JPH10267888A - Biosensor - Google Patents
BiosensorInfo
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- JPH10267888A JPH10267888A JP9107997A JP10799797A JPH10267888A JP H10267888 A JPH10267888 A JP H10267888A JP 9107997 A JP9107997 A JP 9107997A JP 10799797 A JP10799797 A JP 10799797A JP H10267888 A JPH10267888 A JP H10267888A
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- Prior art keywords
- electrode
- enzyme
- approximately
- gold
- antibody
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、所定条件下にて、
液体、特に血液、尿等の体液や測定試料中に含まれる特
定酵素、例えばグルタミン酸−オキザロ酢酸転移酵素
（ＧＯＴ）、および特定基質、例えばコレステロールエ
ステルなどの濃度に対応した電気化学的な応答を示すバ
イオセンサおよび測定試料中の特定酵素及び特定タンパ
ク質の量を測定するバイオセンサに関する。[0001] The present invention relates to a method for producing
Shows an electrochemical response corresponding to the concentration of a specific enzyme, for example, glutamic acid-oxaloacetate transferase (GOT), or a specific substrate, for example, cholesterol ester, contained in a body fluid such as blood or urine or a measurement sample. The present invention relates to a biosensor and a biosensor for measuring the amounts of a specific enzyme and a specific protein in a measurement sample.
【０００２】[0002]
【従来の技術】白金や炭素電極表面に酵素や抗体、微生
物等の生物自体より成る生体機能物質を固定化したバイ
オセンサが種々の化学物質及び生体物質の量や濃度を迅
速かつ連続的に測定できることは既に知られている。2. Description of the Related Art A biosensor in which a biofunctional substance consisting of an organism itself such as an enzyme, an antibody, or a microorganism is immobilized on the surface of a platinum or carbon electrode, quickly and continuously measures the amount and concentration of various chemical substances and biological substances. What we can do is already known.
【０００３】このバイオセンサは、その特長を活かして
様々な分野において利用されているが、特に盛んに利用
されているのは臨床検査の分野においてである。この様
なバイオセンサを化学物質の計測と酵素量の測定につい
て以下詳細に説明する。[0003] The biosensor is utilized in various fields by taking advantage of its features, but it is particularly actively used in the field of clinical tests. Such a biosensor will be described in detail below for measuring a chemical substance and measuring an enzyme amount.
【０００４】化学物質のうち単一酵素を用いて電極活性
物質が生成される場合には、バイオセンサは一般的に平
板状の白金等の電極の表面に固定化酵素膜を装着した構
造を有している。その作製法としては、別途調製した固
定化酵素膜を白金等の電極表面に張り付けるという方法
がしばしば用いられている。例えば、グルコースセンサ
では、酵素固定化膜にはグルコースオキシダーゼが固定
化されており、この酵素が血液、尿等の体液中のグルコ
ースに特異的に働いて、 グルコース＋Ｏ２＋Ｈ２Ｏ −−−→ グルコン酸＋Ｈ２Ｏ２ （１） の反応を引き起こす。この反応により発生した過酸化水
素の量を電気化学的方法により測定することによって体
液中のグルコース量や濃度を測定している。[0004] When an electrode active substance is produced using a single enzyme among chemical substances, a biosensor generally has a structure in which an immobilized enzyme film is attached to the surface of a flat electrode such as platinum. doing. As a production method, a method of attaching a separately prepared immobilized enzyme membrane to an electrode surface such as platinum is often used. For example, in a glucose sensor, glucose oxidase is immobilized on an enzyme-immobilized membrane, and this enzyme acts specifically on glucose in body fluids such as blood and urine, and glucose + O 2 + H 2 O − →→ causing reaction of gluconic acid + H 2 O 2 (1) . The amount and concentration of glucose in body fluids are measured by measuring the amount of hydrogen peroxide generated by this reaction by an electrochemical method.
【０００５】ところで、従来の酵素センサは電極活物質
を生成する場合には上記の電極検知法が有効であるが、
生成物が電極検知できない場合には電極法を採用できな
い。そこで、もうひとつの酵素を用いて、中間生成物を
更に変換して、生成物として電極活物質を得ようとする
方法、いわゆる複合酵素系の採用が試みられている。た
とえば、複数の酵素を膜状に固定し、これを電極に張り
付ける方法などがある。[0005] By the way, in the conventional enzyme sensor, when the electrode active material is generated, the above-described electrode detection method is effective.
If the product cannot be detected by the electrode, the electrode method cannot be adopted. Therefore, a method of further converting an intermediate product using another enzyme to obtain an electrode active material as a product, that is, a so-called composite enzyme system has been attempted. For example, there is a method in which a plurality of enzymes are immobilized in the form of a film, and this is attached to an electrode.
【０００６】[0006]
【発明が解決しようとする課題】上述のような従来技術
のバイオセンサに鑑み、発明が解決しようとする課題は
複数かつ多量の酵素を極めて多孔性に富む金属電極に主
として共有結合によって固定化し、その際の酵素のうち
少なくとも一種類の酵素が体液中の成分を認識分解した
ときの生成物を、同時に固定化した酵素によって電極活
物質に変換し検知しようとするものである。In view of the above-mentioned prior art biosensors, the problem to be solved by the present invention is to immobilize a plurality and a large amount of enzymes on a very porous metal electrode mainly by covalent bonds. At this time, a product obtained when at least one of the enzymes recognizes and decomposes a component in the body fluid is simultaneously converted into an electrode active material by the immobilized enzyme to be detected.
【０００７】更に、多孔性に富む金属電極に特定酵素を
認識する抗体を共有結合し体液中の酵素を多孔性電極に
免疫化学的に間接固定化することによって、この酵素の
生成物が電極活性物質でなくても、同時に固定化した酵
素によって電極活物質に変換されることによって検知し
ようとするものである。Further, by covalently bonding an antibody recognizing a specific enzyme to a porous metal electrode and indirectly immobilizing an enzyme in a body fluid to the porous electrode by immunochemistry, the product of the enzyme is activated by the electrode. Even if it is not a substance, it is intended to be detected by being simultaneously converted into an electrode active material by an immobilized enzyme.
【０００８】この様な方法によって体液中の代謝成分や
タンパク質成分を測定できるバイオセンサを提供でき
る。ここで、タンパク質成分が触媒活性を持たないとき
は一定量の酵素標識タンパク質と試料中の特定タンパク
質との競争的結合法により特定タンパク質を測定でき
る。A biosensor capable of measuring metabolic components and protein components in a body fluid can be provided by such a method. Here, when the protein component has no catalytic activity, the specific protein can be measured by a competitive binding method of a fixed amount of the enzyme-labeled protein and the specific protein in the sample.
【０００９】[0009]
【課題を解決するための手段】上記の課題は、複数の酵
素、あるいは酵素と抗体を局所的空間に配置することに
よって解決されることが見出された。SUMMARY OF THE INVENTION It has been found that the above-mentioned problem is solved by arranging a plurality of enzymes or an enzyme and an antibody in a local space.
【０００１０】即ち、第一のアプローチにおいて、本発
明はポーラスな表面を有する金黒等の電極材料、転移酵
素、合成酵素等の一群の酵素、酸化還元酵素などの電極
活性物質を生成する酵素から成る。更に体液中のアミノ
基転移酵素等の量を測定対象とする際には、その酵素に
対する抗体と当該酵素の生成物を酸化する酵素から成る
抗体及び酵素を固定化した多孔性金属電極を用いる。That is, in the first approach, the present invention relates to an electrode material such as gold black having a porous surface, a group of enzymes such as transferase and synthase, and an enzyme which produces an electrode active substance such as oxidoreductase. Become. Further, when measuring the amount of aminotransferase or the like in a body fluid, an antibody comprising an antibody against the enzyme and an enzyme oxidizing a product of the enzyme and a porous metal electrode on which the enzyme is immobilized are used.
【０００１１】本発明において、多孔性電極とは電極の
表面に塩化金酸等の金属化合物を還元的（電気化学的に
負の電位をかける）に析出させたもので、表面積は平滑
電極に対して、約数百倍から数千倍となったものをい
う。In the present invention, a porous electrode is obtained by depositing a metal compound such as chloroauric acid on the surface of the electrode in a reducing manner (by applying a negative potential electrochemically). About several hundred to several thousand times.
【０００１２】本発明においては複合酵素系を構築する
が、ここで複合酵素系とは直接複合酵素系と間接複合酵
素系から成る。直接複合酵素系は体液などの測定試料に
浸漬すると下記のように二種類の酵素によって電極不活
性の基質Ａ（測定対象）から電極活性物質が生成される
場合をいう。 In the present invention, a complex enzyme system is constructed, wherein the complex enzyme system comprises a direct complex enzyme system and an indirect complex enzyme system. The direct complex enzyme system refers to a case where an electrode active substance is generated from an electrode-inactive substrate A (measurement target) by two kinds of enzymes as described below when immersed in a measurement sample such as a body fluid.
【０００１３】更に、間接複合酵素系では抗体による測
定試料中の特定酵素が結合固定化されることによって、
初めて複合酵素系が形成される。即ち、複合酵素系が形
成されて、下記のように測定試料中の酵素量が測定さ
れ、その酵素量が基質Ａの溶液中で測定される。ここで
測定対象は酵素Ａである。固定化特異抗体＋酵素Ａ−−
→免疫化学的固定化酵素Ａ （３） Furthermore, in the indirect complex enzyme system, a specific enzyme in a measurement sample is bound and immobilized by an antibody,
For the first time, a complex enzyme system is formed. That is, a complex enzyme system is formed, the amount of the enzyme in the measurement sample is measured as described below, and the amount of the enzyme is measured in the substrate A solution. Here, the measurement object is enzyme A. Immobilized specific antibody + enzyme A-
→ Immunochemical immobilized enzyme A (3)
【０００１４】本発明において、酵素（あるいは抗体）
の固定化とは金黒表面等に金−メルカプチド結合法によ
って官能基を導入し、この官能基に酵素や抗体を結合す
ること、及び更にグルタルアルデヒドで多量の酵素や抗
体を結合することである。更に白金黒電極やカーボン電
極では直接タンパク質を固定化することである。In the present invention, an enzyme (or an antibody)
Immobilization means introducing a functional group to a gold-black surface or the like by a gold-mercaptide bonding method, binding an enzyme or antibody to this functional group, and further binding a large amount of enzyme or antibody with glutaraldehyde. . Further, a platinum black electrode or a carbon electrode directly fixes proteins.
【０００１５】例えば、体液中の代謝物質を測定する一
例としてコレステロールエステルの量を測定する場合で
は、上記反応は以下のようになる。 （６）の反応も同時に起きるので、生成される過酸化水
素を測定することによって総コレステロールの測定が可
能になる。For example, in the case of measuring the amount of cholesterol ester as an example of measuring a metabolite in a body fluid, the above reaction is as follows. Since the reaction (6) also occurs at the same time, the total cholesterol can be measured by measuring the generated hydrogen peroxide.
【０００１６】体液ではないが、農産物中の澱粉の測定
も可能であり、この場合には以下のような複合酵素系を
用い、生成される過酸化水素を電極検知すれば澱粉を直
接測定できる。 Although not a body fluid, it is also possible to measure starch in agricultural products. In this case, starch can be directly measured by using the following complex enzyme system and detecting the produced hydrogen peroxide as an electrode.
【０００１７】本発明において、測定試料中の酵素の量
を測定するためには一例として下記のような抗体−酵素
系を採用する。なお抗体はモノクローナル抗体あるいは
ポリクロナル抗体何れであっても良い。例えば、ＧＯＴ
の直接測定を行う場合には抗ＧＯＴ抗体を固定化する。
従って測定試料のＧＯＴを抗原抗体反応によって間接的
に固定化した電極を基質溶液に浸漬することによってＧ
ＯＴの量を測定することになる。 抗ＧＯＴ抗体＋ＧＯＴ（試料中）−−−→抗ＧＯＴ抗体・ＧＯＴ複合体 （８） In the present invention, the following antibody-enzyme system is employed as an example to measure the amount of the enzyme in the measurement sample. The antibody may be a monoclonal antibody or a polyclonal antibody. For example, GOT
When direct measurement is performed, an anti-GOT antibody is immobilized.
Therefore, by immersing the electrode in which the GOT of the measurement sample is indirectly immobilized by an antigen-antibody reaction in a substrate solution,
The amount of OT will be measured. Anti-GOT antibody + GOT (in sample) --- → anti-GOT antibody-GOT complex (8)
【０００１８】本発明において、電気化学的測定方法と
は、上述のような反応において生じる電流量（又は電荷
量）を測定し、それに基づいて基質の量あるいは酵素の
量、従って濃度を求める方法を意味する。In the present invention, the electrochemical measurement method refers to a method of measuring the amount of electric current (or the amount of charge) generated in the above-described reaction, and obtaining the amount of the substrate or the amount of the enzyme, that is, the concentration based on the measured amount. means.
【０００１９】本発明の電極は、電気化学的測定法にお
いて作用電極（測定電極）として使用することができ、
本発明の電極（バイオセンサ）を作用電極として用いる
電気化学的測定方法において汎用される。参照電極とし
ても作用する対極から成る二電極系、並びに参照電極と
対極を分離した三電極系を構成し、反応により生じる電
流量を測定する。作用電極に所定電位を印加するが、本
発明のバイオセンサ以外の要素は周知であり、これ以上
の説明は必要ない。The electrode of the present invention can be used as a working electrode (measurement electrode) in an electrochemical measurement method,
It is widely used in an electrochemical measurement method using the electrode (biosensor) of the present invention as a working electrode. A two-electrode system including a counter electrode that also functions as a reference electrode and a three-electrode system including a reference electrode and a counter electrode are configured, and the amount of current generated by the reaction is measured. A predetermined potential is applied to the working electrode, but elements other than the biosensor of the present invention are well known and need not be further described.
【０００２０】本発明において、電極活性物質とは電気
化学的に容易に酸化もしくは還元できるものであって、
このときに電子メディエータを用いて更に容易に酸化還
元できるものを含む。In the present invention, the electrode active substance is a substance which can be easily oxidized or reduced electrochemically,
At this time, those which can be more easily redox-reduced using an electron mediator are included.
【０００２１】本発明において、採用する複合酵素系と
は少なくとも一種類の酵素が電極活性物質でない生体物
質を別種の電極不活性物質に変換し、少なくとも一つの
酵素が最終的に電極活性物質に変換するものから構成さ
れる。この様な複合酵素系から成る金黒電極を模式的に
図１に示す。図１の（Ａ）は金黒のポーラスな孔の内部
に２つの酵素が固定されている様子を示し、（Ｂ）は金
メルカプチド結合によって２種類の酵素が共有結合され
ている状態を模式的に示す。[0002] In the present invention, the complex enzyme system used is that at least one kind of enzyme converts a biological material that is not an electrode active substance into another kind of electrode inactive substance, and at least one enzyme finally converts it into an electrode active substance. It consists of what you do. FIG. 1 schematically shows a gold-black electrode composed of such a complex enzyme system. FIG. 1A shows a state in which two enzymes are immobilized inside a gold-black porous hole, and FIG. 1B schematically shows a state in which two kinds of enzymes are covalently bonded by a gold mercaptide bond. Shown in
【０００２２】本発明において、採用する抗体−酵素系
のうち、抗体とは測定試料中の測定対象となる特定酵素
と結合するものを示し、酵素とは特定酵素の生成物を電
極活性物質に変換するものをいう。この抗体−酵素系の
模式図を図２に示す。ここに抗体、酵素の何れも共有結
合によって固定化されている。抗体−酵素系は図３に示
す如く目的の酵素と結合することによって始めて複合酵
素系となるものである。[0002] In the present invention, among the antibody-enzyme systems employed, an antibody refers to an antibody that binds to a specific enzyme to be measured in a measurement sample, and an enzyme converts a product of the specific enzyme into an electrode active substance. To do. A schematic diagram of this antibody-enzyme system is shown in FIG. Here, both the antibody and the enzyme are immobilized by covalent bonds. The antibody-enzyme system becomes a complex enzyme system only after binding to the target enzyme as shown in FIG.
【０００２３】[00023]
【実施例１】 （グルコアミラーゼ及びグルコースオキシダーゼから成
る複合酵素系を金黒電極に共有結合法によって固定化し
たスターチセンサ）平滑金電極（直径１．６ｍｍ）を硫
酸（３０％）一過酸化水素（７０％）混液中で洗浄し、
電極表面をアルミナ研磨した。この電極を酢酸鉛を含む
塩化金酸溶液中で負の電位を印加すると、金が析出し金
黒電極が調製できた。なお金黒析出量は通電電気量で制
御した。即ち、電気量を制御することによって、金黒の
多孔性を制御できる。作製した金黒電極を１０ｍＭアミ
ノエタンチオール水溶液に２時間浸漬し、金黒表面にア
ミノ基を金−メルカプチド結合を介して導入した。エタ
ノールで洗浄後、１％グルタルアルデヒド水溶液に３０
分間浸漬して金黒表面にアルデヒド基を導入した。リン
酸緩衝液で洗浄後、グルコースオキシダーゼ（３０ｍｇ
／ｍｌ）溶液に３０分間浸漬し、洗浄後グルコアミラー
ゼ（３０ｍｇ／ｍｌ）溶液に浸漬することによって金黒
表面にバイエンザイムセンサを作製した。なお、孔の内
部の２つの酵素の固定化を強固にする必要がある場合
は、更にアルブミン溶液（１％）に浸漬した後、グルタ
ルアルデヒドで架橋処理した。Example 1 (A starch sensor in which a complex enzyme system composed of glucoamylase and glucose oxidase was immobilized on a gold black electrode by a covalent bond method) A smooth gold electrode (diameter 1.6 mm) was treated with sulfuric acid (30%) hydrogen peroxide. (70%) wash in the mixture,
The electrode surface was polished with alumina. When a negative potential was applied to this electrode in a chloroauric acid solution containing lead acetate, gold was deposited and a gold black electrode was prepared. The amount of deposited gold black was controlled by the amount of electricity supplied. That is, the porosity of gold black can be controlled by controlling the amount of electricity. The prepared gold black electrode was immersed in a 10 mM aqueous solution of aminoethanethiol for 2 hours to introduce an amino group to the gold black surface via a gold-mercaptide bond. After washing with ethanol, 30% in 1% glutaraldehyde aqueous solution
The aldehyde group was introduced into the gold black surface by immersion for minutes. After washing with a phosphate buffer, glucose oxidase (30 mg
/ Ml) solution for 30 minutes, and after washing, immersion in a glucoamylase (30 mg / ml) solution to produce a bienzyme sensor on the gold black surface. If it is necessary to strongly fix the two enzymes inside the pores, they were further immersed in an albumin solution (1%) and then crosslinked with glutaraldehyde.
【０００２４】作製したバイエンザイムセンサを作用電
極とし、対極に白金板電極、そして参照電極に銀・塩化
銀電極を用いてセンサ特性を明らかにした。基質として
スターチ溶液を用いた。センサに印加する電位は＋９０
０ｍＶとし、スターチ濃度を０．０１〜２．５（ｗ／
ｗ）％とした。The sensor characteristics were clarified using the produced bienzyme sensor as a working electrode, a platinum plate electrode as a counter electrode, and a silver / silver chloride electrode as a reference electrode. A starch solution was used as a substrate. The potential applied to the sensor is +90
0 mV, and the starch concentration was 0.01 to 2.5 (w /
w)%.
【０００２５】図４に示すのは、スターチを添加したと
きの経時変化である。スターチ濃度の増加とともにセン
サ出力も大きくなっていることが分かる。これを検量線
として示したのが図５である。この図からスターチ濃度
０．０１〜０．５（ｗ／ｗ）％の範囲で直線関係が認め
られることが解った。FIG. 4 shows the change with time when starch is added. It can be seen that the sensor output increases as the starch concentration increases. FIG. 5 shows this as a calibration curve. From this figure, it was found that a linear relationship was observed in the starch concentration range of 0.01 to 0.5 (w / w)%.
【０００２６】[00026]
【実施例２】 （総コレステロール測定用バイエンザイムセンサ）同様
にして、コレステロールエステラーゼとコレステロール
オキシダーゼから成る複合酵素センサを作製し、総コレ
ステロールセンサとして評価した。ここではコレステロ
ール及びコレステロールエステルを可溶化するためにＴ
ｒｉｔｏｎＸ−１００（１０％）を含むリン酸（０．１
Ｍ）緩衝液を用いた。印加電位を５００ｍＶとした。コ
レステロールエステルとしてコレステロールパルミチン
酸を添加したときの経時変化を図６に示す。更に図７に
同一センサにそれぞれコレステロール、コレステロール
リノール酸、そしてコレステロールパルミチン酸を添加
したときの基質濃度とセンサ出力との関係を示す。この
図からコレステロール及びその誘導体に補正係数を掛け
れば総コレステロールの推定が可能であることが解る。Example 2 (Bienzyme sensor for measuring total cholesterol) Similarly, a composite enzyme sensor comprising cholesterol esterase and cholesterol oxidase was prepared and evaluated as a total cholesterol sensor. Here, T is used to solubilize cholesterol and cholesterol esters.
phosphoric acid containing 0.1% of ritonX-100 (0.1%)
M) Buffer was used. The applied potential was 500 mV. FIG. 6 shows the change over time when cholesterol palmitic acid was added as a cholesterol ester. FIG. 7 shows the relationship between the substrate concentration and the sensor output when cholesterol, cholesterol linoleic acid, and cholesterol palmitic acid were added to the same sensor. From this figure, it can be seen that total cholesterol can be estimated by multiplying cholesterol and its derivatives by a correction coefficient.
【０００２７】[00027]
【実施例３】 （測定試料中のＧＯＴ量の測定センサ）グルタミン酸オ
キシダーゼ溶液（１０ｍｇ／ｍＬ）中に上述の処理で表
面にアルデヒド基を導入した金黒電極を３０分浸漬した
後、抗ウシＧＯＴ抗体溶液（１０ｍｇ／ｍＬ）中に３０
分間浸漬し抗体−酵素系を構築した。Example 3 (Measurement Sensor of GOT Amount in Measurement Sample) A gold black electrode having an aldehyde group introduced into the surface by the above-described treatment was immersed in a glutamate oxidase solution (10 mg / mL) for 30 minutes. 30 in antibody solution (10 mg / mL)
The sample was immersed for 5 minutes to construct an antibody-enzyme system.
【０００２８】上述の抗体−酵素系から成る金黒電極を
ＧＯＴウシ溶液（１０ｍｇ／ｍＬ）に浸し、抗原抗体反
応を攪拌下で１時間行った。このときウシＧＯＴ溶液量
を１ｍＬ、２ｍＬ、４ｍＬ、６ｍＬ、８ｍＬそして１０
ｍＬとした。反応後、アルパラギン酸及び２−オキソグ
ルタミン酸（各々１０ｍＭ）中で免疫化学的に調製した
複合酵素系を評価した。その結果ウシＧＯＴ溶液の量と
センサ出力との間に６ｍＬ以下の範囲で液量と出力との
間に直線性が得られた。A gold-black electrode comprising the above-mentioned antibody-enzyme system was immersed in a GOT bovine solution (10 mg / mL), and an antigen-antibody reaction was carried out for 1 hour under stirring. At this time, add 1 mL, 2 mL, 4 mL, 6 mL, 8 mL and 10 mL of bovine GOT solution.
mL. After the reaction, the complex enzyme system prepared immunochemically in aspartic acid and 2-oxoglutamic acid (10 mM each) was evaluated. As a result, linearity was obtained between the amount of the bovine GOT solution and the output in the range of 6 mL or less between the amount of the bovine GOT solution and the sensor output.
【０００２９】以上のことは抗体がウシＧＯＴを結合
し、このＧＯＴがアルパラギン酸と２−オキソグルタミ
ン酸の間のアミノ基を転移し、グルタミン酸とオキサロ
酢酸を生成することを示している。このグルタミン酸を
グルタミン酸酸化酵素がα−ケトグルタミン酸、アンモ
ニア、および過酸化水素に変換し、この過酸化水素が電
極で検知されることを示している。The above indicates that the antibody binds bovine GOT, which transfers the amino group between aspartic acid and 2-oxoglutamic acid to produce glutamic acid and oxaloacetic acid. Glutamate oxidase converts this glutamic acid into α-ketoglutamic acid, ammonia, and hydrogen peroxide, indicating that the hydrogen peroxide is detected at the electrode.
【０００３０】ウシＧＯＴを含む溶液量（ウシＧＯＴ
量）とセンサ出力との間に直線性があることから、抗体
に結合されるウシＧＯＴは測定試料の量に依存している
ことが解る。即ち、溶液量一定のもとでウシＧＯＴ濃度
に依存することを示している。The volume of the solution containing bovine GOT (bovine GOT
The linearity between the (volume) and the sensor output indicates that the amount of bovine GOT bound to the antibody depends on the volume of the measurement sample. In other words, it shows that it depends on the bovine GOT concentration under a constant solution amount.
【０００３１】[00031]
【発明の効果】本発明のバイオセンサ、及びその作製法
を使用することによって、従来電気化学デバイスで検知
できなかった代謝物質及び疾患特有のマーカー酵素やマ
ーカータンパク質の計測が可能となる。この事によっ
て、測定試料中の多種多様な物質の計測デバイスが実現
できる。By using the biosensor of the present invention and the method for producing the same, it becomes possible to measure metabolites and marker enzymes and marker proteins specific to diseases that could not be detected by the conventional electrochemical device. This makes it possible to realize a measurement device for various kinds of substances in a measurement sample.
【図１】多孔性表面への複合酵素系（酵素Ａおよび酵素
Ｂ）の固定化されている様子（Ａ）、及びそれぞれの酵
素が金メルカプチド結合によって固定化されている模式
図（Ｂ）である。FIG. 1 shows a state in which a complex enzyme system (enzyme A and enzyme B) is immobilized on a porous surface (A), and a schematic diagram (B) in which each enzyme is immobilized by a gold mercaptide bond. is there.
【図２】多孔性金表面に抗体（Ａｂ）及び酵素（Ｅ）が
金メルカプチド結合によって固定化されている模式図で
ある。FIG. 2 is a schematic diagram in which an antibody (Ab) and an enzyme (E) are immobilized on a porous gold surface by a gold mercaptide bond.
【図３】金メルカプチド結合によって固定化されている
抗体が対応する酵素ＧＯＴを認識結合している図であ
る。FIG. 3 is a diagram in which an antibody immobilized by a gold mercaptide bond recognizes and binds to a corresponding enzyme GOT.
【図４】グルコアミラーゼとグルコースオキシダーゼか
ら複合酵素系を結成させた金黒電極に９００ｍＶの電位
を印加しスターチを添加したときの経時変化である。FIG. 4 is a change with time when a potential of 900 mV is applied to a gold black electrode in which a complex enzyme system is formed from glucoamylase and glucose oxidase and starch is added.
【図５】同上の方法で得られたスターチ濃度とセンサ出
力との関係を示す図である。FIG. 5 is a diagram showing a relationship between a starch concentration and a sensor output obtained by the above method.
【図６】金黒電極にコレステロールエステラーゼ及びコ
レステロールオキシダーゼから成る複合酵素系を形成
し、コレステロールパルミチン酸を添加したときのコレ
ステロールエステル濃度とセンサ応答の経時変化を示す
図である。FIG. 6 is a graph showing the change over time in cholesterol ester concentration and sensor response when a complex enzyme system comprising cholesterol esterase and cholesterol oxidase is formed on a gold black electrode and cholesterol palmitic acid is added.
【図７】コレステロールエステラーゼーコレステロール
オキシダーゼ系にコレステロール、コレステロールリノ
ール酸、及びコレステロールパルミチン酸を適応したと
きの濃度（ｍＭ）とセンサ出力（ｎＡ）との関係を示す
図である。FIG. 7 is a graph showing the relationship between the concentration (mM) and the sensor output (nA) when cholesterol, cholesterol linoleic acid, and cholesterol palmitic acid are applied to the cholesterol esterase-cholesterol oxidase system.
１．コレステロールの測定曲線 ２．コレステロールリノール酸の測定曲線 ３．コレステロールパルミチン酸の測定曲線 1. 1. Cholesterol measurement curve 2. Measurement curve of cholesterol linoleic acid Measurement curve of cholesterol palmitic acid
Claims (8)
て貴金属電極の表面に多孔性に富む表面を形成し、その
内部に二種類（以上）の酵素を固定化する。このとき酵
素Ａの生成物を酵素Ｂの触媒作用により、電極活物質に
変換し、多孔性電極自身によって検知る複合酵素センサ
及びその作製法1. A noble metal electrode is electrolytically reduced to form a porous surface on the surface of a noble metal electrode, and two (or more) types of enzymes are immobilized inside the surface. At this time, a composite enzyme sensor that converts a product of the enzyme A into an electrode active material by the catalytic action of the enzyme B and detects the active material by the porous electrode itself, and a method for producing the same
ルカプチド結合によって多孔性（表面積大）表面に主と
して化学的に結合させる方式の複合酵素センサ及びその
作製法。2. A composite enzyme sensor according to claim 1, wherein in the case of a gold electrode, the composite enzyme sensor is mainly chemically bonded to a porous (large surface area) surface by gold mercaptide bonds.
の場合には表面処理によって化学的に結合させる方式の
複合酵素センサ及びその作製法。3. A composite enzyme sensor according to claim 1, wherein a platinum electrode, a carbon electrode or the like is chemically bonded by surface treatment.
の酵素を架橋した構造の複合酵素センサ及びその作製法4. A composite enzyme sensor having a structure in which a larger amount of an enzyme is crosslinked to an enzyme immobilized by a covalent bond, and a method for producing the same.
カーの抗体及びその抗体に結合されたマーカー酵素によ
って生成される物質を代謝する酵素を同時に固定化した
酵素免疫電極を作製し、血清（血液）中のマーカーと一
定時間共存させることによって、抗体で固定されたマー
カー酵素とその生成物を電極活物質に変換する酵素シス
テムから成る複合酵素系を創出し、マーカー酵素量を測
定する方法及びそのための電極。5. An enzyme immunoelectrode in which an antibody of an enzyme marker of interest and an enzyme that metabolizes a substance produced by a marker enzyme bound to the antibody are simultaneously immobilized on a porous electrode, and a serum (blood Method for measuring the amount of marker enzyme by creating a complex enzyme system consisting of an enzyme system that converts the marker enzyme immobilized with the antibody and its product to an electrode active material by coexisting with the marker in) for a certain period of time, and Electrodes.
ゼ類、アルカリ性フォスファターゼ、アミラーゼ、γ−
ＧＴＰ、クレアチンキナーゼ、ロイシンアミノペプチダ
ーゼ、コレステロールオキシダーゼ等の疾病マーカーの
みならず、前立腺特異抗原やＣ反応性タンパク質等を含
む。このうち酵素活性のないタンパク質の場合には酵素
標識タンパク質を用いた競争法、サンドイッチ法等の免
疫化学的方法によって測定する。この様な方式の酵素免
疫電極及びその作製法。6. As enzyme markers, transferases, alkaline phosphatase, amylase, γ-
It includes not only disease markers such as GTP, creatine kinase, leucine aminopeptidase, cholesterol oxidase, but also prostate specific antigen and C-reactive protein. Among them, a protein having no enzyme activity is measured by an immunochemical method such as a competition method using an enzyme-labeled protein and a sandwich method. An enzyme immunoelectrode of such a method and a method for producing the same.
であるときは、適当なメディエータを用いる請求項１〜
５のいずれかの易酸化（あるいは易還元）型電極及びそ
の作製法。7. The final product is hydrogen peroxide or NAD (P) H
, When an appropriate mediator is used.
5. The easily oxidizable (or easily reduced) electrode according to any one of 5 and a method for producing the same.
ためのパルス電位検出法などの電気化学的測定法、その
デバイス及び作製法8. An electrochemical measuring method such as a pulse potential detecting method for enhancing detection sensitivity, a device and a method for producing the same according to the above claim.
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| JP10799797A JP3393361B2 (en) | 1997-03-24 | 1997-03-24 | Biosensor |
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| JP10799797A JP3393361B2 (en) | 1997-03-24 | 1997-03-24 | Biosensor |
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