JP2004212393A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor having a simple structure, capable of quickly, precisely measuring a substrate in a sample liquid, without being affected by AIC (Adsorptive Interfering Compounds) and OIC (Oxidizable Interfering Compounds) contained in the sample liquid. <P>SOLUTION: A first reaction layer is provided in an electrode system, including at least one pair of electrodes, on at least one insulating substrate for supporting the electrode system, and on at least the acting electrode of the electrode system. A second reaction layer includes a first reaction layer, containing an organic compound which contains a functional group that can be bonded or adsorbed to the electrode and a hydrophobic hydrocarbon group; and an amphipatic lipid, that is provided on the first reaction layer and can be bonded or adsorbed to the hydrophobic portion in the first reaction layer. The biosensor has the second reaction layer; and a reagent family that is a reagent family, carried by a two-component film composed of the first and second reaction layers and includes at least a membrane-bonded type pyrroloquinolinequinone-dependent type glucose dehydrogenase and an electron carrier. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a biosensor for measuring a substrate (a measurement target) contained in a sample solution. In particular, the present invention relates to a biosensor for measuring the concentration of glucose contained in a sample solution.

  The measurement error in the measured value such as the substrate concentration obtained by the biosensor is caused by the influence of substances other than the measurement object (substrate) contained in the sample liquid.

  For example, when a current detection type electrochemical sensor is used to measure the glucose concentration in a blood sample, oxidizable chemical substances such as ascorbic acid (vitamin C), uric acid, and acetaminophen contained in blood. However, since the current generated by electrochemical oxidation at the electrode (working electrode) of the sensor is superimposed on the current for glucose, a positive error occurs in the measured value of the glucose concentration.

  The blood concentrations of these compounds vary from person to person, and even from the same person, change daily, so it is difficult to estimate and correct the measurement errors that occur in advance.

  Chemical substances that cause such errors are called oxidizable interfering compounds (hereinafter abbreviated as OICs), and various measures have been tried in the art to eliminate the effects of these substances. I have.

  One of such measures is, for example, a method disclosed in Patent Document 1, in which a third electrode for measuring an OIC is provided on a biosensor substrate in addition to a working electrode and a counter electrode. In order to correct the influence of the OIC, a device is devised.

  In addition, as a method for removing the influence of the OIC, a method for suppressing a current caused by the OIC by providing a film on the working electrode to block diffusion of the OIC to the working electrode and a biosensor have been developed. . An example of such a biosensor is, for example, a biosensor using a poly (o-phenylenediamine) film disclosed in Non-Patent Document 1.

  On the other hand, peptides such as blood cells and proteins contained in blood are easily adsorbed on the electrode surface, and therefore, the current is also inhibited by the adsorption of these peptides on the electrode surface. When these adsorptive inhibitory substances (Adsorbive Interfering Compounds; hereinafter abbreviated as AIC) are adsorbed on the electrodes (working electrodes) of the glucose sensor, the effective electrode area is reduced, and as a result, oxidation-reduction reactions involving glucose are reduced. The resulting current is reduced, resulting in a negative error in the measurement. The degree of the current decrease varies depending on the degree of adsorption of the AIC to the electrode surface. Furthermore, since the degree of the adsorption varies depending on the concentration of the AIC in the sample solution, it is very difficult to predict the degree of the decrease in the current and correct the error that occurs.

  Various strategies have been attempted to eliminate the effects of AIC. For example, a method is disclosed in which a filter paper for separating blood cells is provided on an electrode system of a biosensor to thereby physically and efficiently remove blood cells and the like (for example, see Patent Document 2).

Further, by applying a hydrophilic polymer such as carboxymethylcellulose to the surface of an electrode system of a biosensor, adsorption of AIC such as blood cells and proteins is also suppressed (for example, see Patent Document 3).
U.S. Pat. No. 6,340,428 U.S. Pat. No. 6,033,866 JP-A-3-202768 Wang, J .; Et al., "Enhanced selectivity and sensitivity of first-generation enzyme electrodes based on the coupling of rhodinized carbon paste transducers and permselective poly (o-phenylenediamine) coatings", Electroanalysis, 8 vol., 1996, p1127-1130

  However, in the conventional biosensor, when a fluid containing OIC or AIC is used as a sample liquid, suppression of oxidation of the OIC at the working electrode and suppression of adsorption of the AIC to the electrode (working electrode) surface are not always perfect. However, there still remains a problem that a measurement error occurs in the measurement of the substrate concentration, and the concentration of the substrate in the sample solution is estimated to be lower or higher than the actual concentration. Alternatively, there is a problem that the structure of the sensor becomes complicated in order to correct the oxidation current of the OIC and to filter the AIC.

  The present invention is to solve such a problem of the related art, and provides a biosensor with a simple structure that can eliminate a measurement error in the biosensor and can quickly and accurately measure a substrate in a sample solution. The purpose is to do.

  In order to solve the conventional problems, the present invention provides a biosensor for accurately and reproducibly measuring a substrate concentration in a sample, the biosensor comprising an electrode system including at least a pair of electrodes, At least one insulating substrate for supporting the electrode system, a first reaction layer provided on at least a working electrode of the electrode system, and a second reaction provided on the first reaction layer And layers.

  In the biosensor, the first reaction layer contains an organic compound containing a functional group capable of binding or adsorbing to the electrode and a hydrophobic hydrocarbon group. The second reaction layer includes an amphipathic lipid that can bind or adsorb to the hydrophobic portion of the first reaction layer.

  Furthermore, the biosensor of the present invention includes a reagent system supported on a two-component membrane constituted by a first reaction layer and a second reaction layer, and the reagent system includes at least a membrane-bound pyrroloquinoline. It contains quinone-dependent glucose dehydrogenase and an electron carrier.

  In a preferred embodiment, the first reaction layer and the second reaction layer each form a monolayer.

  In a preferred embodiment, the organic compound is a compound represented by the following general formula (1), (2), or (3):

_{HS- (CH 2) n -X (} 1)
_{X- (CH 2) n -S-} S- (CH 2) n -X (2)
_{S- (CH 2) n -X (} 3)
(However, in the above general formulas (1), (2), and (3), n is an integer of 1 to 20, and X is a methyl group, a benzyl group, an aminobenzyl group, a carboxybenzyl group, or a phenyl group.) . n is more preferably an integer of 5 to 15.

  In a preferred embodiment, the amphiphilic lipid is L-α-phosphatidylcholine, β-oleoyl-γ-palmitoyl.

  In a preferred embodiment, the electron carrier is 1-methoxy-5-methylphenazinium.

  In a preferred embodiment, the working electrode contains gold, platinum, or palladium.

  In a preferred embodiment, the counter electrode of the electrode system does not contain any of gold, platinum, or palladium.

  In a preferred embodiment, the entire surface of the working electrode of the electrode system and a part of the surface of the counter electrode are covered with the two-component film. More preferably, the area of the portion of the counter electrode not covered with the two-component film is larger than the area of the working electrode.

  In a preferred embodiment, only the surface of the working electrode of the electrode system is covered with a two-component film.

  In a preferred embodiment, each of the pair of electrodes is supported on an opposing surface of two insulating substrates disposed so as to face each other so as to form the supply path for the sample liquid therebetween.

  In a preferred embodiment, the reagent system further comprises a pH buffer.

  The present invention provides, in still another aspect, a method for producing a biosensor. The method for producing a biosensor of the present invention includes a step of forming an electrode system including at least a pair of electrodes on an insulating substrate, and a step of providing a first reaction layer on at least a working electrode of the electrode system. Contacting a solution containing an organic compound containing a functional group and a hydrophobic hydrocarbon group capable of binding or adsorbing to the working electrode, providing a second reaction layer on the first reaction layer. Contacting a solution containing the amphipathic lipid with the first reaction layer, wherein the solution containing the organic compound and the solution containing the amphipathic lipid are mixed. At least one of them contains an electron carrier, and the solution containing the amphipathic lipid further contains a membrane-bound pyrroloquinoline quinone-dependent glucose dehydrogenase.

  In a preferred embodiment of the method for producing a biosensor of the present invention, the first reaction layer and the second reaction layer each form a monomolecular layer.

  In a further preferred embodiment, the organic compound is a compound represented by the following general formula (1), (2), or (3):

_{HS- (CH 2) n -X (} 1)
_{X- (CH 2) n -S-} S- (CH 2) n -X (2)
_{S- (CH 2) n -X (} 3)
(However, in the above general formulas (1), (2), and (3), n is an integer of 1 to 20, and X is a methyl group, a benzyl group, an aminobenzyl group, a carboxybenzyl group, or a phenyl group.) . n is more preferably an integer of 5 to 15.

  In a preferred embodiment of the method for producing a biosensor of the present invention, the amphipathic lipid is L-α-phosphatidylcholine, β-oleoyl-γ-palmitoyl.

  In a preferred embodiment of the method for producing a biosensor according to the present invention, the electron carrier is 1-methoxy-5-methylphenazinium.

  In a preferred embodiment of the biosensor manufacturing method according to the present invention, the working electrode contains gold, platinum, or palladium.

  In a preferred embodiment of the biosensor manufacturing method according to the present invention, the counter electrode of the electrode system does not contain any of gold, platinum, and palladium.

  In a preferred embodiment of the biosensor manufacturing method of the present invention, the entire surface of the working electrode of the electrode system and a part of the surface of the counter electrode are covered with the first and second reaction layers. More preferably, the area of the portion of the counter electrode that is not covered with the first and second reaction layers is larger than the area of the working electrode.

  In a preferred embodiment of the method for producing a biosensor of the present invention, only the surface of the working electrode of the electrode system is coated with the first and second reaction layers.

  In a preferred embodiment of the method for producing a biosensor of the present invention, each of the pair of electrodes is provided on an opposing surface of two insulating substrates arranged to face each other so as to form the supply path for the sample liquid therebetween. Each is supported.

  In a preferred embodiment of the method for producing a biosensor of the present invention, at least one of the solution containing the organic compound and the solution containing the amphipathic lipid further contains a pH buffer.

  The present invention provides a film of an organic compound containing a sulfur atom bonded to an electrode surface on a substrate, wherein a pyrroloquinoline quinone-dependent glucose dehydrogenase (hereinafter abbreviated as PQQ-GDH) is formed on the film of the organic compound. The present inventors have found that the use of a two-component membrane composed of the provided amphipathic lipid membrane enables stable immobilization on the electrode surface.

  As a means for effectively preventing the effect of OIC on the oxidation-reduction reaction occurring on the electrode surface and the effect of AIC adsorbed on the electrode surface, and selectively performing only the oxidation-reduction reaction involving glucose with the electrode, The present invention uses the above two-component membrane provided on an electrode and an enzyme bound or adsorbed to the two-component membrane, that is, a membrane-bound PQQ-GDH. As far as the present inventors know, there was no example of applying such a two-component membrane and membrane-bound PQQ-GDH to a glucose sensor before the present invention.

  Thus, by using a biosensor electrode in which PQQ-GDH is immobilized in a two-component film formed on the electrode, and using an appropriate electron carrier in the two-component film, Thus, a system that enables highly selective electrochemical oxidation of glucose as a substrate is realized.

  In general, enzymes that catalyze the selective oxidation reaction of glucose are roughly classified into glucose oxidase (GOx) and glucose dehydrogenase (GDH) according to the type of the reaction. Furthermore, it is known that GDH includes nicotinamide adenine dinucleotide (NAD) -dependent GDH (NAD-GDH) and PQQ-GDH.

  Furthermore, it is known that PQQ-GDH includes a water-soluble PQQ-GDH, which has been conventionally known to be applied to a biosensor, and a membrane-bound PQQ-GDH. , Oublie et al., The EMBO Journal Vol. 18 No. 19 pp. 5187-5194, 1999, and Matsushita et al., Biochemistry 1989, 28 (15), 6276-80). The membrane-bound PQQ-GDH has a hydrophobic domain on the surface of its protein molecule, and the hydrophobic domain is enhanced by hydrophobic interaction with a hydrophobic portion of a lipid bilayer such as a cell membrane. It is PQQ-GDH in which stable binding to a biological membrane is realized in vivo by binding with affinity. On the other hand, such membrane-bound GOx and NAD-GDH are not known, and only water-soluble types are currently available.

  Among these enzymes, that is, GOx, NAD-GDH, and PQQ-GDH, a membrane-bound type is used for the immobilization to the sulfur-containing organic compound / amphiphilic lipid-based two-component membrane in the present invention. Most preferably, PQQ-GDH is used. This is because, in the case of a membrane-bound type, an enzyme that catalyzes a selective oxidation reaction of glucose can be stably bound or adsorbed to the hydrophobic portion of the two-component membrane. On the other hand, in the case of the water-soluble type, since there is substantially no hydrophobic domain on the surface of the enzyme molecule, it is difficult to bind with high affinity to the hydrophobic portion of the two-component membrane.

  The present invention provides a biosensor in which a fluid containing OIC or AIC is used as a sample liquid, a measurement error generated when the OIC is oxidized at a working electrode, and the AIC adsorbed on an electrode (working electrode) surface. Provided is a biosensor having a simple structure that can eliminate a measurement error that occurs and can quickly and accurately measure a substrate in a sample solution.

  The biosensor of the present invention prevents oxidation at the working electrode of the OIC and adsorption of the AIC to the electrode (or working electrode) surface by the two-component membrane, and furthermore, the biosensor between the working enzyme and the working electrode is used. Achieving electron transfer eliminates measurement errors in substrate concentration measurement, which has been an issue so far. Significantly, the present invention has the advantage that since oxygen does not accept electrons from PQQ-GDH, the effect of dissolved oxygen on glucose oxidation as with conventional glucose oxidase also does not occur. .

  A biosensor according to one embodiment of the present invention is provided on at least an electrode system including at least a pair of electrodes, at least one insulating substrate for supporting the electrode system, and at least a working electrode of the electrode system. A first reaction layer, a first reaction layer containing a functional group capable of binding or adsorbing to the electrode and an organic compound containing a hydrophobic hydrocarbon group, and a first reaction layer provided on the first reaction layer. A second reaction layer, comprising a second reaction layer containing an amphipathic lipid capable of binding or adsorbing to the hydrophobic portion of the first reaction layer, and the first and second reaction layers A reagent system supported on a two-component membrane, comprising at least a membrane-bound pyrroloquinoline quinone-dependent glucose dehydrogenase and an electron mediator.

  Hereinafter, the present invention will be described with reference to the drawings.

  FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, from which a two-component film and a reagent system contained therein have been removed. The working electrode 2 and the counter electrode 3 are provided on the substrate 1. To form such an electrode, an electrode made of a resin or the like for covering the substrate surface other than the portion on the substrate on which an electrode pattern is to be formed on an electrically insulating substrate 1 such as glass. A pattern mask may be placed and a metal such as gold may be sputtered thereon. Such a method is commonly used in the art. In the present application, the term “working electrode” mainly refers to an electrode (anode) that causes an oxidation reaction of an electron carrier, and the term “counter electrode” mainly refers to an electrode (cathode) that causes another reaction. Used as a pointer. However, when the reduction reaction is used for detecting and / or quantifying the substrate, the anode and the cathode are reversed.

  Alternatively, as described in Patent Document 1, for example, a metal paste may be printed on a resin substrate 1 by screen printing to form an electrode pattern. In order to improve the adhesion between the gold and the glass, a chromium layer may be formed between the gold and the glass to increase the adhesion between the two. The working electrode 2 and the counter electrode 3 are electrically connected to measurement terminals outside the biosensor by leads 4 and 5, respectively. In this specification, the working electrode 2 and the counter electrode 3 may be simply referred to as electrodes without particular distinction depending on the context. Also, as can be seen from FIG. 1, the electrode and the lead are often integrally formed of the same material. In such a case, in the present invention, the electrode is a sample of such a structure. It refers to the part that comes into contact with the liquid.

  Next, a description will be given with reference to FIGS. FIG. 2 is a longitudinal sectional view of a biosensor according to one embodiment of the present invention, and FIG. 3 schematically shows a part of a biosensor manufacturing scheme according to one embodiment of the present invention and the principle of the present invention. FIG.

  On the working electrode 2 formed on the substrate 1, a film 10 of an organic compound containing a sulfur atom, and further thereon a film 11 of an amphipathic lipid are formed. As a reagent system, an enzyme such as PQQ-GDH and an electron carrier such as 1-methoxy-5-methylphenadium are contained in a two-component membrane composed of the membrane 10 and the membrane 11.

  As shown in FIG. 3, the organic compound film 10 containing a sulfur atom is bonded to the working electrode 2 via a sulfur atom (indicated by S in the figure). This organic compound represented by the general formula described above has a hydrophobic portion (indicated by a single broken line in FIG. 3). In order to form a uniform two-component layer with the amphipathic lipid on the substrate, it is preferable to use the organic compound having a hydrophobic portion having a uniform length. Thereby, a monomolecular layer of the organic compound is easily formed on the substrate, and as a result, the two-component layer becomes uniform and a stable blocking effect is obtained. The length of the hydrocarbon chain forming the hydrophobic portion depends on the value of n in the general formula (1), (2) or (3). n is preferably an integer of 1 to 20, more preferably an integer of 5 to 15. The optimal n can be appropriately selected from the above range from the viewpoint of eliminating the interfering current by the OIC and easily detecting the current based on the oxidation-reduction of glucose.

  The organic compound containing a sulfur atom is preferably a compound represented by the above general formula (1), (2) or (3). These include, for example, ethylthiol, propylthiol, butylthiol, pentylthiol, hexylthiol, heptylthiol, octylthiol, nonylthiol, decanethiol, undecanethiol, dodecanethiol, tridecanethiol, tetradecanethiol, pentadecanethiol, hexadecane Thiol, heptadecanethiol, octadecanethiol, nonadecanethiol, icosanthiol, and disulfides corresponding to each of them (having a structure in which the thiol having the same structure is subjected to S—S coupling), and a benzyl group at the terminal thereof; Thiols and disulfides such as an aminobenzyl group, a carboxybenzyl group, or a phenyl group are exemplified. These organic compounds are commercially available from suppliers well known to those skilled in the art. The above-mentioned thiol compound and disulfide compound are preferred because they strongly and irreversibly adsorb and bond to the metal surface and substantially form a monomolecular film. Furthermore, since such a thin film composed of a monomolecular film is arranged with regularity, the film of the amphiphilic lipid is formed thereon by utilizing the affinity of the surface of the film and the amphiphilic lipid. This is convenient for further forming.

  Referring again to FIG. 3, after the working electrode 2 on the substrate 1 is coated with the film 10 of the organic compound containing a sulfur atom, a hydrophobic portion (shown by two broken lines in FIG. 3) is further formed thereon. An amphiphilic lipid having a hydrophilic portion (indicated by a white ellipse in FIG. 3) forms a membrane 11 in which a monolayer is uniformly formed. The procedure will be described later. As can be seen from FIG. 3, the hydrophobic part of the amphipathic lipid binds to the hydrophobic part of the organic compound containing a sulfur atom by hydrophobic interaction to form a two-component membrane (10, 11). The electron carrier 12 is scattered and included in the two-component film. Furthermore, membrane-bound PQQ-GDH binds with high affinity to the hydrophobic portion of the two-component membrane by hydrophobic interaction and is embedded in the membrane. In the embodiment, L-α-phosphatidylcholine and β-oleoyl-γ-palmitoyl are used as the amphipathic lipid, but other amphiphilic lipids may be used. The length of the hydrophobic portion is selected to be long enough to embed the enzyme when the two-component film is formed. Its length depends on the size of the enzyme used and its hydrophobic domain.

  As described above, the amphiphilic lipid used in the present invention is preferably L-α-phosphatidylcholine, β-oleoyl-γ-palmitoyl. Since L-α-phosphatidylcholine and β-oleoyl-γ-palmitoyl have relatively uniform lengths of the two hydrophobic chains, the thickness of the amphiphilic lipid membrane 11 becomes uniform and the OIC becomes more stable. 15 and AIC 14 are obtained. Examples of other amphiphilic lipids suitable for use in the present invention include, for example, L-α-phosphatidic acid, L-α-phosphatidylcholine, L-α-phosphatidylethanolamine, And phospholipids such as dioleoyl, dipalmitoyl, distearoyl, dilauroyl, dimyristoyl, and dilinoleoyl derivatives of L-α-phosphatidyl-DL-glycerol, glycolipids, and bile acids. Such amphiphilic lipids are commercially available from suppliers well known to those skilled in the art.

  Using a two-component membrane as described above, the diffusion of OIC 15 to the biosensor electrode surface can be completely (or almost completely) blocked and the electron mediator following the reaction of PQQ-GDH with glucose. Only a series of reactions, i.e., the reaction between the working electrode 12 and the working electrode 2, can be selectively performed. As a result, a measurement error caused by oxidizing the OIC 15 at the working electrode 2 is eliminated, and a highly accurate measurement can be performed. Further, adsorption of the AIC 14 to the surface of the working electrode 2 can be completely (or almost completely) blocked. In addition, since the affinity of AIC 14 for amphipathic lipid 11 is significantly lower than the affinity of AIC 14 for metal, measurement error due to adsorption of AIC 14 on the surface of working electrode 2 is reduced and high accuracy is achieved. Measurement can be performed.

  Here, the two-component film used in the present invention will be further described.

First, regarding the film 10 of an organic compound containing a sulfur atom, the sulfur atom plays a role in securing adhesion to an electrode in the organic compound. Similarly, the general formula (1), (2), or (3) As apparent from the above organic compound, - (CH ₂₎ has a hydrophobic moiety represented by _n -X. The hydrophobic portion serves to stably maintain the two-component membrane by bonding with the hydrophobic portion of the amphiphilic membrane 11 with high affinity by the hydrophobic interaction as described above. . Therefore, according to such considerations, the compounds that can be used to form the film 10 in the present invention are not limited to the above-described organic compounds containing a sulfur atom, and those that are functionally equivalent thereto are also the same. It can be appreciated that That is, any compound having a functional group for realizing stable adhesion to an electrode and a hydrophobic part capable of realizing high-affinity binding to an amphipathic lipid forms the two-component film of the present invention. Can be used to Further, as described above, such a functionally equivalent compound preferably has a structure capable of forming a monolayer on the electrode surface. Thereby, the thickness of the layer becomes uniform, and a more stable OIC blocking effect can be obtained. However, the film 10 does not necessarily need to be a monolayer. For example, the film 10 has a state in which the functional groups of some of the organic compound molecules are not in contact with the electrode, and the hydrophobic portions of the molecules are inserted into the gaps of the substantial monolayer. It may be a layer. Alternatively, a structure in which the layer constituting the film 10 is wavy as a whole due to the influence of the surface roughness of the substrate and the electrode, or a structure in which a part or all of the layer forms a multimolecular layer is taken. May be possible. Even in the case described above, it can be considered that the effects of the present invention can be obtained. In this specification, the term "first reaction layer" is used to refer to the concept of the film 10 of an organic compound containing a sulfur atom and the film functionally equivalent thereto formed on the electrode. use.

  Further, with respect to the amphiphilic lipid membrane 11 formed on the membrane 10, the hydrophobic portion of the amphiphilic lipid is higher due to hydrophobic interaction with the hydrophobic portion of the compound forming the first reaction layer. It plays a role in binding with affinity. Here, the film 11 does not necessarily have to be a monolayer as shown in FIG. For example, in the membrane 11, the tip of the hydrophobic group of some of the amphipathic lipid molecules is not in contact with the membrane 10, and the hydrophobic portion of the molecule is inserted into the gap of a substantially monolayer. It may be a layer having such a state. Alternatively, due to the surface roughness of the substrate and the electrode, or the structure of the film 10, the structure of the film 11 as a whole is wavy, or a part or all of the layer forms a polymolecular layer. In some cases, it may be possible to adopt such a structure. Even in such a case, it can be considered that the effects of the present invention can be obtained. In the present specification, the term “second reaction layer” refers to a concept that comprehensively refers to the amphiphilic lipid membrane 10 formed on the first reaction layer and a membrane functionally equivalent thereto. Use

  The term “two-component film” is a concept that basically consists of such a first reaction layer and a second reaction layer formed on an electrode. Typically, it provides an environment similar to a lipid bilayer found in cell membranes and the like, as schematically shown in FIGS. Thereby, the membrane-bound enzyme can be embedded in the two-component membrane as seen in a natural cell membrane or the like. However, as described above, since the film 10 and the film 11 do not necessarily have to be monolayers, the two-component film is necessarily a bilayer composed of a combination of two monolayers as shown in FIG. No need. Furthermore, the vicinity of the boundary between the first reaction layer and the second reaction layer has a structure in which the molecule of the organic compound having the functional group and the amphiphilic lipid molecule are mixed and dispersed in a complicated manner. May be. The two-component membrane of the present invention effectively prevents the effect of OIC on the oxidation-reduction reaction of glucose occurring on the electrode surface and the effect of AIC adsorbed on the electrode surface, and furthermore, the oxidation-reduction involving glucose with the electrode. The function of selectively performing only the reaction is realized. In the present invention, the two-component membrane further includes an electron carrier 12 for realizing electron transport between the enzyme and the working electrode 2.

  As the electron carrier 12 used in the present invention, a compound that realizes electron transport from PQQ-GDH to the working electrode 2 is used. As the electron carrier 12 used in the present invention, a compound such as 1-methoxy-5-methylphenadium is inserted near the working electrode because the molecule has high planarity and is inserted into the film. It is preferable for efficient electron transfer. Further, a phenazine derivative, a phenothiazine derivative (such as azul and thionin), a quinone derivative, and the like also have high molecular planarity, and are thus preferable. These compounds are commercially available from suppliers well known to those skilled in the art.

  Further, the electron carrier 12 may be in a form bonded to a polymer backbone (see FIG. 4A) or a form in which part or all of itself forms a polymer chain (see FIG. 4B). . Such electron carriers are commercially available from suppliers well known to those skilled in the art.

  One or more of these electron carriers 12 are used. Further, these are merely examples, and the electron mediator 12 used in the embodiment of the present invention is not limited to the above example.

  Next, a method of forming a two-component film on the electrode will be described.

  As schematically shown in FIG. 2, it is preferable that substantially the entire surface of the working electrode 2 is covered with the organic compound film 10 containing a sulfur atom. The coating of the film 10 of the organic compound containing a sulfur atom is achieved by a method of immersing the surface of the working electrode 2 in a solution of the organic substance or a method of dropping the solution on the surface of the working electrode 2. Alternatively, the same coating can be performed by exposing the surface to the vapor of the organic substance. However, when the working electrode 2 and the counter electrode 3 are arranged on the same substrate as shown in FIGS. 1 and 2, only the working electrode surface, or a part of the counter electrode and the whole or almost the entire surface of the working electrode are formed. In the case of coating, in order to accurately define the area to be coated with the solution of the organic compound, the following is preferable.

  That is, the surface of a resin rod-shaped member having a surface having substantially the same shape as the working electrode 2 and, if necessary, the counter electrode 3 and having a surface capable of forming a desired contact area with the working electrode 2, is provided with the organic compound. A small amount of the above solution, and bringing the surface of this member into contact with the surface of the working electrode 2 and a desired region including the counter electrode 3 as necessary, to thereby obtain a solution of the organic compound applied on the surface of the member. Can be transferred onto the surface of the working electrode 2. In this way, the area on the electrode covered with the organic compound film is accurately defined, and the measurement accuracy of the biosensor can be maintained. Such a so-called transfer method is widely used in the art and is a technique well known to those skilled in the art.

  To further coat the sulfur-containing organic compound film 10 covering the working electrode surface with an amphipathic lipid, a vesicle (liposome) dispersion of the amphipathic lipid has the above-mentioned surface having a sulfur atom. What is necessary is just to immerse the working electrode covered with the film of the organic compound containing.

  In order to embed PQQ-GDH and the electron carrier in the above amphiphilic lipid membrane, it is effective to dissolve PQQ-GDH and the electron carrier in the process of forming each membrane. .

  The sample solution containing the substrate to be measured in the practice of the present invention is preferably a sample solution containing glucose. Examples include blood, plasma, serum, interstitial fluid, biological sample solutions such as saliva and urine, and food and drinking water. Furthermore, it is also possible to use an electrolytic solution used at a normal laboratory level, a liquid used for environmental measurement, and the like. In particular, in the measurement of glucose, a liquid containing OIC or AIC in addition to glucose, such as whole blood, plasma, or urine, is often used as a sample liquid.

  Here, the index in the measurement of the substrate contained in the sample liquid may be an output that changes due to an electrochemical reaction, and for example, an electric current or a charged amount of electricity can be used.

  In the biosensor of the present invention, the working electrode 2 preferably contains gold, platinum or palladium. In this way, the potential applied to the working electrode 2 is stabilized, so that more accurate measurement is realized.

  Further, from the viewpoint that the adsorption of the organic compound containing a sulfur atom is strong, it is preferable that the working electrode 2 contains a noble metal such as gold, palladium, and platinum, and another transition metal such as silver, copper, and cadmium. . Therefore, for the purpose of substantially covering only the working electrode 2 with the organic compound film 10, it is preferable that the counter electrode 3 does not contain these metals.

  Further, it is preferable that at least a part of the surface of the counter electrode 3 is not covered with the film 10 of the organic compound containing a sulfur atom and the film 11 of the amphipathic lipid. In this way, on the surface of the counter electrode 3, the substance that is reduced in the sample liquid is more likely to reach in a part that is not covered with the film than in a part that is covered with the film, The reduction reaction (cathode reaction) at the counter electrode 3 easily proceeds, and more stable measurement can be performed. It is more preferable that the area of the portion of the counter electrode 3 that is not covered with the organic compound film 10 containing a sulfur atom and the amphiphilic lipid film 11 is larger than the area of the working electrode 2. Furthermore, it is preferable that the film 10 of the organic compound containing a sulfur atom and the film 11 of an amphipathic lipid are not substantially present on the counter electrode 3. Furthermore, it is preferable that substantially only the working electrode 2 is coated with the film 10 of the organic compound containing a sulfur atom and the film 11 of an amphipathic lipid. In this way, the reduction reaction at the counter electrode 3 proceeds more smoothly, and more stable measurement can be performed.

  Referring to FIG. 1, a spacer 7 having a slit 6 and a cover 9 having an air hole 8 are adhered to the substrate 1 prepared as described above in a positional relationship as shown by a dashed line in FIG. Thus, the biosensor of the present invention is manufactured. A sample liquid supply path is formed at the slit 6 of the spacer 7. The open end of the slit 6 at the end of the biosensor serves as a sample liquid supply port to the sample liquid supply path.

  When the sample liquid is brought into contact with the open end of the slit 6 serving as the sample liquid supply path of the biosensor having the structure shown in FIG. The reaction proceeds. As described above, when the sample liquid supply path is formed by combining the cover member including the spacer 7 and the cover 9 on the substrate 1 provided with the electrode system, the supply amount of the sample liquid including the substrate to be measured to the biosensor is reduced. Since it can be made constant, the accuracy of measurement can be improved. Further, in a biosensor provided with a sample liquid supply path, a pH buffer may be provided on the cover member side.

  As shown in FIG. 7, an insulating second substrate formed with either the counter electrode 3 or the working electrode 2 together with the corresponding lead 5 or 4 may be used instead of the cover 9. . Also in this case, since the sample liquid supply path is formed by the substrate 1, the spacer 7, and the second substrate, the amount of the sample liquid supplied to the biosensor can be made constant, and the accuracy of measurement can be improved. it can. An example of such a form of biosensor is described in, for example, US Pat. No. 6,458,258.

  Further, the biosensor can be configured only with the substrate 1 without forming the sample liquid supply path as described above. In this case, the reagent system is provided on or near the electrode system. An example of such a biosensor is described in, for example, Patent Document 3 described above.

  Hereinafter, the present invention will be described with reference to Examples, but these Examples are exemplifications of the present invention and do not limit the present invention.

(Preparation of biosensor of the present invention)
(A. Preparation of proteosome suspension)
The following preparation method is an example, and is not limited thereto.

  First, liposomes of L-α-phosphatidylcholine, β-oleoyl-γ-palmitoyl (hereinafter abbreviated as PCOP) (obtained from Wako Pure Chemicals (Wako Pure Chemical Industries, Ltd.)) were prepared as follows. . PCOP was dissolved in trichloromethane in a round bottom flask to a concentration of 10 mM. The solvent was completely evaporated under reduced pressure using a rotary evaporator. Then, the PCOP in the flask was redissolved using 2-propanol so that the PCOP concentration was 40 mM, and 0.5 mL of the obtained solution was added to a 20 mM Tris-HCl buffer solution containing 0.15 M NaCl ( pH = 7.3) was added to 10 mL. This solution was vigorously stirred for 10 minutes to obtain a suspension of liposomes of PCOP.

  Then, in order to incorporate the enzyme and the electron carrier into the liposome, PQQ-GDH (purified from Acinetobacter calcoaceticus as described in Matsushita et. Al. (1989) above) was added to 10 mL of the above liposome suspension of PCOP. 1 mg and 30 mg of 1-methoxy-5-methylphenazinium (hereinafter abbreviated as MMP) as an electron carrier (obtained from Dojindo Laboratories (Dojindo Laboratories Co., Ltd.)) were added, and the solution was added for 10 minutes. Stir vigorously. The liposome suspension of PCOP containing PQQ-GDH and MMP thus obtained is hereinafter referred to as MMP-proteosome suspension.

(B. Preparation of substrate)
On the other hand, in order to fabricate the working electrode and the counter electrode on the substrate, first, a resin electrode pattern mask is placed on the glass electrically insulating substrate 1 and chromium is sputtered thereon to form a chromium layer. Formed. Further, a working electrode 2 and a lead 4 and a counter electrode 3 and a lead 5 were formed thereon by sputtering gold.

(C. Electrode processing)
The working electrode 2 thus formed was coated with n-octylthiol (hereinafter referred to as OT) as follows according to the above-described transfer method. First, a small amount of an OT ethanol solution (concentration: 5 mM) was applied to a flat surface of a rod-shaped resin device having a plane having substantially the same shape as the working electrode 2 on the substrate 1. Then, the surface on which the OT was applied was carefully adjusted so as to overlap almost the entire surface of the working electrode 2 and then superposed. Next, the rod-shaped device was separated from the working electrode 2 so that the OT solution remained on the working electrode 2, and the OT was left until the OT was adsorbed on the surface of the working electrode.

  Thus, a film 10 of an organic compound containing a sulfur atom in the molecule, that is, a film of OT was formed. After one hour, the working electrode 2 was rinsed with ethanol and ultrapure water in this order. The obtained substrate 1 was immersed in an aqueous solution of 10 mM MMP for 1 hour to promote the infiltration of the MMP into the OT film. After sufficiently rinsing the substrate 1 using ultrapure water, the substrate 1 was immersed in the above MMP-proteosome suspension for 8 hours to fuse the OT film 10 on the working electrode 2 with the MMP-proteosome 17 ( See FIG. 3).

  After rinsing the above substrate 1 with ultrapure water and drying it, a spacer 7 and a cover 9 were combined with the substrate 1 to produce a biosensor as shown in FIG.

  As a comparative example, 1 μL of a solution of 1 mg of PQQ-GDH and 30 mg of MMP dissolved in 10 mL of 20 mM Tris-HCl buffer (pH = 7.3) was dropped on the surface of the working electrode 2 on the substrate 1 and dried. A biosensor was fabricated by combining the spacer 1 and the cover 9 with the biosensor 1.

(Effect of AIC on Biosensor of the Present Invention)
Using the blood containing a certain amount of D-glucose (400 mg / dL) as a sample liquid, the biosensor according to the embodiment of the present invention prepared as described above and the opening of the sample liquid supply path of the biosensor of the comparative example, That is, it was supplied to the open end of the slit 8 of the spacer 7. Note that a sample liquid having a red blood cell volume ratio (hematocrit, hereinafter abbreviated as Hct) in blood of 25, 40, or 60% was used. After a lapse of a fixed time (reaction time: 25 seconds), a voltage of 500 mV was applied to the working electrode 2 with respect to the counter electrode 3, and a current value flowing 5 seconds later was measured. As shown in FIG. 5, in the case of the biosensor of the comparative example, a tendency was observed that the current decreased as Hct increased.

  This suggests that the amount of red blood cells adsorbed on the surface of the working electrode tends to increase with an increase in Hct, and that the electrode reaction is inhibited in accordance with the tendency. As a result, even if the glucose concentration is the same, it is considered that the current value changes due to the presence of Hct, and a measurement error has occurred.

  On the other hand, in the biosensor of this example, almost the same current value was obtained regardless of the presence of Hct. Therefore, it is considered that the adsorption of red blood cells to the surface of the working electrode 2 was suppressed by the OT and PCOP films present on the surface of the working electrode 2. The physical properties of the surface of the electrode covered with the organic compound OT and PCOP films are greatly changed from the uncoated gold surface. Alternatively, the electrode interface is positively or negatively charged depending on the terminal group of the coating film.

  It can be considered that the adsorption of blood cells has been suppressed by both or one of these effects. In addition, it was found that the MMP incorporated in the OT and PCOP films had the ability to achieve electron transfer between PQQ-GDH and the working electrode. In this way, by covering with the OT and PCOP films, it was possible to eliminate measurement errors due to AIC adsorption.

(Effect of OIC on Biosensor of the Present Invention)
Further, ascorbic acid, which is one of OICs, was added to blood with an Hct of 40%, and the total concentration was adjusted to 1, 1.5, and 2 mM, including the amount originally contained in the blood. Using these blood, the current value was measured in the same manner as described above. As shown in FIG. 6, in the case of the biosensor of the comparative example, there was a tendency that the current increased as the ascorbic acid concentration increased. This suggests that the oxidation reaction of ascorbic acid is progressing at the working electrode. As a result, even if the glucose concentration was the same, the current value changed depending on the ascorbic acid concentration, and it is considered that a measurement error occurred.

  On the other hand, in the biosensor of this example, almost the same current value was obtained regardless of the ascorbic acid concentration. This is because the surface of the gold electrode of the working electrode 2 is covered with a film of OT and PCOP, which are organic compounds having a long molecular chain, and thus the approach of ascorbic acid to the surface of the gold electrode is suppressed by this film. It is considered that the result was obtained. In this way, by coating with the OT and PCOP films, it was possible to eliminate not only errors due to AIC adsorption but also measurement errors due to electrochemical oxidation of the OIC.

(Use of reference electrode)
In this example, immediately after the sample solution was supplied to the biosensor according to one embodiment of the present invention manufactured by the same procedure as in Example 1, silver / silver chloride was passed through a salt bridge composed of potassium chloride and agar. The electrode was brought into contact with the sample liquid near the sample liquid supply port. The silver / silver chloride electrode has a stable potential and can be used as a reference electrode.

  Various Hct blood containing a certain amount of D-glucose (400 mg / dL) was supplied as a sample liquid to the opening of the sample liquid supply path of the biosensor, that is, the open end of the slit 8 of the spacer 7. After a lapse of 25 seconds, a voltage of 500 mV was applied to the working electrode 2 with respect to the silver / silver chloride electrode, and the current flowing after 5 seconds was measured.

  As a result, almost the same current value was obtained regardless of Hct. The variation in the current value under the same condition was smaller than the result obtained in Example 2. Therefore, it was found that the stability of the measured value was further improved by introducing the reference electrode into the biosensor system.

  The current value was measured in the same manner as above using blood having an Hct of 40% and an ascorbic acid concentration of 1, 1.5, or 2 mM. As a result, almost the same current value was obtained regardless of the ascorbic acid concentration. The variation in the current value under the same conditions was smaller than the result obtained in Example 3. It was found that the introduction of the reference electrode into the biosensor system further improved the stability of the measured value against a change in ascorbic acid concentration.

(Use of n-octyl disulfide)
In this example, a biosensor was produced by the method shown in Example 1 using n-octyl disulfide instead of OT. The response to glucose in blood was measured in the same manner as described in Examples 2 and 3.

  As a result, in this example, almost the same current value was obtained regardless of Hct and ascorbic acid concentration, as in Examples 2 and 3. OT is a compound obtained by cleaving the SS bond of n-octyl disulfide. It is known that thiol and disulfide having such a correspondence form a similar film.

(Working electrode and counter electrode formed on different substrates)
In this embodiment, the counter electrode 3 is formed on the cover 9. The surface of the working electrode 2 was modified as described in Example 1. The response to blood glucose was measured in the same manner as described in Examples 2 and 3.

  As a result, in this example, almost the same current value was obtained regardless of Hct and ascorbic acid concentration, as in Examples 2 and 3. Therefore, it was found that a similar effect can be obtained when electrodes are formed on a plurality of substrates. In this embodiment, the working electrode 2 and the counter electrode 3 exist on different substrates. Therefore, when producing a sensor in which the working electrode 2 is substantially covered with a film without covering the counter electrode 3 with a film, the substrate 1 on which the working electrode 2 is arranged is immersed in an OT solution, It is possible to use a method of exposing the pole 2 surface to OT vapor. As described above, according to the embodiment, compared to a sensor in which the working electrode and the counter electrode are provided on the same surface, a sensor in which substantially only the working electrode is coated with the film can be easily manufactured. it can.

(Use of platinum or palladium)
In this example, the working electrode 2 and the counter electrode 3 were manufactured using platinum or palladium. An electrode pattern mask made of a resin was placed on an electrically insulating substrate 1 made of glass to form a chromium layer, and each electrode was formed by sputtering platinum or palladium. Further, the surface of the working electrode 2 was coated in the same manner as described in Example 1. The response to blood glucose was measured in the same manner as described in Examples 2 and 3.

  As a result, when platinum was used, the current value showed a slight dependence on Hct, but showed almost the same current value even when the ascorbic acid concentration was changed. On the other hand, in the biosensor fabricated using platinum instead of gold in the same manner as in the comparative example described in Example 1, the current was larger and depended on the Hct and ascorbic acid concentrations.

  From this, it was found that even when platinum was used as the material for the working electrode, the effect of suppressing the effects of OIC and AIC by coating with the film of the present invention was obtained. Further, when palladium was used as the material for the working electrode, the same degree of independence of Hct and ascorbic acid concentration was observed as compared with the results obtained with gold. It was found to be a suitable electrode material.

(Effect of pH buffer)
The biosensor characteristics when a pH buffer was further included in the biosensor system were evaluated. The biosensor prepared in the present example has a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate as a pH buffer as a reagent system and is supported in the amphiphilic lipid membrane 11 containing PQQ-GDH and an electron carrier. Other than that, it is the same as that used in Example 1.

  Blood containing a certain amount of D-glucose (400 mg / dL) and having different Hct was supplied to the space of the biosensor as a sample solution. After a certain period of time, a voltage of 500 mV was applied to the working electrode 2 with respect to the counter electrode 3, and the value of the current flowing at that time was measured. As a result, the obtained current value did not depend on Hct.

  As compared with the results obtained in Examples 2 and 3, the dependence of the current value on the ascorbic acid concentration was almost unchanged, but the Hct dependence was further reduced. That is, a constant current value more independent of Hct was obtained for the same glucose concentration.

  The reason why such a result is obtained can be considered as follows. By incorporating a pH buffer in the biosensor system, the pH of the sample solution in the biosensor is stabilized. Therefore, it is considered that the charge state of the terminal group of the membrane present on the electrode was stabilized, and the effect of preventing adsorption of AIC in blood became constant for each sample liquid.

  Further, it is considered that the enzyme activity was also stabilized by such a stable pH, and the amount of the reduced form of the electron carrier generated after a certain period of time became constant for each sample solution. It is considered that the dependency of the current value on Hct was reduced by the stabilizing effect of these two or both by the pH stabilization. In the present example, a particularly stabilizing effect was observed when the pH of the measurement solution was 4 to 9. Therefore, the preferred pH range is pH 4-9. From the viewpoint of the most stable enzyme activity, a more preferable pH range is in the range of pH 5 to 8.

  Although the current values were measured in Examples 2 to 8, the same tendency as the above-described current values was also observed when the charge amount was measured instead of the current value.

  Further, in the above embodiment, the applied voltage to the electrode system was set to 500 mV, but the applied voltage is not limited to this value. Any voltage may be used as long as the electron carrier is oxidized at the working electrode.

  In the above embodiment, the reaction time was set to 25 seconds or 55 seconds, and the voltage application time and the current detection time were set to 5 seconds after the reaction time. However, the present invention is not limited to this. I just need.

  Further, if necessary, it is preferable to immobilize one or more of the reagent system or the reagents contained in the reagent system on the working electrode to insolubilize or elute the enzyme and the electron carrier. In this case, use a method that utilizes interaction with the membrane due to van der Waals force, a covalent bonding method, a cross-linking fixing method, or an immobilization method using coordination bond or specific binding interaction. Can be. In particular, in practicing the present invention, a method of covalently immobilizing the reagent on a film of an organic compound containing a sulfur atom on an electrode is also preferable.

  Although membrane-bound PQQ-GDH has been described as a preferred enzyme in the practice of the present invention, this is not intended to mean that the enzyme to be used in the present invention is necessarily limited to only membrane-bound PQQ-GDH. Absent. Even enzymes other than membrane-bound PQQ-GDH have a hydrophobic domain on at least a part of the molecular surface, and bind with high affinity to the hydrophobic portion of the two-component membrane as described above. Any enzyme can be used as long as it is an enzyme obtained (or an enzyme capable of realizing a form embedded in a two-component membrane) and catalyzes a selective oxidation reaction of glucose. Of course, the combination of components that make up a suitable two-component membrane can vary depending on such enzymes.

  By including an enzyme other than PQQ-GDH in the reagent system, it is also possible to measure a substrate other than glucose. For example, sucrose can be quantified by adding invertase having a function of decomposing sucrose into glucose and fructose to a reagent system.

  In the above embodiments, the sputtering method using a mask was used as a method for forming the electrodes and their patterns, but the method is not limited thereto, and for example, any of sputtering method, ion plating method, evaporation method, and chemical vapor deposition method A pattern may be produced by combining the metal film produced in the above with photolithography and etching. Pattern formation can also be performed by metal trimming with a laser. Screen printing may be performed on the substrate using a metal paste to form an electrode pattern. Further, the patterned metal foil may be directly adhered to the insulating substrate.

  The shape, arrangement, number, and the like of these electrode systems are not limited to those described in the above embodiments. For example, the working electrode and the counter electrode may be formed on different insulating substrates (see FIG. 7), or a plurality of the working electrode and the counter electrode may be formed. Further, the shapes, arrangements, numbers, and the like of the leads and terminals are not limited to those described in the above embodiment.

  For the purpose of improving the measurement accuracy, the amount of the solution containing the substrate to be measured can be easily regulated to a constant amount. Therefore, it is preferable that a spacer is included as a component of the biobiosensor. However, when the biosensor of the present invention is used in combination with a device capable of collecting a fixed volume of sample solution, a cover member including a spacer and a cover is not necessarily required. The material used for these spacers, covers, or substrates is not limited to the glass used as the material for the substrates in the examples, but also includes, for example, inorganic materials such as silicon and its oxides, polyethylene terephthalate (PET), and polypropylene. Electrically insulating materials such as resins such as (PP) can be used in the practice of the biosensor of the present invention as well.

  As described above, the biosensor of the present invention is not affected by AIC and OIC contained in a sample solution, and has a simple structure and the like that can quickly and accurately measure a substrate in a sample solution. Are suitable. In particular, the biosensor of the present invention is suitable as a disposable type biosensor for detecting the glucose concentration in a sample solution.

FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, excluding a reagent system. 1 is a longitudinal sectional view of a biosensor according to an embodiment of the present invention. Schematic diagram showing a part of a biosensor production scheme according to an embodiment of the present invention and an outline of the principle of the present invention. The figure which shows the example of the electron carrier used in the biosensor of this invention: (A) The form which the electron carrier (indicated by A in the figure) was couple | bonded with the polymer backbone; (B) The electron carrier (all) mutually Form in which polymer chains are bonded to each other FIG. 4 is a diagram showing Hct dependence of a current obtained in one embodiment of the present invention. The figure which shows the ascorbic acid concentration dependence of the electric current obtained in one Embodiment of this invention. FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, excluding a reagent system.

Explanation of reference numerals

DESCRIPTION OF SYMBOLS 1 Substrate 2 Working electrode 3 Counter electrode 4, 5 Lead 6 Slit 7 Spacer 8 Air hole 9 Cover 10 Film of organic compound containing sulfur atom 11 Film of amphiphilic lipid containing PQQ-GDH and electron carrier as reagent system 12 Electron Transmitter 13 PQQ-GDH
14 AIC
15 OIC
16 Glucose 17 Proteosome

Claims (24)

An electrode system including at least a pair of electrodes,
At least one insulating substrate for supporting the electrode system;
A first reaction layer provided on at least the working electrode of the electrode system, a first reaction layer containing a functional group capable of binding or adsorbing to the electrode and an organic compound containing a hydrophobic hydrocarbon group,
A second reaction layer provided on the first reaction layer, a second reaction layer containing an amphipathic lipid capable of binding or adsorbing to the hydrophobic portion of the first reaction layer,
A reagent system supported on a two-component membrane constituted by the first and second reaction layers, the reagent system comprising at least a membrane-bound pyrroloquinoline quinone-dependent glucose dehydrogenase and an electron carrier. , Biosensors.   The biosensor according to claim 1, wherein the first reaction layer and the second reaction layer each form a monolayer. The biosensor according to claim 1, wherein the organic compound is a compound represented by the following general formula (1), (2), or (3):
_{HS- (CH 2) n -X (} 1)
_{X- (CH 2) n -S-} S- (CH 2) n -X (2)
_{S- (CH 2) n -X (} 3)
(However, in the general formulas (1), (2), and (3), n is an integer of 1 to 20, and X is a methyl group, a benzyl group, an aminobenzyl group, a carboxybenzyl group, or a phenyl group).   The biosensor according to claim 1, wherein the amphiphilic lipid is L-α-phosphatidylcholine, β-oleoyl-γ-palmitoyl.   The biosensor according to claim 1, wherein the electron carrier is 1-methoxy-5-methylphenazinium.   The biosensor according to claim 1, wherein the working electrode contains gold, platinum, or palladium.   The biosensor according to claim 1, wherein the counter electrode of the electrode system does not contain any of gold, platinum, and palladium.   The biosensor according to claim 1, wherein the entire surface of the working electrode of the electrode system and a part of the surface of the counter electrode are covered with the two-component film.   The biosensor according to claim 8, wherein an area of a portion of the counter electrode that is not covered with the two-component film is larger than an area of the working electrode.   The biosensor according to claim 1, wherein only the surface of the working electrode of the electrode system is covered with the two-component film.   2. The bio-engine according to claim 1, wherein each of the pair of electrodes is supported on an opposing surface of two insulating substrates disposed to face each other so as to form the supply path for the sample liquid therebetween. 3. Sensors.   The biosensor according to claim 1, wherein the reagent system further comprises a pH buffer. A method for producing a biosensor, comprising:
Forming an electrode system including at least a pair of electrodes on an insulating substrate,
A step of providing a first reaction layer on at least the working electrode of the electrode system, wherein a solution containing a functional group capable of binding or adsorbing to the electrode and an organic compound containing a hydrophobic hydrocarbon group is brought into contact with the working electrode. Including the steps,
Providing a second reaction layer on the first reaction layer, comprising contacting a solution containing an amphipathic lipid with the first reaction layer,
Here, at least one of the solution containing the organic compound and the solution containing the amphipathic lipid contains an electron carrier, and the solution containing the amphipathic lipid further contains a membrane-bound pyrroloquinoline quinone-dependent solution. A method comprising glucose dehydrogenase.   14. The method of claim 13, wherein the first reaction layer and the second reaction layer each form a monolayer. The method according to claim 13, wherein the organic compound is a compound represented by the following general formula (1), (2), or (3):
_{HS- (CH 2) n -X (} 1)
_{X- (CH 2) n -S-} S- (CH 2) n -X (2)
_{S- (CH 2) n -X (} 3)
(However, in the general formulas (1), (2), and (3), n is an integer of 1 to 20, and X is a methyl group, a benzyl group, an aminobenzyl group, a carboxybenzyl group, or a phenyl group).   14. The method of claim 13, wherein the amphiphilic lipid is L- [alpha] -phosphatidylcholine, [beta] -oleoyl- [gamma] -palmitoyl.   14. The method according to claim 13, wherein said electron carrier is 1-methoxy-5-methylphenazinium.   14. The method according to claim 13, wherein the working electrode contains gold, platinum, or palladium.   14. The method of claim 13, wherein the counter electrode of the electrode system does not contain any of gold, platinum, or palladium.   14. The method of claim 13, wherein the entire surface of the working electrode and a portion of the surface of the counter electrode of the electrode system are coated with the first and second reaction layers.   21. The method according to claim 20, wherein an area of a portion of the counter electrode that is not covered with the first and second reaction layers is larger than an area of the working electrode.   14. The method according to claim 13, wherein only the surface of the working electrode of the electrode system is coated with the first and second reaction layers.   14. The method according to claim 13, wherein each of the pair of electrodes is supported on opposing surfaces of two insulative substrates disposed to face each other so as to form the supply path for the sample liquid therebetween. .   14. The method of claim 13, wherein at least one of the solution comprising the organic compound and the solution comprising the amphipathic lipid further comprises a pH buffer.

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