JP2004163386A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the following problems: a biosensor utilizing an electrolytic polymerization film is more subject to the influence of interference components; stability in long-term use is low; and hence measurement precision is decreased and the lifetime of the sensor becomes short. <P>SOLUTION: In the biosensor having a hydrogen peroxide electrode and a hydrogen peroxide selection permeable film and an enzyme film that are laminated onto the hydrogen peroxide electrode, the hydrogen peroxide selection permeable film is a polymerization film made of an electrolytic polymerization polymer and a nonelectrolytically polymerized polymer, thus reinforcing the film, easily improving adhesion force with the enzyme film, and hence achieving high stability in the sensor. Additionally, the high stability in the sensor is achieved without being affected by the interference components, even if the interference components coexist with high concentration, thus achieving high measurement precision and a long sensor use lifetime. <P>COPYRIGHT: (C)2004,JPO

Description

表１から、膜の付着力または膜強度は電解液中のナフィオン添加量と相関しており、ナフィオン濃度が0.5％付近に膜がもっとも剥離しにくいことが分かる。図１９に示すセンサの性能データと合わせて考慮すると、ナフィオン濃度範囲は0.2〜1％の範囲にあることがもっとも好ましい。
以上膜１の電解重合条件検討について非電解重合高分子がＢＳＡとナフィオンの場合を例として説明したが、他の高分子についても類義したな方法で検討してよい。
【００７８】
なお、膜１の組成や構造はＩＲ、ＸＰＳ、ＳＥＭなど、周知の分析方法で分析可能である。
【００７９】
電解重合後、膜１を含む基体１０を洗浄し次の工程に移行する。ポリピロールやポリアニリンなど形成された直後の状態では導電性を有し、測定時高いベース電流を生じる膜に対しては、さらに酸化処理を行なう。酸化処理せずセンサとして試料を測定すると、高いベース電流により、特に初期では測定できないか精度が悪くなる。一般的な処理方法として、電極活成分を含まない電解液（例えば、塩化カリウムを含むリン酸緩衝液）中において、作用極に一定の電位を印加した定電位酸化で行なう。酸化処理はベース電流が実際の試料測定に際してベース電流が測定精度に影響を与えないレベルになるまで行なうことが望ましい。具体的な酸化時間は膜厚や電極の状態および印加電位によって異なるが、２００−１２００ｍＣ／ｃｍ２のポリピロールの場合、０．６−０．７Ｖ（Ａｇ／ＡｇＣｌ）の印加電位では約１ないし６時間が妥当な処理時間である。
なお、酸化処理工程は、膜１を形成した直後ではなく、その後工程の後（例えば酵素膜３の形成後）で行なっても良い。
【００８０】
４）酵素膜３の形成：酵素膜３の成膜法は選定される材料および予定構造などを勘案して周知の膜形成法から選定して適宜決定すれば良いが、好ましい成膜方法の例として、酵素膜の材料を一定濃度に調整した溶液（原液）を、膜１が形成された作用極表面を含めた領域に層状に展開し、その後溶媒を蒸発させ、酵素膜３を形成する方法が挙げられる。
原液の調整に用いられる溶媒として水が好ましいが、材料の性質等を勘案して有機溶媒を含ませても良い。また、原液の濃度は膜２の材料、溶液粘度、目標膜厚、塗布方法など、種々の条件を勘案して決定すれば良く、特に限定されないが、膜厚を調整する重要ファクターのひとつとして位置づけるとよい。
【００８１】
原液を基体１０の表面に層状に展開して酵素膜３を成膜する方法の好ましい例として、基体１０が静止した状態での滴下塗布、流延塗布、及びスピンコーターなどの成膜装置による回転塗布、膜原液と接触させた後で基体１０を引き上げる方法等が挙げられる。回転塗布法や引き上げ法は薄い酵素膜を有するセンサを大量に製造するのに適しているが、生産量が少ない場合、またはセンサ寿命を確保するために一定量以上の酵素量を作用極の表面に担持させる必要がある場合には、一般的に高価である酵素を節約するために、作用極を中心とする表面に酵素原液をドロップして乾燥させる方法が好ましい。この方法によれば、酵素膜３が作用極及びその周辺領域のみ一定の厚さをもって被覆するので、酵素のロスが無く、酵素使用量を最小限に押さえ安価に製造することができる。被覆される周辺領域の範囲は、原液の使用量および／またはドロップされた直後の液滴操作により調整される。
【００８２】
以上、酵素膜３まで成膜したバイオセンサの製造方法について説明したが、更に図４に示す様に酵素膜３の表面に膜５を必要とする場合、後工程で続けて膜５を成膜すれば良い。
【図面の簡単な説明】
【図１】本発明によるバイオセンサの基本構造図である。
【図２】本発明による一実施例のバイオセンサの構造図である。
【図３】本発明による他例のバイオセンサの構造図である。
【図４】本発明による他例のバイオセンサの構造図である。
【図５】本発明による他例のバイオセンサの構造図である。
【図６】本発明による他例のバイオセンサの構造図である。
【図７】本発明による他例のバイオセンサの構造図である。
【図８】膜１の構造を例示する図である。
【図９】過酸化水素電極をセラミック基板上に作成した基体を示す図である。
【図１０】膜厚とセンサの感度および選択比との関係を示す図である。
【図１１】本発明による一実施例のバイオセンサの製造工程を示す図である。
【図１２】本発明による他例のバイオセンサの製造工程を示す図である。
【図１３】本発明による他例のバイオセンサの製造工程を示す図である。
【図１４】本発明による他例のバイオセンサの製造工程を示す図である。
【図１５】本発明による他例のバイオセンサの製造工程を示す図である。
【図１６】ピロールの電解重合の反応スキームを示す図である。
【図１７】ピロールの定電流電解重合における電位−時間曲線を示す図である。
【図１８】ピロールの定電流電解重合における電位−時間曲線を示す別の図である。
【図１９】電解液中におけるナフィオンの濃度と膜の選択比との関係を示す図である。
【図２０】バイオセンサを評価するフローインジェクション評価装置の概略図である。
【図２１】従来のバイオセンサの構造図である。
【図２２】従来の別のバイオセンサの構造図である。
【符号の説明】
１…膜１
２…作用極
３…酵素膜
４…リンカー層
５…膜５
６…支持体
７…参照極
８…対極
１０…基体
１２…電解重合ポリマー
１４…高分子
２０…ポリピロール−ＧＯＤ複合膜
２２…ポリピロール
２４…ＧＯＤ
２６…ポリピロール膜
２８…ＧＯＤ膜[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a biosensor for detecting and measuring a specific component from a medium in which many components are mixed, and more particularly to a current detection type electrochemical biosensor.
[0002]
[Prior art]
An enzyme electrode combining a hydrogen peroxide electrode and an oxidoreductase is one of the most frequently used types of biosensors.
A hydrogen peroxide electrode, which is a type of current measuring transducer, responds to a reducing substance such as uric acid or ascorbic acid (hereinafter referred to as an interfering component) in addition to hydrogen peroxide to generate a current. When a sample in which such interfering components coexist is to be measured, it is necessary to suppress the influence. The most frequently used technique is to provide a permselective membrane on or near the electrode surface that selectively or preferentially transmits hydrogen peroxide by eliminating coexisting interference components.
[0003]
Electropolymerized membranes such as a polypyrrole membrane can form a membrane directly on the electrode from the monomer, and have a size exclusion selective permeability to low molecular substances by adjusting the membrane formation conditions (for example, Therefore, biosensors using these electropolymerized films have been proposed.
[0004]
For example, a sensor in which a polypyrrole-enzyme composite film is formed on an electrode (working electrode) using a solution containing pyrrole and an enzyme as an electrolytic solution has been studied (for example, see Non-Patent Document 2). FIG. 21 conceptually shows the structure of a glucose sensor using glucose oxidase (GOD) as an example. Glucose contained in the sample in contact with the surface of the composite film 20 made of polypyrrole 22-GOD24 diffuses into the composite film 20 and is converted into hydrogen peroxide by the GOD24. The generated hydrogen peroxide passes through the composite membrane 20 and reaches the surface of the electrode 1. However, interfering components having a molecular weight larger than that of hydrogen peroxide, for example, ascorbic acid are mostly removed from the electrode surface by the size exclusion effect of the composite membrane 20. Is prevented from reaching. As a result, the sensor has specificity for the target component (glucose). Further, a sensor using a pyrrole derivative having a reactive substituent instead of pyrrole has been studied (for example, see Non-Patent Document 3). In this case, the enzyme is immobilized on the membrane through a covalent bond or the like.
[0005]
Further, there has been proposed a sensor having a double membrane structure in which an enzyme film is provided thereon after a polypyrrole film is formed (for example, see Non-Patent Document 4). FIG. 22 shows the structure. That is, the polypyrrole film 26 containing no enzyme and the GOD film 28 are sequentially laminated on the surface of the working electrode 2. In brief, the manufacturing method is as follows. First, a polypyrrole film 26 is formed on a surface of a platinum electrode (disk having a diameter of 4 mm) cleaned by nitric acid washing or the like by a constant potential (0.7 V vs. Ag / AgCl) electrolytic method. Form. Subsequently, the formed film 26 is peroxidized at the same potential, and after drying, a solution containing an enzyme (GOD) and bovine serum albumin (BSA) is applied on the film 26 and dried to form a GOD film 28. .
[0006]
[Non-Patent Document 1] J. Electroanal. Chem, 273 (1989) 231-242
[Non-Patent Document 2] Fresenius J. Anal. Chem., 342 (1992) 729-733
[Non-Patent Document 3] Anal. Sci., 15 (1989) 1175-1176
[Non-Patent Document 4] Biosensors & Bioelectronics, 13 (1998) 103-112
[0007]
[Problems to be solved by the invention]
Such a conventional technique has the following problems. In the sensor having the structure shown in FIG. 21, the sensitivity of the sensor is low, and the sensor is susceptible to interference components. The reason is that, for example, glucose, which is a component to be measured, has a molecular size similar to that of interfering components such as ascorbic acid and uric acid, so that the reaction does not penetrate into the membrane, and the reaction concentrates on the surface of the membrane. The amount is small and the sensitivity is low. In order to maintain a certain level of sensitivity, it is necessary to make the film thinner, but since the permeability to the interference component increases, the film is more susceptible to the interference component. This type of sensor is not suitable for measuring a sample in which coexisting components such as urine are present at a high concentration. On the other hand, in the sensor having a double membrane structure shown in FIG. 22, since the enzyme membrane is permeable to the substrate and a sufficient reaction amount can be ensured, both sensitivity and selectivity can be achieved, and the stability over time can be improved. Can be improved. However, in this type of sensor, there is a problem that the selectivity deteriorates with time due to the weak adhesive force of the film 26 and the weak film strength.
[0008]
In view of such a current situation, an object of the present invention is to provide a high-sensitivity biosensor that is hardly affected by interfering components and does not deteriorate selectivity even when used for a long period of time.
[0009]
[Means for Solving the Problems and Their Functions and Effects]
According to a first aspect of the present invention, there is provided a biosensor comprising a hydrogen peroxide electrode and a hydrogen peroxide selective permeable membrane and an enzyme membrane laminated thereon. The hydrogen peroxide permselective membrane is characterized by being a composite membrane composed of an electropolymerized polymer and a non-electropolymerized polymer. Since the adhesive force between them can be easily improved, the stability of the sensor is high.
The biosensor according to the present invention according to claim 2, wherein the biosensor comprises a hydrogen peroxide electrode and a hydrogen peroxide selective permeable membrane and an enzyme membrane laminated thereon, wherein the hydrogen peroxide selective permeable membrane is: It is characterized by being formed so as to cover the working electrode surface of the electrode by electrolytic polymerization from an electrolytic polymerizable compound which is a monomer forming an electrolytic polymer and a non-electrolytic polymer solution. The selectively permeable membrane of hydrogen peroxide thus formed is strengthened by blending another polymer in the electropolymerized polymer, and the adhesion between the membrane and the enzyme membrane can be easily improved. Therefore, the stability of the sensor is high.
[0010]
According to a third aspect, in the biosensor according to the first or second aspect, the polymer is an anionic polymer. Generally, many electropolymerized polymers having selective permeability to hydrogen peroxide are uncharged or positively charged. The anionic polymer is strongly bonded to such an electropolymerized polymer, whereby the membrane is effectively strengthened. Furthermore, in addition to the size exclusion for the anionic interference component, the effect of charge exclusion is realized.
[0011]
A biosensor according to a fourth aspect is the biosensor according to the third aspect, wherein the anionic polymer is albumin. Albumin, a type of protein, has the effect of increasing the affinity for the enzyme formed on the hydrogen peroxide permselective membrane.
[0012]
The biosensor according to claim 5 is the biosensor according to claim 4, wherein the concentration of albumin is in the range of 0.2 to 1%. This provides a sensor having a high-quality film in which albumin is efficiently incorporated into the film without hindering the uniformity of the film.
[0013]
According to a sixth aspect of the present invention, in the biosensor of the third aspect, a sulfonic acid resin having a perfluorocarbon in a skeleton of an anionic polymer is used. Sulfonic acid resin with perfluorocarbon in the skeleton hardly swells in aqueous solution.Since the sulfonic acid group is a strong electrolyte, the amount of charge is large and constant without being affected by the environmental pH. You.
[0014]
The biosensor according to claim 7, wherein the biosensor according to claim 6, wherein the hydrogen peroxide selective permeable membrane is formed from an electrolytic solution containing a sulfonic acid resin having perfluorocarbon in the skeleton and an electropolymerizable compound. In the case of the above, the concentration of the sulfonic acid resin having perfluorocarbon in the skeleton was in the range of 0.1-3%. This concentration range is suitable for efficiently incorporating a sulfonic acid resin having perfluorocarbon in the skeleton into the membrane during electrolytic polymerization.If the concentration is low, the amount of incorporation is insufficient, and if the concentration is high, the electropolymerizable compound is dispersed. This makes it difficult to form an electrolytic polymerized film.
[0015]
The biosensor according to claim 8 is the biosensor according to claim 7, wherein the concentration of Nafion is in the range of 0.2 to 1%. A film formed in this range is more preferable because its strength is most enhanced and hydrogen peroxide selective permeability is good.
[0016]
9. The biosensor according to claim 9, wherein the hydrogen peroxide electrode is formed on an insulating support, wherein the enzyme membrane is used. Is formed so as to cover at least the working electrode and its peripheral region. By bringing the enzyme membrane into contact with the surface of the support with a constant area, the adhesion between the membrane and the substrate (the hydrogen peroxide electrode and the support are referred to as the substrate) is further improved, and a highly stable sensor is provided. Is done.
[0017]
According to a tenth aspect, in the biosensor according to the ninth aspect, before or after forming the hydrogen peroxide selective permeable membrane, at least the surface including the peripheral region of the working electrode is subjected to silanization treatment with a silane coupling agent. I decided. This provides a sensor in which the silane coupling agent acts as a linker to further improve the adhesive force between at least the enzyme film and the substrate.
[0018]
An eleventh aspect is the biosensor according to the first to tenth aspects, wherein the electropolymerized polymer is polypyrrole. Polypyrrole is conductive at least at the time of formation, and when a hydrogen peroxide selective permeable membrane is formed in situ by electrolytic polymerization, a film having a sufficient rejection force can be easily formed. .
[0019]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail with reference to the drawings and the like.
[0020]
The biosensor according to the present invention has a basic film structure shown in FIG. FIG. 1 is a sectional view showing a film structure of a working electrode portion of a hydrogen peroxide electrode. A film 1 made of a non-electrolytic polymerized polymer and an electrolytic polymerized polymer is formed on the surface of a working electrode 2, and an enzyme film 3 is formed thereon. Here, the non-electrolytic polymer is a natural polymer or a polymer synthesized by a method other than electrolytic polymerization, and mainly refers to a polymer having a molecular weight of 1,000 or more. Hereinafter, the non-electrolytic polymer in the present invention is simply referred to as a polymer.
[0021]
Hereinafter, a planar sensor in which a hydrogen peroxide electrode is formed on an insulating support will be described in more detail as an example.
[0022]
In the biosensor of the first embodiment shown in FIG. 2, a membrane 1 and an enzyme membrane 3 are sequentially laminated on a surface of a support 6 including a working electrode 2 of a hydrogen peroxide electrode. The membrane 1 covers only the surface of the working electrode, and the enzyme membrane 3 is formed so as to cover a larger area than the membrane 1, completely cover and hide the membrane 1, and further cover the peripheral area. Here, the peripheral region of the working electrode refers to a range within a certain distance from the outer periphery of the working electrode 2. The lower limit of the size is not particularly limited as long as the necessary adhesive force is realized between the enzyme membrane and the surface of the support, but as a general preferable example, the distance from the outer periphery of the working electrode 2 is 1 to 5 mm. Range. The upper limit of the size of the peripheral region is not particularly limited, but is appropriately determined in consideration of the situation such as the ease of manufacturing the sensor and the cost as long as the lower limit is satisfied.
[0023]
The support 6 is made of an insulating substrate such as glass or ceramic. As the hydrogen peroxide electrode, an electrode formed on the surface of the support 6 by a method such as screen printing or vapor deposition is exemplified. FIG. 9 shows an example of a hydrogen peroxide electrode composed of a working electrode 2, a reference electrode 7, and a counter electrode 8 formed by screen printing on a ceramic substrate. Each electrode has a lead wire so that it can be connected to an electric circuit such as a potentiostat. Well-known electrode materials, for example, platinum, gold, and carbon are used as the material of the working electrode and the counter electrode. Hereinafter, the support 6 and the hydrogen peroxide electrode formed thereon are collectively referred to as a base 10. The base 10 also refers to the support 6 and a part (for example, a working electrode) of a hydrogen peroxide electrode.
[0024]
The membrane 1 covers only the working electrode surface (including the side surface when the working electrode protrudes from the peripheral support as shown in FIG. 2).
[0025]
The role of the polymer in the membrane 1 is to mix well with the electropolymerized polymer and to strengthen the membrane 1 as a permselective membrane, the adhesive force between the membrane 1 and the enzyme membrane, or between the membrane 1 and the working electrode. For example, in the case of an anionic polymer, by giving a negative charge to the membrane 1, it is possible to raise the exclusion performance particularly for anionic interfering components such as ascorbic acid and uric acid.
[0026]
These roles will first be described in detail regarding the strengthening of the film by the polymer. Generally, an electropolymerized polymer is a single-chain molecule without branching, and often does not have a side chain. Also, there is often no or only one type of reactive or polar functional group inside the molecule. Since a film made of these molecules has a weak intermolecular force due to a hydrogen bond, an ionic bond, or the like, the strength is often poor. In the case of polymers with strong hydrophobicity such as polystyrene, intermolecular force due to hydrophobic interaction acts, and in some cases the film is strong. Has a weak membrane strength preserving action due to hydrophobic interaction (since the sensor according to the present invention is often used in contact with an aqueous solution, hydrophilicity of the membrane often leads to weakness of the membrane). By mixing different kinds of polymers into these electropolymerized polymers, the intermolecular interaction can be strengthened. This strengthens the film. For example, when polar hydrogen such as polypyrrole or polyaniline is contained, an in-film hydrogen bond can be formed by adding a polymer containing oxygen. As another example, when the electropolymerized polymer is charged, an electrostatic intermolecular force such as an ionic bond can be formed by mixing an oppositely charged polymer. Furthermore, in the case of an electropolymerized polymer having a hydrophilic property, the hydrophobicity is improved by entering a polymer having a strong hydrophobicity into the membrane, so that the membrane is strengthened.
[0027]
Next, the improvement of the adhesion of the film will be described. As described above, in general, an electropolymerized polymer does not have a reactive functional group or a polar functional group, and thus often has a weak adhesive force between a working electrode and an enzyme membrane. Therefore, for example, by putting a polymer having a reactive functional group into the membrane 1, it is possible to easily improve the adhesive force between the membrane 1 and the enzyme membrane (which is generally rich in reactive functional groups). it can. For the same reason, if a functional group (reactive by silanization treatment or the like) is present on the surface of the working electrode, the adhesive force between the film 1 and the working electrode is also improved. Adhesion can also be improved by selecting a polymer so that the membrane 1 has a charge opposite to that of the enzyme membrane or strengthens it.
[0028]
It is considered that the permselectivity of hydrogen peroxide of a membrane made of an electropolymerized polymer having no charge or a positive charge such as polypyrrole or polyaniline is due to size exclusion for coexisting components. On the other hand, uric acid and ascorbic acid, which are typical coexisting interference components, are negatively charged. Therefore, if the membrane has a similar negative charge, an elimination effect due to charge repulsion occurs. Therefore, in particular, when the membrane 1 contains an anionic polymer, both the mechanism of size exclusion and the mechanism of charge repulsion work, thereby further enhancing the exclusion effect on interfering components.
[0029]
Next, a polymer material will be described. The material and type of the polymer may be appropriately determined in consideration of various conditions such as the type and properties of the electropolymerized polymer and the role that the polymer should play. When the membrane 1 is formed directly on the surface of the working electrode 2 by electrolytic polymerization from an electrolyte solution containing a polymer and an electropolymerizable compound, it is generally soluble in an electrolyte solution in which the electropolymerizable compound is dissolved. Desirably. In this case, since the polymer is taken into the film during the process of film growth by electrolytic polymerization, it is generally preferable that the electrolyte solution has a net negative charge. The reason is that the electropolymerization of the electropolymerizable compound has many oxidation reactions. That is, since the polymerization is carried out by applying a positive potential to the working electrode, the negatively charged polymer of the net is efficiently taken into the film by the electric attraction action.
[0030]
From this viewpoint, the polymer is more preferably an anionic polymer. Here, an anionic polymer is defined as having a certain negative charge in a neutral pH environment. In the case of a polymer having an amphoteric ion such as a protein or an amphoteric electrolyte, an isoelectric point can be used as an index indicating the degree of the negative charge. Generally, those having an isoelectric point of 6 or less are desirable. Since the anionic polymer has a negative charge even in a neutral pH environment, one of the advantages is that the electrolyte solution does not need to be adjusted to an extremely low pH. Examples of the anionic polymer include an ion exchange resin such as a sulfonic acid resin having perfluorocarbon in the skeleton, a polyamino acid such as polyglutamic acid, a nucleic acid such as DNA, a polysaccharide such as carboxymethylcellulose, and an acidic protein such as albumin. Among these anionic polymers, a sulfonic acid resin having albumin and perfluorocarbon is more preferable. The reason is that albumin, which is a protein, has the effect of increasing the affinity for an enzyme film formed on the hydrogen peroxide film. On the other hand, a membrane containing a sulfonic acid resin having perfluorocarbon in the skeleton is unlikely to swell in an aqueous solution, and since the sulfonic acid group is a strong electrolyte, the charge amount is large and constant without being affected by the environmental pH. The membrane is reinforced efficiently. Examples of albumin include bovine serum albumin (BSA) and human serum albumin. Examples of the sulfonic acid resin having perfluorocarbon in the skeleton are those commercially available under the trade name of Nafion.
[0031]
The size (molecular weight) of the polymer is not particularly limited, but it is generally preferable that the molecular weight is in the range of 1,000 to 100,000. If the molecular weight is small, the polymer may be detached from the membrane 1 during the use period. On the other hand, if it is large, it may be difficult to take in the film due to diffusion resistance or the like, or the mixing with the electropolymerized polymer may be poor, so that the strength of the film may be reduced.
[0032]
The electropolymerized polymer, which is the other material constituting the membrane 1, is the main material responsible for the size exclusion performance as a hydrogen peroxide selective permeable membrane. Preferred examples of the electropolymerizable compound for synthesizing the electropolymerized polymer include pyrrole, aniline, phenylenediamine, phenol, toluidine, and styrene. Among them, pyrrole, which does not increase the resistance even when a film is formed and can easily synthesize a film having a certain thickness, is more preferable. Another feature of polypyrrole is that it has a certain degree of hydrophobicity and good stability as a film in an aqueous solution.
[0033]
The ratio between the polymer and the electropolymerized polymer in the membrane 1 may be appropriately determined in consideration of the type of the material and the required sensor performance (sensitivity, interference component elimination performance, etc.). Is small, and that of the electropolymerized polymer is large. As described above, the size exclusion performance of the membrane 1 as a permselective membrane is mainly based on the electropolymerized polymer. Therefore, when the amount is reduced, the lower limit of the excluded molecular size increases, and the hydrogen peroxide selection The performance as a permeable membrane may be reduced. On the other hand, in order to realize the above role of the polymer, a certain amount is necessary, but if it is contained more than necessary, there is a risk of causing a decrease in the selectivity of the membrane, and in the case of a hydrophilic polymer, in particular, The strength and stability of the membrane may be reduced due to the significant improvement in the hydrophilicity of the membrane. As an example of a general preferable ratio, the mass ratio of the polymer is in the range of 5 to 30%, and the mass ratio of the electropolymerized polymer is in the range of 95 to 70%. In general, the membrane 1 is formed by electrolytic polymerization directly on the surface of the working electrode 2 from an electrolytic solution containing a simple substance of an electropolymerized polymer and a polymer. It can be adjusted by the concentration of the polymer in the inside.
[0034]
A desirable example of the structure of the membrane 1 is that the polymer is uniformly dispersed in the electropolymerized polymer. When the polymer is in the form of particles (for example, a spherical protein such as albumin), an example shown in FIG. FIG. 8 is an enlarged cross section of the film 1. The electropolymerized polymer 12 is a substantially continuous film, and the polymer 14 having a spherical structure is dispersed so as to be embedded in the electropolymerized polymer 12. In the case of a conjugated polymer such as polypyrrole, it is positive in the oxidized state (the hydrogen peroxide electrode is operated by applying a positive potential to the working electrode, so it is always placed in an oxidizing environment when used). They are charged and generally have anionic dopants bound in the film. Therefore, in the case of the polypyrrole film alone, the carrier liquid in contact with the sensor or an anionic component in the sample is incorporated, but generally only low-molecular-weight components are introduced, so that the dopant changes from the film due to a change in oxidation state. Separation and rebonding can cause variations in membrane performance. In this sense, an anionic polymer is more desirable. In the membrane 1 according to the present invention which contains an anionic polymer in advance, the anionic polymer is strongly adsorbed to the cationic electropolymerized membrane and exists as a dopant which cannot move in a sense, and thus serves to strengthen the membrane.
[0035]
The thickness of the film 1 may be appropriately determined in consideration of the material and composition of the film, required sensor performance (sensitivity, interference component elimination performance, and the like), and the like. I do. When the output for a unit concentration of a component to be measured is defined as sensitivity and used as a ratio (selection ratio) of the sensitivity to the interference component and the component to be measured as an index of the interference component elimination performance, the film thickness, the sensitivity, and the selection are generally obtained. The relationship shown in FIG. That is, as the film thickness increases, both the sensitivity and the selectivity decrease, but the degree of decrease in the selectivity gradually decreases, and when the thickness exceeds a certain thickness, the sensitivity hardly decreases. Decreases continuously. In practice, the upper limit of the selectivity to be achieved is set based on the amount (concentration) of the coexisting interference component expected from the sample to be measured, and the film thickness is determined so that the selectivity is equal to or less than the upper limit. .
In cases where it is difficult or inaccurate to directly evaluate the film thickness due to irregularities on the working electrode surface, sensor performance such as selectivity is directly used as a parameter to determine the film production conditions as an example of the film thickness. You may. In the case where the film 1 is formed directly on the surface of the working electrode 2 by electrolytic polymerization from a solution containing an electrolytic polymerizable compound and a polymer, the amount of electrolysis per unit electrode area (for example, the unit is mC / cm ² ) May be used as a parameter for the film thickness. When expressed in terms of the amount of electrolysis, the conversion coefficient to the film thickness varies depending on the film density, the size of the monomer (molecular weight), the number of electrons per unit generated during polymerization, and the content of the anionic polymer. When the types of films are formed under certain conditions, a substantially linear relationship holds between the amount of electrolysis and the film thickness. A preferable range of the general film thickness is, for example, 0.1 to 10 μm.
[0036]
Further, since the film density and the structure vary depending on the film formation conditions, it goes without saying that not only the film thickness but also the film formation conditions are factors to be considered in determining the film 1.
[0037]
Next, the enzyme film 3 will be described. The enzyme membrane 3 is formed so as to cover at least the membrane 1 on the working electrode and the support 6 in the peripheral area thereof.
[0038]
The enzyme membrane 3 preferably contains a hydrogen peroxide producing enzyme and a polymer for immobilizing or stabilizing the enzyme as main components. If necessary, a bifunctional crosslinking agent such as glutaraldehyde is included. In the case of a glucose sensor using GOD, proteins such as albumin and any carbohydrate of chitosan are preferable examples of the polymer. Particularly, albumin is more preferable because it has a function of stabilizing the enzyme. In this case, glutaraldehyde may be added as a crosslinking agent. The cross-linking agent connects the enzyme and albumin in the enzyme membrane with a covalent bond and suffices only by insolubilizing. For example, a cross-linking agent forms a covalent bond between the membrane 1 containing an anionic protein and the enzyme membrane. Also plays the role of bringing close contact. The specific composition and thickness of the enzyme film may be appropriately determined depending on the enzyme used, the required performance for the sensor, the type of the silane coupling agent used for the treatment of the support, and the like.
[0039]
In the biosensor of the second embodiment shown in FIG. 3, the basic structures of the substrate 10, the membrane 1, and the enzyme membrane 3 are the same as those of the above embodiment, but before the membrane 1 is formed, at least the working electrode 2 and The surface of the support in the peripheral region is silanized with a silane coupling agent, and the enzyme film 3 is adhered to the support by a linker layer 4 made of a silane coupling agent. Here, the peripheral region of the working electrode refers to a range within a certain distance from the outer periphery of the working electrode. The lower limit of the size is the coverage of the enzyme film 3, and the upper limit is not particularly limited.
[0040]
The linker layer 4 made of a silane coupling agent is formed on the surface 6 of the support in the working electrode 2 and its peripheral region. Although FIG. 3 shows a continuous layer to exemplify its existence, the layer is not necessarily a continuous layer as long as it serves as a linker connecting the surface of the support 6 and the enzyme membrane 3. There is no.
[0041]
The type of the silane coupling agent may be appropriately selected in consideration of the situation, such as the material of the support 6 and the enzyme membrane 3, but as general preferable examples, an amino group, a carboxyl group, an epoxy group, an alkene group, a halogen group, Those containing a reactive functional group such as a vinyl group are desirable, and most preferred are silane coupling agents containing an amino group and an epoxy group.
[0042]
Specific examples of silane coupling agents include allyltrichlorosilane, allyltriethoxysilane, allyltrimethylsilane, 3- (2-aminoethylaminopropyl) trimethoxysilane, 3-aminopropyltrimethoxysilane, and 3-aminopropyltrimethoxysilane. Aminopropyltriethoxysilane, chloromethyldimethylchlorosilane, chloromethyltrimethylsilane, 3-chloropropyltrimethoxysilane, dimethoxymethylchlorosilane, dimethylaminotrimethylsilane, methylchlorosilane, ethoxydimethylvinylsilane, ethyldichlorosilane, 3-glycidoxypropyl Trimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, hydroxymethyltrimethylsilane, 3-methacryloxypropyltrimethoxysilane, 3- Data heterocyclyloxy dimethoxy silane, methyl vinyl dichlorosilane, trichlorovinylsilane, triethoxy vinyl silane, trimethoxy vinyl silane, and material selected from the group consisting of trimethyl vinyl silane. Among them, 3-aminopropyltriethoxysilane and 3-glycidoxypropyltrimethoxysilane are most preferred as inexpensive silane agents for efficiently introducing an amino group or an epoxy group onto the substrate surface, respectively.
[0043]
In this embodiment, since the silane coupling agent is also bonded to the surface of the working electrode 2, the presence of the linker layer 4 made of the silane coupling agent on the surface does not hinder the formation of the film 1. It is necessary to confirm. The amount of silane coupling abundance is supported because the number of functional groups such as hydroxyl groups required for the silane coupling agent to bind is smaller than the surface of a support such as platinum or a metal made of glass or ceramic. Less than body surface. However, the silane coupling agent present on the surface of the working electrode has the function of increasing the adhesion between the film 1 and the electrode.
[0044]
In Example 3 shown in FIG. 4, the linker layer 4 exists on the surface of the support 6 in the peripheral region excluding the surface of the working electrode 2. In this embodiment, since the silane coupling agent is not bonded to the surface of the working electrode 2, it is not necessary to consider the influence on the film 1 when selecting the silane coupling agent as in the first embodiment. .
[0045]
In the biosensor of the fourth embodiment shown in FIG. 5, since the silanization treatment is performed after the film 1 is formed, a linker layer made of a silane coupling agent is formed on the surface of the support around the working electrode 2 and the surface of the film 1. There are four. In general, since the film 1 is in close contact with the surface of the working electrode and the film 1 has a size exclusion function, the surface of the working electrode 1 has no or no silane coupling agent. The amount of the silane coupling agent bonded is smaller than that of No. 1. On the other hand, particularly when the molecular weight of the silane coupling agent is small, the silane coupling agent may bond not only to the surface of the film 1 but also to the surface of molecules inside the film 1. In this case, the silane coupling agent is distributed not only on the surface of the linker layer as shown in FIG. In such a case, the silane coupling agent acts as a linker for attaching the enzyme membrane 3 and the membrane 1, and at the same time, crosslinks the molecular chains of the membrane 1 to improve the strength and the membrane density. effective. The amount of the silane coupling agent varies depending on the type of the electropolymerized film.
[0046]
Therefore, in the present embodiment, when examining the material selection and formation method of the silane coupling agent, not only the influence on the performance of the membrane 1 but also the performance of the membrane 1 is taken into consideration.
[0047]
As another example of the biosensor according to the present invention, a sensor in which the enzyme film 3 covers only the surface of the film 1 can be considered. FIG. 5 shows an example. That is, this is an example in which the enzyme film does not exist on the surface of the support 6. In addition, the enzyme film 3 may cover the side surface of the film 1 by the forming method.
[0048]
Further, as a further embodiment of the biosensor according to the present invention, there is a biosensor in which a membrane 5 is further provided on the enzyme membrane 3. FIG. 7 shows an example. The biosensor of the present embodiment shown in FIG. 7 is obtained by further providing a membrane 5 on the enzyme membrane 3 of the biosensor of Embodiment 1 shown in FIG. Although not shown, the biosensor shown in FIGS. 3 to 6 may be further provided with a membrane 5 on the enzyme membrane 3.
[0049]
The role of the membrane 5 formed on the enzyme membrane 3 is to protect the enzyme membrane 3 from an external sample, or to limit the arrival of the component to be measured to the enzyme membrane 3 to support the measurement of a sample with a higher concentration. Doing so is an example. As an example of the former, a sensor that analyzes components in blood can be considered. Since blood contains a large number of macromolecules such as proteins that degrade the sensor by adhering to the surface of the enzyme film 3, the film 5 is provided to prevent these components from reaching the enzyme film 3. Measurement accuracy and stability can be improved. As an example of the latter, when measuring urinary components or online monitoring of a manufacturing process in the industrial field, a test sample is used without dilution or at a low dilution factor (for example, 5 to 10 times) to support a wide range of concentration measurement. Sensors.
[0050]
Next, a method for manufacturing a biosensor according to the present invention will be described in detail based on the above-described embodiment.
[0051]
First, the base 10 is prepared. Although the shape of the base 10 is not limited, a hydrogen peroxide electrode system including a working electrode is preferably formed on a flat insulating substrate. As the insulating substrate, a glass plate, a silicon wafer, a ceramic plate, or the like is used.
[0052]
The hydrogen peroxide electrode system on the insulating substrate only needs to include a working electrode.However, from the viewpoint of manufacturing costs of the sensor and simplification of the measurement system using the sensor, the hydrogen peroxide electrode is formed on the substrate by patterning. As an example, as shown in FIG. 9, a three-electrode base is formed on a ceramic substrate by a screen printing technique.
[0053]
Next, if necessary, the substrate 10 is pretreated for the purpose of cleaning and activating the surface of the substrate 10. Water or an acid can be used for washing, but washing with an acid is particularly preferable because the surface of the base 10 can be activated at the same time as removing stains. Preferred examples of the acid species include strong acids such as nitric acid, sulfuric acid, and hydrochloric acid, and weak acids such as phosphoric acid, formic acid, citric acid, and acetic acid. The substrate 10 after the acid cleaning needs to be cleaned with water.
[0054]
The substrate 10 after the cleaning is dried as necessary. The drying conditions are not particularly limited, but preferable drying temperature is 20 to 80 ° C., and drying time is 5 to 120 minutes. In the case where the next step is a step of performing a surface treatment using an aqueous solution, drying can be omitted.
Subsequent manufacturing steps will vary slightly depending on the structure of the sensor being manufactured. In the case of the sensor having the structure of the embodiment shown in FIGS. 2, 6, and 7, there is no silanization step. On the other hand, the sensor having the structure of the embodiment shown in FIGS. 3 to 5 includes a silanization treatment step. In the case of the sensor having the structure of the second embodiment shown in FIG. 3, the film 1 is formed after the base 10 is silanized. In the sensor having the structure of the third embodiment shown in FIG. 4, either the treatment with the silane coupling agent or the formation of the film 1 may be performed first. On the other hand, in the case of the sensor having the structure according to the third embodiment shown in FIG.
[0055]
Here, the manufacturing process of each sensor will be described first based on a flowchart, and then each process will be described in detail.
[0056]
The sensor having the structure of the first embodiment (FIG. 2) is manufactured according to the flow shown in FIG. That is, after the pretreatment, the film 1 and the enzyme film 3 are formed in order. Steps surrounded by dotted lines indicate steps that may be omitted (the same applies hereinafter).
[0057]
The sensor having the structure of the second embodiment (FIG. 3) is manufactured according to the flow shown in FIG. That is, after the pretreatment, the surface of the base including the working electrode 2 is silanized. Subsequently, the film 1 and the enzyme film 3 are sequentially formed.
[0058]
The sensor having the structure of the third embodiment (FIG. 4) is manufactured according to the flow shown in FIG. 13 or FIG. In the step shown in FIG. 13, after the pretreatment, the surface of the working electrode is masked if necessary, and then the silanization treatment is performed. Subsequently, the mask is removed to form the film 1. Finally, an enzyme film 3 is formed. On the other hand, the flow shown in FIG. 14 forms the film 1 after the pretreatment. Subsequently, silanization treatment is performed after masking the surface of the film 1, and the mask is removed after the silanization treatment. Finally, an enzyme film 3 is formed. In FIG. 13, as a case where the mask on the working electrode surface and the subsequent removal treatment may be omitted, there is a case where the silane coupling agent does not adhere or does not substantially adhere even when the silation treatment is performed on the surface of the working electrode. An example is given. In this case, the manufacturing process of the sensor is the same as the manufacturing process of the sensor having the structure of the second embodiment.
[0059]
The sensor having the structure of the fourth embodiment (FIG. 5) is manufactured according to the flow shown in FIG. That is, after the pretreatment, the film 1 is formed, the substrate is silanized, and finally the enzyme film 3 is formed. The manufacture of the sensor of the embodiment shown in FIG. 6 follows the steps shown in FIG. Further, the manufacture of the sensor of the embodiment shown in FIG. 7 is similarly performed according to the process shown in FIG. That is, after the pretreatment, the film 1 is formed, then the enzyme film 3 is formed, and then the film 5 is formed.
[0060]
Next, each step in the above manufacturing flow will be described in detail. Although the order and the number of steps are different depending on the sensor structure or the manufacturing method, the steps with the same name have basically the same processing method regardless of the flow.
[0061]
1) Mask and mask removal on the surface of working electrode 1 or membrane 1: This treatment covered the surface of working electrode 1 or membrane 1 and isolated these surfaces from the silane coupling agent in the silanization treatment and was masked. The purpose is to prevent the silane coupling agent from binding or adsorbing to the part. As long as the above object is achieved, there is no particular limitation on the specific method. Examples of general preferable methods include a method generally used in a semiconductor manufacturing process using a photoresist or the like, and a method of attaching a film or a packing. In the case of a substrate having a plurality of sensors, a patterned mask method may be used. When masking the surface of the film 1, it is needless to say that it is necessary to consider the effect of the masking process on the film 1.
[0062]
2) Silanization treatment: First, a silane coupling agent is selected in consideration of the type of the support, the conditions of the enzyme membrane 3, the membrane 1, etc., and the selected orchid coupling agent is dissolved in an appropriate solvent to have a certain concentration. To prepare a silanization solution. Subsequently, the surface of the substrate 10 (the state of the surface of the working electrode before the silane coupling treatment differs depending on the structure or manufacturing method of the sensor, but is referred to as the substrate 10 for convenience here) is brought into contact with a silane coupling agent solution. Examples of the method of bringing the surface into contact with the silane coupling agent solution include a method of dipping the substrate 10 in the silane coupling agent solution, and a method of applying the silanization solution to the surface with a film forming apparatus such as a spin coater. Next, the base 10 is washed as necessary. Washing removes excess silane coupling agent that is not chemically bound or strongly adsorbed. Note that the cleaning step may be omitted if the effect on the sensor performance is within the allowable range without cleaning.
[0063]
3) Formation of the membrane 1: The formation of the membrane 1 is roughly classified into a method of attaching a previously formed film to the electrode surface and a method of forming a film directly on the electrode surface from a film material dissolved in a liquid or the like. Preferably, it is a method in which a film material is formed directly on the electrode surface. Examples of the latter method include a method of forming a film on the electrode surface from a mixture of a polymer and an electropolymerized polymer, and an electropolymerization including an electropolymerizable compound that is a monomer that forms the electropolymerized polymer and a polymer. There is a method of forming the film 1 on the surface of the working electrode in situ. In the method formed from the solution on the surface of the working electrode 2 by in-situ electrolytic polymerization, the adhesion between the film and the electrode is strong, no film is formed on the surface other than the working electrode surface, and the state of the film as synthesized It is most preferable because it has advantages such as easy control of the structure. . Hereinafter, this method will be described in detail. First, an electrolytic solution containing a polymer and an electropolymerizable compound is prepared. When the polymer and the electropolymerizable compound are water-soluble, it is desirable to use water as a solvent for adjusting the electrolytic solution. In this case, examples of the supporting electrolyte include inorganic salts such as potassium chloride and sodium chloride. Further, the pH is adjusted depending on the charge state of the polymer. On the other hand, when the polymer and the electropolymerizable compound are hardly soluble in water, an organic solvent is used. In this case, not only the polymer and the electropolymerizable compound but also the supporting electrolyte needs to be soluble in the organic solvent. Of course, a mixed solvent of an organic solvent and water may be used. The electrolytic polymerizable compound may be oxidized by oxygen in the air, and if it may affect the performance of the membrane 1, the electrolyte is kept in an inert atmosphere such as nitrogen or argon.
[0064]
Next, the substrate is set in an appropriate electrolytic system so that at least the working electrode and the counter electrode come into contact with the electrolytic solution. The electrolytic system includes a counter electrode and a reference electrode in addition to the working electrode on the substrate. As the counter electrode and the reference electrode, those on a substrate or those separately provided in an electrolytic system are used. When using a counter electrode and a reference electrode separately provided in the electrolytic system, reduce the distance between the working electrode, the counter electrode, and the reference electrode in order to suppress the effect of the ohmic resistance of the electrolyte. It is desirable to keep it constant.
[0065]
As a specific electrolysis method, a constant potential electrolysis method in which a constant potential is applied between a working electrode and a reference electrode, a constant current electrolysis method in which a constant current flows between a counter electrode and a working electrode, or an applied potential Alternatively, the current is changed (scanned) in a constant pattern, for example, a cyclic voltammetry method, or the like. If the insulation of the film is strong, such as polyphenol or polyphenylenediamine, and the resistance increases rapidly with its growth, a method that scans the potential or current, such as cyclic voltammetry, forms a film that is thicker than the constant potential or constant current method. can do. On the other hand, in the case where the resistance does not increase so much even if a film is formed, such as polypyrrole, it is desirable to form the film by a constant potential or constant current method from the viewpoint of simplicity of the method. In either method, the film thickness is controlled by the amount of electrolysis. The amount of electrolysis is determined by time integration of the current curve. In the case of constant current electrolysis, the amount of electrolysis can be determined by the electrolysis time, so that the process management is simpler than other methods. Specifically, it is desirable to determine which method is adopted by comparing film formation by each method or the like.
[0066]
Hereinafter, in the case where the electropolymerizable compound is pyrrole, an experimental study of electrolysis conditions (concentration of a polymer contained in an electrolytic solution) will be described using a constant current electrolysis method as an example.
[0067]
The formation of polypyrrole by electrolytic polymerization is performed according to the scheme shown in FIG. Reaction formula 1) is an electrolysis initiation reaction, and reaction formula 2) is a dimerization reaction. A dimer is synthesized by the deprotonation reaction of the reaction formula 3). In the reaction formula 4), the dimer is further oxidized. Reaction equations 5) and 6) represent further electrolytic and polymerization reactions.
[0068]
One of the features of the polypyrrole electropolymerization reaction is that oxidation of a dimer is easier than oxidation of a simple substance, and oxidation of a highly polymerized polymer is easier than that of a dimer. Since the formed polypyrrole has conductivity, the electrical resistance does not increase so much even when the film is grown. For example, when polymerization is performed at a constant current using an electrolytic solution containing no anionic polymer, the electrolytic potential follows a potential-time curve A shown in FIG. Since the electrolysis speed is controlled to be constant, the electrolysis potential is initially high and then decreases. Stabilizes after a certain period of time.
[0069]
On the other hand, when an anionic polymer is contained in the electrolytic solution, the electrolytic solution follows a time-dependent curve with different electrolytic potential. Curves B and C shown in FIG. 17 are potential-time curves when the anionic polymer is BSA. Curve B is a potential-time curve when the concentration is low (for example, 0.1%), and curve C is a potential-time curve when the BSA concentration is high (for example, 1%). The other electrolysis conditions in this experiment are as follows: The base was a ceramic support shown in FIG. 9 on which a tripolar hydrogen peroxide electrode was formed, and no silanization treatment was performed. Working electrode area is about 3mm ² Met. The counter electrode and the reference electrode at the time of the electrolytic polymerization used those on the support. The substrate was mounted on a flow cell, and an electrolytic solution (0.4 M pyrrole, 50 mM potassium chloride, and a predetermined concentration of BSA dissolved in a 10 mM phosphate buffer at pH 6.8) maintained in a nitrogen atmosphere. ) Was circulated to the flow cell at a constant speed. Room temperature was about 25 ° C. and air conditioned.
[0070]
From FIG. 17, the presence of BSA in the electrolytic solution affects the electropolymerization of pyrrole, and the higher the concentration, the more the potential during constant current electrolysis is shifted upward. Particularly when the concentration is high, the electrolytic potential increases with time. Although not shown, in the case of constant potential electrolysis, the electrolysis current initially increases, but thereafter decreases over time. That is, when BSA is present in the electrolytic solution, the resulting electropolymerized film has an increased electric resistance, and the degree of the increase has a positive correlation with the BSA concentration. Considering that polypyrrole is conductive and BSA is nonconductive, the increase in resistance means the incorporation of BSA into the film during the film growth process, and the amount of incorporation is the concentration of BSA. It can be seen that it is directly proportional to Therefore, the BSA content in the membrane 1 can be controlled by the amount of BSA added in the electrolyte. Of course, in addition to the amount of BSA added, factors such as electrolysis conditions, pyrrole concentration, and supporting electrolyte concentration also affect the membrane composition.
[0071]
Although not shown, when the BSA concentration is high (for example, 2%), the potential increases rapidly with the electrolysis time, and it can be visually confirmed that a uniform film is not formed even after the electrolysis is performed for several minutes. This means that the presence of a large amount of BSA inhibits the diffusion and transfer of hypopyrrole to the electrode surface or the polymerization of pyrrole. On the other hand, when the concentration was 0.1% or less, the amount of BSA incorporated into the membrane was insufficient. From the above results, the concentration of BSA is preferably in the range of 0.1 to 1%.
[0072]
FIG. 18 is a potential-time curve when the anionic polymer is Nafion. The film forming conditions in this example are basically the same as those in the case of the BSA, but before forming the film 1, the surface of the substrate was treated with 3-aminopropyltriethoxysilane. The potential-time curve without Nafion is E. The electric potential of the curve E is higher than that of the curve A of FIG. 17 because the silanizing treatment causes the silanizing agent to adhere to the electrode surface and increases the ohmic resistance.
[0073]
F and G are potential-time curves when the Nafion concentration is 1% and 2%, and the potential is lower than that without Nafion. Thus, unlike BSA, the presence of Nafion in the electrolyte plays a role in promoting the electropolymerization reaction. However, as can be seen from the fact that the potential is higher at 2% than at 1%, it is presumed that when the concentration is increased, a side surface similar to BSA that prevents film formation appears. In addition, even when the Nafion concentration was 2%, an apparently uniform film was formed, but at 3%, nonuniformity of the film could be visually confirmed.
Therefore, the concentration of Nafion in the electrolyte is preferably 3% or less, more preferably 2% or less.
[0074]
FIG. 19 shows the performance (selectivity) of a glucose sensor in which a membrane 1 was formed from electrolyte solutions having different Nafion concentrations, and an enzyme membrane 3 containing glucose oxidase was formed by the dropping method described later. The flow injection analysis shown in FIG. The results are evaluated with the device and the results are shown. Selectivity is defined as the ratio of power at the same concentration to ascorbic acid and glucose, typical co-interfering components:
Selectivity (%) = output for ascorbic acid / output for glucose at the same concentration * 100
[0075]
When the performance of the enzyme membrane is constant, the selectivity can be used as an index for comparing the performance of the hydrogen peroxide selectively permeable membrane. The lower the selectivity, the better the performance of the hydrogen peroxide selectively permeable membrane.
[0076]
From FIG. 19, when the Nafion concentration is 1% or less, the selectivity is almost constant irrespective of the Nafion concentration, but when the Nafion concentration is 1% or more, the selectivity tends to increase. The data in FIG. 19 is an average value of the measured values from four sensors.
In order to further evaluate the sensor, a test was conducted in which the sensor was taken out of the cell, dried, wetted again with deionized water, and dried again, and the state of peeling of the film was observed. Table 1 shows the results. The numerical values in the table are the number of sensors where the film has peeled off such as floating or rupture. In general, when the film is dried, a force for separating the film from the substrate is exerted due to a drying stress or the like. Therefore, the stronger the adhesive force or the strength of the film, the more difficult it is for the film to be separated. The value is 4 when all of the sensors in the four sheets have peeled, and 1 when the film has not peeled. Therefore, the smaller the numerical value, the higher the substrate adhesion or the film strength of the film.
[0077]
table 1. The state of peeling of the sensor film due to drying (number of peels in 4 sheets)

From Table 1, it can be seen that the adhesive force or the film strength of the film is correlated with the amount of Nafion added in the electrolytic solution, and the film is most difficult to peel off when the Nafion concentration is around 0.5%. Considering together with the performance data of the sensor shown in FIG. 19, the Nafion concentration range is most preferably in the range of 0.2 to 1%.
The electrolytic polymerization conditions of the membrane 1 have been described above by way of an example in which the non-electrolytic polymer is BSA and Nafion. However, other polymers may be studied in a similar manner.
[0078]
The composition and structure of the film 1 can be analyzed by a well-known analysis method such as IR, XPS, and SEM.
[0079]
After the electrolytic polymerization, the base 10 including the film 1 is washed, and the process proceeds to the next step. Immediately after the formation of polypyrrole or polyaniline, the film having conductivity and generating a high base current at the time of measurement is further subjected to an oxidation treatment. When a sample is measured as a sensor without oxidation treatment, the measurement cannot be performed or the accuracy is deteriorated particularly at the initial stage due to a high base current. As a general processing method, in an electrolytic solution containing no electrode active component (for example, a phosphate buffer solution containing potassium chloride), a constant potential oxidation is performed by applying a constant potential to the working electrode. It is desirable to perform the oxidation treatment until the base current reaches a level at which the base current does not affect the measurement accuracy in actual sample measurement. The specific oxidation time varies depending on the film thickness, the state of the electrode, and the applied potential, but in the case of polypyrrole of 200 to 1200 mC / cm2, about 1 to 6 hours at an applied potential of 0.6 to 0.7 V (Ag / AgCl). Is a reasonable processing time.
The oxidation treatment step may be performed not immediately after the formation of the film 1 but after the subsequent step (for example, after the formation of the enzyme film 3).
[0080]
4) Formation of the enzyme film 3: The method of forming the enzyme film 3 may be appropriately determined by selecting from well-known film formation methods in consideration of the selected material, the planned structure, and the like. A method in which a solution (stock solution) in which the material of the enzyme film is adjusted to a constant concentration is spread in a layered manner on the region including the surface of the working electrode on which the film 1 is formed, and then the solvent is evaporated to form the enzyme film 3 Is mentioned.
Water is preferred as the solvent used for preparing the stock solution, but an organic solvent may be included in consideration of the properties of the material and the like. The concentration of the undiluted solution may be determined in consideration of various conditions such as the material of the film 2, the solution viscosity, the target film thickness, the coating method, etc., and is not particularly limited. Good.
[0081]
As preferable examples of the method of forming the enzyme film 3 by spreading the undiluted solution on the surface of the substrate 10 in a layered form, drop coating, casting coating, and rotation by a film forming apparatus such as a spin coater while the substrate 10 is stationary. A method of pulling up the substrate 10 after coating and contacting with a stock solution of the film may be used. The spin-coating method and the pull-up method are suitable for mass production of sensors with thin enzyme films.However, when the production volume is small, or when a certain amount of enzyme is When it is necessary to carry the enzyme, a method of dropping and drying the enzyme stock solution on the surface mainly of the working electrode is preferable in order to save the generally expensive enzyme. According to this method, since the enzyme film 3 covers only the working electrode and its peripheral region with a certain thickness, there is no loss of the enzyme, the amount of the enzyme used can be minimized, and the production can be performed at low cost. The extent of the peripheral area to be covered is adjusted by the amount of stock solution used and / or the drop operation immediately after dropping.
[0082]
As described above, the method for manufacturing the biosensor having the enzyme film 3 formed thereon has been described. However, if the film 5 is required on the surface of the enzyme film 3 as shown in FIG. Just do it.
[Brief description of the drawings]
FIG. 1 is a basic structural diagram of a biosensor according to the present invention.
FIG. 2 is a structural view of a biosensor according to an embodiment of the present invention.
FIG. 3 is a structural diagram of another example of a biosensor according to the present invention.
FIG. 4 is a structural diagram of another example of a biosensor according to the present invention.
FIG. 5 is a structural diagram of another example of a biosensor according to the present invention.
FIG. 6 is a structural diagram of another example of a biosensor according to the present invention.
FIG. 7 is a structural diagram of another example of a biosensor according to the present invention.
FIG. 8 is a diagram illustrating the structure of a film 1;
FIG. 9 is a view showing a substrate in which a hydrogen peroxide electrode is formed on a ceramic substrate.
FIG. 10 is a diagram showing a relationship between a film thickness, a sensor sensitivity, and a selection ratio.
FIG. 11 is a view showing a manufacturing process of the biosensor of one embodiment according to the present invention.
FIG. 12 is a diagram showing a manufacturing process of another example of a biosensor according to the present invention.
FIG. 13 is a diagram showing a manufacturing process of another example of a biosensor according to the present invention.
FIG. 14 is a view showing a process of manufacturing another example of a biosensor according to the present invention.
FIG. 15 is a view showing a manufacturing process of another example of a biosensor according to the present invention.
FIG. 16 is a diagram showing a reaction scheme of electrolytic polymerization of pyrrole.
FIG. 17 is a view showing a potential-time curve in galvanostatic polymerization of pyrrole.
FIG. 18 is another diagram showing a potential-time curve in galvanostatic polymerization of pyrrole.
FIG. 19 is a graph showing the relationship between the concentration of Nafion in an electrolytic solution and the selectivity of a membrane.
FIG. 20 is a schematic diagram of a flow injection evaluation device for evaluating a biosensor.
FIG. 21 is a structural diagram of a conventional biosensor.
FIG. 22 is a structural diagram of another conventional biosensor.
[Explanation of symbols]
1 ... Membrane 1
2 ... working electrode
3. Enzyme membrane
4: Linker layer
5 ... Membrane 5
6 ... Support
7 ... Reference electrode
8 ... Counter electrode
10 ... Base
12. Electropolymerized polymer
14… Polymer
20: Polypyrrole-GOD composite membrane
22 Polypyrrole
24… GOD
26: Polypyrrole film
28 ... GOD film

Claims (11)

In a biosensor comprising a hydrogen peroxide electrode and a hydrogen peroxide selective permeable membrane and an enzyme membrane laminated thereon, the hydrogen peroxide selective permeable membrane is a composite membrane comprising an electropolymerized polymer and a non-electrolyzed polymer. A biosensor characterized in that: In a biosensor comprising a hydrogen peroxide electrode and a hydrogen peroxide selective permeable membrane and an enzyme membrane laminated thereon, the hydrogen peroxide selective permeable membrane is an electropolymerizable compound which is a monomer forming an electropolymerized polymer. And a solution containing a non-electrolytic polymer and a non-electrolytic polymer. The biosensor according to claim 1, wherein the non-electrolytic polymer is an anionic polymer. The biosensor according to claim 3, wherein the anionic polymer is albumin. The biosensor according to claim 4, wherein the concentration of albumin in the solution containing the electropolymerizable compound and albumin is in the range of 0.1 to 1%. 4. The biosensor according to claim 3, wherein the anionic polymer is a sulfonic acid resin having perfluorocarbon in a skeleton. The concentration of the sulfonic acid resin having perfluorocarbon in the skeleton in the solution containing the electropolymerizable compound and the sulfonic acid resin having perfluorocarbon in the skeleton is in a range of 0.1-3%. The biosensor as described. The concentration of the sulfonic acid resin having perfluorocarbon in the skeleton in the solution containing the electropolymerizable compound and the sulfonic acid resin having perfluorocarbon in the skeleton is in the range of 0.2-1%. 8. The biosensor according to 7. 9. The method according to claim 1, wherein the hydrogen peroxide electrode is formed on an insulating support, and the enzyme film is formed so as to cover at least the working electrode and its peripheral region. The biosensor according to any one of the above. 10. A silanization treatment using a silane coupling agent is performed on at least a surface including a peripheral region of a working electrode before or after forming the hydrogen peroxide selective permeable membrane (membrane 1). The biosensor as described. The biosensor according to any one of claims 1 to 10, wherein the electropolymerized polymer is polypyrrole or a derivative thereof.

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