

  When measuring glycerol, it is necessary to set the reaction time, and it is usually set as the time when the reaction reaches equilibrium. For example, in Example 12, it can be set to 45 seconds. However, in Comparative Example 3, it is difficult to set the reaction time because the equilibrium state is not reached. Moreover, even if it is set at an arbitrary time, the current value continues to increase, resulting in variations in measurement. From these results, it is considered that γ-cyclodextrin can improve the accuracy in glycerol measurement by suppressing the background current value.

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 (10)

A biosensor having an insulating substrate, an electrode including at least a working electrode and a counter electrode formed on the insulating substrate, and a sample supply unit formed on the electrode,
The sample supply unit is
Oxidoreductase,
An electron carrier;
At least one of cyclodextrin and cyclodextrin derivatives;
A biosensor having a reaction layer comprising:   The biosensor according to claim 1, wherein at least one of the cyclodextrin and the cyclodextrin derivative is contained in an amount of 0.01 to 0.8 mol with respect to 1 mol of the electron carrier.   The oxidoreductase has pyrroloquinoline quinone (PQQ), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine nucleotide phosphate (NADP) as prosthetic groups. The biosensor according to claim 1 or 2, which is an oxidoreductase containing at least one selected from the group consisting of:   The biooxid according to any one of claims 1 to 3, wherein the oxidoreductase is an oxidoreductase containing at least one of pyrroloquinoline quinone (PQQ) and flavin adenine dinucleotide (FAD) as a prosthetic group. Sensor. The cyclodextrin is γ-cyclodextrin;
The biosensor according to any one of claims 1 to 4, wherein the cyclodextrin derivative is a β-cyclodextrin derivative or a γ-cyclodextrin derivative.   The biosensor according to claim 5, wherein the cyclodextrin derivative is a γ-cyclodextrin derivative.   The electron carrier is potassium ferricyanide, sodium ferricyanide, ferrocene, ferrocene derivative, phenazine methosulfate, phenazine methosulfate derivative, p-benzoquinone, p-benzoquinone derivative, 2,6-dichlorophenolindophenol, methylene blue, nitrotetrazolium The biosensor according to any one of claims 1 to 6, which is at least one selected from the group consisting of blue, osmium complex, and ruthenium complex. The reaction layer has a first reaction layer formed on the electrode and a second reaction layer formed separately from the first reaction layer;
The first reaction layer includes one of the enzyme and the electron carrier; and
The biosensor according to claim 1, wherein the second reaction layer includes the other.   The biosensor according to any one of claims 1 to 8, which is a neutral fat sensor.   A method for improving the accuracy of a biosensor by using the biosensor according to claim 1 and suppressing a background current of the biosensor.

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