JP5419779B2 - Biosensor - Google Patents

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.

  Various biosensors have been proposed as a method for easily quantifying a specific component in a sample. As a typical method, an electrode system comprising at least a working electrode and a counter electrode is formed on an insulating substrate. On this electrode system, a reaction layer containing a hydrophilic polymer, an oxidoreductase and an electron carrier is formed in contact with the electrode system.

  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 (see, for example, Patent Document 1).

  For example, when glucose is used as a sample, the following method is known. Glucose can be quantified by using glucose oxidase (GOD) as shown by the following formula (1). That is, in the following formula, glucose can be quantified by electrochemically measuring the increased amount of reduced electron mediator.

  However, this method has a problem that it is affected by dissolved oxygen. As a method not affected by dissolved oxygen, there is a method represented by the following formula (2) using NAD-dependent glucose dehydrogenase (NAD-GDH) or NADP-dependent glucose dehydrogenase (NADP-GDH).

  However, this reaction requires the addition of expensive NAD + or NADP. The following method using PQQ-dependent glucose dehydrogenase (PQQ-GDH) or FAD-dependent glucose dehydrogenase (FAD-GDH) is not affected by dissolved oxygen and does not require the addition of an expensive coenzyme. There is a method shown in Equation (3).

  The above reaction has the merit that it is not affected by dissolved oxygen and it is not necessary to add an expensive coenzyme.

  When glycerol is used as a sample, 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 (4) and (5). 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. Moreover, when glycerol-3-phosphate dehydrogenase is used, it is necessary to add expensive NAD +.

  As a method using one type of enzyme without being affected by dissolved oxygen, a method using NAD + -dependent glycerol dehydrogenase (NAD-GLDH) is known as shown in the following formula (6).

However, this reaction requires the addition of expensive NAD + as well as NAD-dependent glucose dehydrogenase.

As a method for quantifying glycerol more easily and cheaply, there is a method using polyol dehydrogenase (PQQ-PDH) containing pyrroloquinoline quinone as a prosthetic group.
Since this method is carried out by the reaction of the following formula (7), there are merits that it is not affected by dissolved oxygen, the reaction is simple, it is not necessary to use a plurality of enzymes, and it is not necessary to add expensive NAD +. is there.

  As a characteristic of an oxidoreductase containing pyrroloquinoline quinone as a prosthetic group (hereinafter also referred to as “PQQ-dependent oxidoreductase”), some hydrophilic ones are known. Since it is an enzyme, it is known that it has strong hydrophobicity and low solubility in water. Therefore, when using this enzyme for a sensor, it is preferable to add a surfactant to the reaction layer. In addition, the surfactant may be added as a component of the biosensor for various reasons such as improving the sample entrance and preventing bubbles from being generated on the electrode surface.

  The above-mentioned hydrophobic membrane-bound oxidoreductase, for example, an oxidoreductase containing pyrroloquinoline quinone as a prosthetic group, needs to be purified before use, but it is as little as possible from the viewpoint of cost reduction in mass production. It is desired to purify in steps.

Japanese Patent No. 2517153

  However, when purifying an oxidoreductase containing pyrroloquinoline quinone (PQQ) as a prosthetic group with a small number of purification steps, it may be purified in a state containing impurities, and the impurities may affect the measurement system. There is a risk of affecting.

  In particular, it should be noted that other oxidoreductases are mixed.

  For example, when purifying GLDH (glycerol dehydrogenase), GDH (glucose dehydrogenase) which is the same oxidoreductase is mixed. Since GDH also performs an electron transfer reaction using a redox mediator (Med) in the same way as GLDH, when GDH is mixed, it reacts with glucose in the blood, and the glucose concentration measured by GLDH uses the glucose concentration. The value will be reflected. For example, a sample with a high glucose concentration results in a glycerol measurement that is higher than actual. This causes variations in measured values, which in turn causes a problem that accurate measurement cannot be performed.

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

  The present inventors have intensively studied to solve the above problems. As a result, it was found that the above problem can be solved by adding an excessive amount of glucose to the reaction layer of the biosensor in advance. In other words, it is found that by setting the sensor in an excessive substrate state, the influence of the glucose concentration is eliminated, and a predetermined measurement value can be obtained regardless of the blood glucose level, and there is almost no variation in the measurement value. The present invention has been completed.

  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. The sensor, wherein the sample supply unit includes an oxidoreductase containing at least pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group, an electron carrier, an interface This is solved by providing a biosensor having a reaction layer comprising an active agent and glucose.

  According to the present invention, a biosensor capable of measuring with high accuracy can be provided.

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. It is a graph showing the measurement result of the neutral fat density | concentration in the whole blood of an Example and a comparative example.

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 includes an oxidoreductase containing at least pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group, an electron carrier, and a surface activity. A biosensor having a reaction layer containing an agent and glucose.

2. Second of the present invention
According to a second aspect of the present invention, an insulating substrate, an insulating substrate formed on the 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, wherein the sample supply unit includes at least a prosthetic group such as pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide ( FMN), a reaction layer containing an electron carrier, a surfactant, and glucose, the reaction layer formed on an electrode, the first reaction layer, A second reaction layer formed separately from one reaction layer, wherein the first reaction layer includes one of the enzyme and the electron carrier, and the second reaction layer includes Including the other , It is a bio-sensor.

  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 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 includes at least an oxidoreductase (hereinafter referred to as “oxidoreductase of the present invention”) containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group. And an electron carrier, a surfactant, and glucose. 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 the oxidoreductase of the present invention and the electron carrier, and the second reaction layer 9 includes the other. In other words, in the second biosensor of the present invention, the oxidoreductase of the present invention and the electron carrier are not simultaneously contained in the same reaction layer. Specifically, if an electron carrier is included in the first reaction layer 8, the oxidoreductase of the present invention is not included, and if the oxidoreductase of the present invention is included in the second reaction layer 9, Does not include electron carriers. 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 (object to be measured) and the oxidoreductase of the present invention. For example, a carbon electrode, a gold electrode, a silver electrode, A platinum electrode, a palladium electrode, etc. are mentioned. From the viewpoint of corrosion resistance and cost, a carbon electrode is preferred.

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

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

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

<Sample supply unit>
As described above, in the first biosensor of the present invention, the sample supply unit has the reaction layer 10 including the electron carrier, the oxidoreductase of the present invention, the surfactant, and glucose.

  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.

  The thickness of the reaction layer 10 is not particularly limited, but is preferably 0.01 to 50 μm, more preferably 0.05 to 40 μm, and particularly preferably 0.1 to 25 μm. 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 the oxidoreductase of the present invention, an electron carrier, a surfactant, and glucose, 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 In addition, one of the oxidoreductase of the present invention and the electron carrier is included, and the second reaction layer 9 includes the other.

  In the embodiment having two reaction layers such as the second biosensor of the present invention, the oxidoreductase and the electron carrier of the present invention can be contained in separate reaction layers. This is preferable in that deterioration during storage due to contact with an oxidoreductase can be prevented.

  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 particularly 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, Especially preferably, it is 0.1-1 mm. If it is the said range, the oxidation-reduction enzyme of this invention and an electron carrier will hardly contact during a preservation | save, a capillary phenomenon will occur easily and a sample will be attracted | sucked to a reaction layer easily. 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)
In the present invention, the reaction layer 10 (first reaction layer 8 and second reaction layer 9) includes pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), as a prosthetic group (also referred to as “coenzyme”), Or an oxidoreductase containing flavin mononucleotide (FMN). In particular, a polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group is preferable. In the present invention, the oxidoreductase of the present invention may be used alone or in the form of a mixture.

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

  Among them, 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”).

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

  Specifically, taking 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 albidas (Gluconobacter albidus) NBRC 3250, 3273, 103509, 103510, 103516, 103520, 103521, 103524; 3276; Gluconobacter frateurii NBRC 3171, 3251, 3253, 3262, 3264, 3265, 3268, 3270, 3285, 3286, 3290, 16669, 103413, 103342, 103427, 103428, 103429, 103437, 103438, 103439, 103440 103441, 103446, 103453, 103454, 103456, 103457, 103458, 103459, 103461, 103462, 103465, 103466, 103467, 103468, 103469, 103470, 103471, 103472, 103347, 103474, 103475, 103476, 103477, 103482, 103487, 103488, 103490, 103491, 103493, 103494, 103499, 103500, 103503, 103502, 103503, 103504, 103506, 103507, 103515, 103517, 103518, 103519, 103523; Gluconobacter japonica ponicus NBRC 3260, 3263, 3269, 3271, 3272; Gluconobacter kanchanaburiensis NBRC 1035887, 103588; Gluconobacter koncter NBRC 3130, 3189, 3244, 3287, 3292, 3293, 3294, 3462, 12528, 14819; Gluconobacter roseus NBRC 3990; Gluconobacter sp NBRC 3259 103508; Gluconobacter sphaericus NBRC 12467; Gluconobacter thalidicus NBRC 3172, 3254, 3255, 3256, 3257, 3258, 3289, 3291, 100600, 601, etc. It is done.

  The medium for cultivating the PQQ-dependent glycerol dehydrogenase may be a synthetic medium or a natural medium as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance, and other necessary nutrients that can be assimilated by the strain used. There may be. As the carbon source, for example, glucose, glycerol, sorbitol and the like are used. 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 preferably 20 ° C to 50 ° C, more preferably 22 ° C to 40 ° C, and most preferably 25 ° C to 35 ° C. The culture pH is preferably 4-9, more preferably 5-8. Even under conditions other than these, it is carried out if the strain to be used grows. The culture period is usually preferably 0.5 to 5 days. By the above culture, oxidoreductase is accumulated in the cells. These oxidoreductases may be enzymes obtained by the above culture or recombinant enzymes obtained by transducing oxidoreductase genes into E. coli and the like.

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

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

  There is no restriction | limiting in particular about content of the oxidoreductase of this invention, It can select suitably by the kind of sample to measure, the addition amount of a sample, the kind of electron carrier, the quantity of the hydrophilic polymer mentioned later, etc. . For example, when a PQQ-dependent glycerol dehydrogenase is used, for example, the amount of enzyme (enzyme activity amount) that rapidly decomposes glycerol and does not decrease the solubility of the reaction layer per sensor. From the viewpoint, preferably 0.01 to 100 U, more preferably 0.05 to 50 U, particularly preferably 0.1 to 10 U of enzyme is present in the reaction layer 10 (first reaction layer 8, second reaction layer 9). It should be included. The definition and measurement method of the activity unit (U) of the PQQ-dependent glycerol dehydrogenase is based on the method described in JP-A-2006-271257. In addition, an oxidoreductase containing a PQQ-dependent glycerol dehydrogenase will be described later, but it is also preferable to prepare it in a buffer solution such as glycylglycine.

(Lipolytic enzyme)
Moreover, when using the biosensor of this invention as a neutral fat sensor, it is preferable to further contain the lipolytic enzyme which hydrolyzes the ester bond which comprises lipid. Specific examples of such lipolytic enzymes 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. For example, from the viewpoint of the amount of enzyme (enzyme activity amount) that rapidly decomposes neutral fat and does not reduce the solubility of the reaction layer, preferably 0.1 to 1000 activity units (U ), More preferably 1 to 500 U, particularly preferably 10 to 100 U (Example is 75 U). In addition, the definition and measuring method of the activity unit (U) of LPL are based on the method as described in international publication 2006/104077 pamphlet. In addition, as will be described later, LPL is preferably prepared with a buffer solution such as glycylglycine.

  The oxidoreductase and the lipolytic enzyme may coexist in one reaction layer, or may exist separately in another layer, but preferably exist separately in another layer. That is, the reaction layer preferably has a layer containing an oxidoreductase and a layer containing a lipolytic enzyme. If it is such a form, the hydrolysis reaction by a lipolytic enzyme will advance efficiently.

  In the second biosensor of the present invention, if the oxidoreductase of the present invention is not included in the same layer as the electron carrier, any reaction of the first reaction layer 8 and the second reaction layer 9 is performed. Although it may be included in the layer, it is preferably included 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. Because of 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 first reaction layer 8 that is not in direct contact with the electrode is used in the present invention. It is generally considered to contain an oxidoreductase. However, in the second biosensor of the present invention, the second reaction layer 9 on the side in contact with the electrode contains the oxidoreductase of the present invention. This is because in the present invention, the oxidoreductase of the present invention is significantly prevented from sticking to the electrode. The reason is considered to be due to the functions of the hydrophilic polymer and surfactant described later. On the contrary, the conversion efficiency of the oxidized electron carrier to the reduced electron carrier in the vicinity of the electrode is increased by containing the redox enzyme of the present invention in the second reaction layer 9, in other words, more There is an advantage that the correlation with the substrate concentration in the sample solution becomes 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 complexes, ruthenium complexes such as hexaammineruthenium (III) chloride, and the like can be suitably 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 per base mass per sensor, preferably 1 to 2000 μg, more preferably 5 to 1000 μg, and particularly preferably 10 to 500 μg of an electron carrier is included. Good. The electron carrier is also preferably prepared in a buffer solution such as glycylglycine, as will be described later.

  In the second biosensor of the present invention, if the electron carrier is not included in the same layer as the oxidoreductase of the present invention, any reaction layer of the first reaction layer 8 and the second reaction layer 9 is used. May be contained in the first reaction layer 8. The reason is that, as described above, the second reaction layer 9 preferably contains the oxidoreductase of the present invention. 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 preferably included in the first reaction layer 8 (that is, the one not in contact with the electrode).

(Surfactant)
In the biosensor of the present invention, the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9) has 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 of the present invention to be used is not lowered. For example, a nonionic surfactant, an amphoteric surfactant, a positive surfactant is used. An ionic surfactant, an anionic surfactant, a natural surfactant and the like can be appropriately selected and used. These may be used alone or in the form of a mixture. Preferably, it is at least one of a nonionic surfactant and an amphoteric surfactant from the viewpoint of not affecting the enzyme activity of the oxidoreductase of the present invention.

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

  The polyoxyethylene-based nonionic surfactant is not particularly limited, but polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-octylphenol; Triton (registered) Trademark) X-100)], polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Polyoxyethylene sorbitan monopalitanate; Tween oxyethylene 40) Sorbitan Monosteate; Tween 60 , Polyoxyethylene sorbitan monooleate (Polyoxyethylene Sorbitan Monooleate; Tween80), polyoxyethylene polyoxypropylene glycol (Emulgen PP-290 (manufactured by Kao Corporation)) and the like are preferable. Among them, from the viewpoint of increasing the solubility of the oxidoreductase of the present invention, polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-octylphenol; Triton (registered trademark)) X-100)], polyoxyethylene polyoxypropylene glycol (Emulgen PP-290 (manufactured by Kao Corporation)) is preferred.

  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, alkylthioglycoside is very preferable to alkylglycoside from the viewpoint of spread and wettability. These may be used alone or in the form of a mixture.

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

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

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

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

  Among the above surfactants, from the viewpoint of further improving the accuracy of the biosensor, it is preferable to use a low hemolytic surfactant when using whole blood as a sample. When a specific example is given, said CHAPS, Tween, and Emulgen PP-290 (made by Kao Corporation) (polyoxyethylene polyoxypropylene glycol) are preferable.

  In the second biosensor of the present invention, the surfactant may be contained in any reaction layer of the first reaction layer 8 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 the oxidoreductase of the present invention. For example, as the oxidoreductase of the present invention, PQQ-dependent glycerol dehydration is possible. When elemental enzymes are contained, since they are highly hydrophobic, it is preferable that at least the second reaction layer 9 contains a surfactant. In this case, as a kind of surfactant, from the viewpoint of further improving the accuracy of the biosensor, when using whole blood as a sample, a low hemolytic surfactant (for example, CHAPS, Tween, Emulgen PP-290, etc.) ) Is preferably used. On the other hand, as described above, the first reaction layer 8 preferably contains an electron carrier (for example, hexaammon ruthenium (III) chloride), but improves the spread and wettability. In view of further improving the accuracy of the biosensor, it is more preferable that the first reaction layer 8 also contains a surfactant. Also in this case, it is preferable to use a low hemolytic surfactant (for example, CHAPS, Tween, Emulgen PP-290, etc.) from the viewpoint of spreading and wettability. By applying such a device, the accuracy as a biosensor is further improved.

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

  When using amphoteric surfactants, it is preferable from the viewpoint of increasing the solubility of the oxidoreductase of the present invention per sensor, not deactivating the enzyme activity, and being easy to apply in the production process. 0.01 to 100 μg, more preferably 0.05 to 50 μg, and particularly preferably 0.1 to 10 μg may be contained. In addition, as described later, such a surfactant is preferably prepared in a buffer solution such as glycylglycine. When two or more kinds of surfactants are contained in one sensor, the content means the total amount.

  When a nonionic surfactant is used as the surfactant, the solubility of the oxidoreductase of the present invention is increased per sensor, the enzyme activity is not deactivated, and it is easy to apply in the production process. From the viewpoint, preferably 0.01 to 100 μg, more preferably 0.05 to 50 μg, and particularly preferably 0.1 to 10 μg is included. Such a surfactant is also preferably prepared in a buffer solution such as glycylglycine.

(glucose)
The reaction layer 10 (first reaction layer 8 and second reaction layer 9) in the present invention contains glucose. As described above, by containing glucose in the reaction layer 10 (the first reaction layer 8 and the second reaction layer 9), the influence of glucose in the blood on the measured value can be almost eliminated or not at all. Therefore, a predetermined measurement value can be obtained at any blood glucose level, and the measurement value hardly varies.

  There is no restriction | limiting in particular about content of glucose, According to the addition amount of a sample, etc., it can adjust suitably. For example, 1 sensor is preferably contained in an amount of 1 to 100 μg, more preferably 2 to 50 μg, and particularly preferably 5 to 25 μg. More specifically, when the sample to be supplied is whole blood, the whole blood preferably contains 0.5 to 50 mg, more preferably 1 to 25 mg, particularly preferably 2.5 to 12.5 mg per 1 ml. It is good to be done. If it is this range, even if the decomposition reaction of the neutral fat by an enzyme is complete | finished, it is a base mass sufficient for GDH and glucose to continue reacting. When the amount of glucose is small, the reaction between GDH and glucose is completed within the measurement time of the neutral fat concentration, which may affect the measured value.

  In the second biosensor of the present invention, glucose may be contained in one of the first reaction layer 8 and the second reaction layer 9 or in both layers. May be. In addition, when the reaction layer has a layer containing an oxidoreductase and a layer containing a lipolytic enzyme as described above, glucose contains an oxidoreductase from the viewpoint of efficiently performing a reaction between GDH and glucose. Preferably included in the layer.

  In actual measurement, the current value corresponding to the reaction of glucose can be canceled by the measuring instrument as background, and there is no possibility of affecting the measured value.

  It is also preferable to prepare the glucose in a buffer solution such as glycylglycine.

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

  The hydrophilic polymer has a function of immobilizing the oxidoreductase or 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 (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 and the 2nd reaction layer 9) contains a hydrophilic polymer, it may have a form in which a hydrophilic polymer is contained in the reaction layer, It may have a form in which a hydrophilic polymer layer containing a hydrophilic polymer is formed so as to cover the layer 10 (the first reaction layer 8 and the second reaction layer 9). Moreover, you may have the form which formed the hydrophilic polymer layer on the action part of an electrode, and also formed the layer containing an oxidoreductase and an electron carrier on it.

  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 of the present invention and having high solubility. These may be used alone or in the form of a mixture.

  In addition, the blending amount of such a hydrophilic polymer is preferably 0.01 to 100 μg from the viewpoint that an enzyme or an electron carrier can be immobilized per sensor and the solubility of the reaction layer is not lowered. More preferably, it is 0.05-50 micrograms, Most preferably, it is 0.1-10 micrograms. As will be described later, the hydrophilic polymer is preferably prepared in a buffer solution such as glycylglycine.

  The hydrophilic polymer may be contained in either the first reaction layer 8 or the second reaction layer 9 in the second biosensor of the present invention, but preferably in the second reaction layer 9. Since the oxidoreductase of the present invention is included, considering the immobilization effect, it is preferable that the second reaction layer 9 containing the oxidoreductase contains a hydrophilic polymer.

<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 is mentioned.

  A raw material for forming the reaction layer 10 containing the oxidoreductase of the present invention, an electron carrier, a surfactant, glucose, and, if necessary, a lipolytic enzyme, is obtained with a glycylglycine buffer or the like. A predetermined amount of the prepared raw material is dropped onto 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. 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, hydrophilic polymer etc.) mentioned above as needed. Moreover, you may add volatile organic solvents, such as ethanol, as needed. By adding a volatile organic solvent, it is easy to dry quickly and crystallization is small. 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.

  About the 1st reaction layer 8, the raw material for forming the 1st reaction layer 8 containing an electron carrier (for example, hexaammineruthenium (III) chloride) and surfactant as needed is used. The glycylglycine buffer solution is prepared, and a predetermined amount of the prepared raw material is dropped on the cover 7. 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. 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. 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, the oxidoreductase of the present invention (for example, PQQ-dependent glycerol dehydrogenase), a surfactant (for example, emulgen), glucose, and lipoprotein lipase (LPL). ) And, if necessary, a hydrophilic polymer, a raw material for forming the second reaction layer 9 is prepared with a glycylglycine buffer or the like, and the prepared raw material is applied to the electrode in a predetermined amount. Dripping. 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.

  In addition, for example, a raw material containing the oxidoreductase of the present invention (for example, PQQ-dependent glycerol dehydrogenase), a surfactant (for example, emulgen), glucose, and a hydrophilic polymer as necessary. A layer (GLDH layer) is formed by preparing a glycylglycine buffer or the like, dropping a predetermined amount of the prepared solution onto an electrode and drying it. Thereafter, lipoprotein lipase (LPL) is prepared with a glycylglycine buffer or the like, and a predetermined amount of the prepared liquid is dropped onto the GLDH layer and dried to form a layer (LPL layer). It may be 9. 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. Moreover, you may have the form which formed the surfactant containing layer on the electrode action part, and also formed the layer containing oxidoreductase, surfactant, and glucose on it. Note that an adhesive may be installed on the substrate 1 in advance.

  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 of the present invention.

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

  The form in particular of supplying a sample to a sample supply part is not restrict | limited, For example, 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 for disposable use, 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 glucose is contained in the reaction layer containing a specific oxidoreductase, the inside of the sensor is in an excessive substrate state, and the influence of the glucose concentration on the measured value is almost eliminated or not at all. Therefore, a predetermined measurement value can be obtained regardless of the blood glucose level, the measurement value hardly varies, and the measurement accuracy of the biosensor is further 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 neutral fat concentration in whole blood>
(Example)
As the electrode, DEP Chip EP-N (manufactured by Biodevice Technology Co., Ltd.) was used. In DEP Chip EP-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 carbon is sandwiched between insulating layers 5. , A reference electrode action part 3-1 made of silver-silver chloride and a counter electrode action part 4-1 made of carbon are formed.

  The second reaction layer (enzyme layer) was formed by the following procedure.

  Per sensor (volume of 2 μl of sample “whole blood” to be supplied) at a final concentration, 1.5 U of PQQ-dependent glycerol dehydrogenase and 5 mM of glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) 65 μg), Emulgen PP-290 (manufactured by Kao Corporation) 0.025% (0.5 μg), glucose (manufactured by Wako Pure Chemical Industries, Ltd.) 500 mg / dl (10 μg) and mixed to obtain a solution (GLDH Solution). The obtained GLDH solution was dropped so as to cover the working electrode working part, the reference electrode working part, and the counter electrode working part of EP-N, and dried at 30 ° C. for 5 minutes to form a GLDH layer.

  Overlaid on the formed GLDH layer, 75 μL of lipoprotein lipase (LPL, manufactured by Asahi Kasei Co., Ltd.) and 5 mM glycylglycine (manufactured by Wako Pure Chemical Industries, Ltd.) per sensor (sample volume 2 μl) (0.65 μg) was mixed to obtain a solution (LPL solution). The obtained LPL solution was dropped so as to cover the working electrode working part, reference electrode working part, and counter electrode working part of ER-N, and dried at 30 ° C. for 5 minutes to form an LPL layer.

  In this way, an enzyme layer in which the LPL layer was overlaid on the GLDH layer was obtained.

  The first reaction layer (mediator layer) was formed by the following procedure. 100 mM (62 μg) of hexaammineruthenium (III) chloride (Wako Pure Chemical Industries, Ltd.) and Emulgen PP-290 (Kao) at a final concentration per sensor (2 μl of sample “whole blood” to be supplied) 0.05% (2 μg) and glycylglycine (Wako Pure Chemical Industries, Ltd.) were mixed to 25 mM (3.25 μg) to obtain a mediator solution. The obtained mediator solution was dropped into a gap formed by bonding an adhesive (double-sided tape) to a cover made of PET and then dried at 50 ° C. for 5 minutes to form a second reaction layer (mediator layer).

  A neutral fat sensor is produced by bonding the substrate on which the first reaction layer is formed and the adhesive (double-sided tape) adhered to the cover on which the second reaction layer is formed to each other. Evaluation was performed. At this time, the thicknesses of the first reaction layer and the second reaction layer were 5 μm, respectively, and the separation distance was 0.15 mm.

  60 seconds after inhaling 2 μl of the sample solution (whole blood), a potential of +200 mV is applied between the working electrode and the counter electrode with reference to the reference electrode, and a current value flowing between the working electrode and the counter electrode after 5 seconds. Was measured. This current value is proportional to the concentration of the reduced electron carrier, that is, the neutral fat concentration in the whole blood, and the neutral fat concentration in the whole blood can be obtained from this current value.

  Four blood samples with different triglyceride concentrations shown in Table 1 were used as sample solutions, and each measurement was performed three times. The results are shown in Table 2 and FIG.

(Comparative example)
A neutral fat sensor was prepared and measured in the same manner as in the example except that glucose was not added to the GLDH layer. The results are shown in Table 2 and FIG.

  As is clear from FIG. 5, in the comparative example, the measured value varies greatly due to the influence of the glucose concentration in the whole blood, whereas in the example, the overall current value increases, but good linearity is obtained. It can be seen that there is no influence of glucose concentration in each sample, and there is almost no variation in measured values.

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 (7)

Bio for measuring a substrate (excluding glucose) 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. A sensor,
The sample supply unit is
An oxidoreductase containing at least pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group;
An electron carrier;
A surfactant,
Glucose and
A biosensor for measuring a substrate (excluding glucose) having a reaction layer containing. The biosensor for measuring a substrate (excluding glucose) according to claim 1, wherein the reaction layer further contains a lipolytic enzyme. The biosensor for measuring a substrate (excluding glucose) according to claim 1 or 2, wherein the glucose is contained in an amount of 5 to 25 µg per sensor. The biosensor for measuring a substrate (excluding glucose ) according to any one of claims 1 to 3, wherein the oxidoreductase is a polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group. The biosensor for measuring a substrate (excluding glucose ) according to any one of claims 2 to 4, wherein the reaction layer has a layer containing the oxidoreductase and a layer containing the lipolytic enzyme. 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 for measuring a substrate (excluding glucose ) according to any one of claims 1 to 5, wherein the second reaction layer includes the other. The biosensor for measuring a substrate (excluding glucose ) according to any one of claims 1 to 6, wherein the reaction layer further contains a hydrophilic polymer.