JPH0640086B2 - Biosensor - Google Patents

Biosensor

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JPH0640086B2
JPH0640086B2 JP59030544A JP3054484A JPH0640086B2 JP H0640086 B2 JPH0640086 B2 JP H0640086B2 JP 59030544 A JP59030544 A JP 59030544A JP 3054484 A JP3054484 A JP 3054484A JP H0640086 B2 JPH0640086 B2 JP H0640086B2
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electrode
dye
glucose
biosensor
blood
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JPS60173459A (en
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史朗 南海
真理子 河栗
孝志 飯島
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松下電器産業株式会社
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001Enzyme electrodes

Description

【発明の詳細な説明】 産業上の利用分野 本発明は、種々の生体試料中の特定成分を迅速かつ容易に定量することのできるバイオセンサに関するものである。 DETAILED DESCRIPTION OF THE INVENTION INDUSTRIAL FIELD The present invention relates to a biosensor capable of quantifying quickly and easily identify components of various biological samples.

従来例の構成とその問題点 近年、酵素の有する特異的触媒作用を利用した種々のバイオセンサが開発され、特に臨床検査分野への応用が試みられている。 Recently conventional configuration and problems, various biosensors utilizing a specific catalytic action have been developed with the enzyme, which has been attempted particularly applicable to the field of clinical tests. 検査項目及び検体数が増加している現在、迅速に精度よく測定できるバイオセンサが望まれている。 Currently test items and the number of specimens is increased, and a biosensor capable of rapidly and accurately measured is desired.

グルコースセンサに例をとると、糖尿病の増加が激しい今日、血液中の血糖値を測定し管理するには、以前のように血液を遠心分離し血漿にして測定するのでは非常に時間がかかるため、全血で測定できるセンサが要求されている。 Taking an example glucose sensor, today intense increase in diabetes, to manage and measure the blood sugar level in blood, because of being measured in the blood was centrifuged plasma as before very time consuming , sensors that can be measured with whole blood is required. 簡易型としては、尿検査の時に使用されている検査紙と同様に、スティック状の支持体に糖(グルコース)にのみ反応する酵素および酵素反応時又は酵素反応の生成物により変化する色素を含有する担体を設置したものがある。 The simplified, similarly to the test paper that is used when the urine test, containing a dye that varies with the product of the enzymatic and enzymatic reactions during or enzymatic reaction to react only to a sugar (glucose) in the stick-shaped support there are those set up carrier to. この担体の血液を添加し、一定時間後の色素の変化を目又は光により測定する方式であるが、血液中の色素による妨害が大きく精度は低い。 Adding the support of the blood, is a method to measure the eye or light changes in dye after a certain time, greater accuracy is interference by the dye in the blood is low.

そこで、第1図のような多層式の分析担体が開発されている。 Therefore, multilayered analysis carriers such as Figure 1 has been developed. 透明な支持体1の上に試薬層2,展開層3,防水層4,過層5が順に積層した構造となっている。 Reagent layer 2 on a transparent support 1, the developing layer 3, the waterproof layer 4 has a structure in which over-layer 5 are laminated in this order. 血液サンプルを上部から滴下すると、まず過層5により血液中の赤血球,血小板などの固形成分が除去され、防水層4にある小孔4aから展開層3へ均一に浸透し、試薬層2において反応が進行する。 When dropping a blood sample from the top, first, red blood cells in the blood by over-layer 5, the solid components such as platelets are removed, uniformly permeates from the small holes 4a in the waterproof layer 4 to the developing layer 3, the reaction in the reagent layer 2 but to proceed. 反応終了後、透明な支持体を通して矢印の方向から光をあて、分光分析により基質濃度を測定する方式である。 After completion of the reaction, illuminated from the direction of the arrow through the transparent support is a method of measuring the substrate concentration by spectrophotometry. 従来の簡易なスティック状の担体にくらべ、複雑な構造であるが、血球除去などにより精度は向上した。 Compared to the conventional simple stick-like carrier, it is a complex structure, the accuracy was improved by including blood cells removed. しかし、血液の浸透および反応に時間がかかるため、サンプルの乾燥を防ぐ防水層4が必要となったり、反応を速めるために高温でインキュベートする必要があり、装置および担体が複雑化するという問題がある。 However, it takes time to blood penetration and reaction, or become waterproof layer 4 is required to prevent drying of the sample, it is necessary to incubate at an elevated temperature to accelerate the reaction, a problem that the apparatus and the carrier is complicated is there.

最近、酵素反応と電極反応を結びつけて基質濃度を測定するバイオセンサが開発されている。 Recently, bio-sensor for measuring the substrate concentration in conjunction with the enzyme reaction and electrode reaction has been developed. グルコースセンサに例をとると、第2図のように、グルコースオキシダーゼ固定化電極6を容器7に入れ、緩衝液8で満たし、スターラ9で撹拌している中に試料液を添加する。 Taking an example glucose sensor, as in the second figure, put glucose oxidase immobilized electrode 6 into the container 7, filled with buffer 8, the addition of the sample solution into the stirred at stirrer 9. グルコースオキシダーゼ固定化電極6には定電圧が印加されており、試料中のグルコースと反応して生成した過酸化水素を検知して電流が流れ、グルコース濃度が測定できる。 The glucose oxidase immobilized electrode 6 and the constant voltage is applied, current flows to detect the hydrogen peroxide reacts with the glucose in a sample can be measured glucose concentration. この方式を用いれば、血液中の色素などに妨害されず迅速に測定できる。 Using this method, it can be rapidly determined without interference such as a dye in the blood. しかし、撹拌装置が不可欠なため泡が発生したり、液の乱れが精度に影響するという問題があった。 However, stirrer bubbles may occur because essential disturbance of the liquid there is a problem that affects the accuracy. 又希釈しているため、緩衝液の量や試料の添加量に精度が要求され、操作が複雑化する不都合があった。 Also because of the dilution, the accuracy is required amount of added amount and the sample buffer, there is a disadvantage that the operation is complicated.

発明の目的 本発明は、上記の問題点を克服し、生体試料中の特定成分を小型で簡易に、しかも安定に精度よく測定できるバイオセンサを得ることを目的とする。 Object of the Invention The present invention overcomes the above problems, in a simple a specific component in a biological sample in a small, yet an object of the present invention to provide a biosensor that can be stably and accurately measured.

発明の構成 本発明のバイオセンサは、絶縁性の基板上に測定極と対極および参照極からなる電極系を有し、前記電極系を酸化還元酵素および酸化還元酵素と共役する酸化型色素を含有する多孔体で被覆したことを特徴とする。 Configuration biosensor of the present invention the invention comprises an electrode system consisting of measuring electrode and a counter electrode and a reference electrode on an insulating substrate, containing said electrode system oxidoreductase and oxidoreductase and conjugated to oxidized dye characterized by being coated with the porous body.

本発明のバイオセンサは、簡易に製造でき、かつこのバイオセンサを用いることにより、生体試料を適当量添加するだけで、試料液の特定成分を高感度に精度よく測定することができる。 The biosensor of the present invention can be prepared easily, and by using this biosensor, just an appropriate amount of biological sample can be measured accurately a specific component in a sample liquid with high sensitivity.

実施例の説明 バイオセンサの1つとして、グルコースセンサを例に説明する。 One explanation biosensor examples illustrate the glucose sensor as an example. 酸化還元酵素としてグルコースオキシダーゼを、酸化還元酵素と共役する酸化型色素としてフェリシアン化カリウムを用い、電極系を構成する材料として安定な白金を用いた。 Glucose oxidase as the oxidoreductase, using potassium ferricyanide as the oxidizing dye conjugate to oxidoreductase, with stable platinum as the material constituting the electrode system. 第3図にグルコースセンサの一実施例の模式図を示す。 It shows a schematic diagram of one embodiment of the glucose sensor in Figure 3. 塩化ビニル樹脂からなる絶縁性の基板10に白金を埋めこみ測定極11と対極12および参照極13とした。 Was measured electrode 11 and the counter electrode 12 and reference electrode 13 embeds platinum substrate 10 of insulating made of vinyl chloride resin. 前記電極系を覆うようにナイロン不織布14を設置した。 It was placed nylon nonwoven 14 so as to cover the electrode system. このナイロン不織布14は、あらかじめグルコースオキシダーゼとフェリシアン化カリウムを溶解した液を含浸し、乾燥して作製したものである。 The nylon non-woven fabric 14 is to be impregnated with a solution prepared by dissolving previously glucose oxidase and potassium ferricyanide was prepared and dried.

このナイロン不織布14上にグルコース標準液を添加し、充分浸透させた後、参照極13を基準に測定極11 With glucose standard solution on the nylon nonwoven fabric 14, after sufficiently permeate, measured reference electrode 13 to reference electrode 11
の電圧を0〜+0.5Vの間で鋸歯状に0.1V/秒で変化させた。 It was varied in 0.1 V / sec the voltages serrated between 0 to + 0.5V. 添加されたグルコースがナイロン不織布1 Nylon nonwoven fabric 1 the added glucose
4に担持されているグルコースオキシダーゼ15により酸化される際、酵素−色素共役反応によりフェリシアン化カリウム16が還元され、この反応によって生成されるフェロシアン化カリウムを測定極11の電圧を掃引することにより酸化し、その時酸化電流が流れる。 4 when it is oxidized by glucose oxidase 15 carried on the enzyme - is potassium ferricyanide 16 by the dye conjugate reaction reduced, oxidized by sweeping the voltage of the measuring electrode 11 the potassium ferrocyanide produced by the reaction, At that time oxidation current flows. この酸化電流は色素の変化量に比例し、色素が充分に存在すれば色素の変化量は基質濃度に対応するため、電流値を測定すると基質であるグルコースの濃度が検知できる。 The oxidation current is proportional to the change amount of the dye, the change amount of the dye if present dye is sufficiently in order to correspond to the substrate concentration, it is possible to detect the concentration of glucose is a substrate when a current value is measured. 得られた電流値と添加したグルコース濃度は、500mg/ The obtained current value and the glucose concentration was added, 500 mg /
dlまで非常により直線性を示した。 It showed linearity with very until dl. 又ナイロン不織布1 The nylon non-woven fabric 1
4は測定のたびに交換したが、再現性も良好であった。 4 was replaced every measurement, but was also good reproducibility.
又、グルコース標準液の添加量を20〜140μlの範囲で変化させたが、添加量に関係なく一定の値を示した。 Although the amount of glucose standard solution was varied in the range of 20~140Myueru, it showed a constant value irrespective of the added amount.

測定極及び対極からなる2電極系においても測定が可能であるが、対極が少なくとも測定極の2倍以上の面積にしないと安定した電流値が得られなかった。 Although it is possible to measure even in the measuring electrode and the second electrode system comprising a counter electrode, counter electrode unless more than double the area of ​​at least measuring electrode stable current value is not obtained. これは、基準となる対極の電位が電流を流すことにより動いてしまうからである。 This is the potential of the counter electrode as a reference is because thus moved by flowing a current. 又、銀塩化銀を対極に用いると電位は安定するが、製造する手間および組み込みの点で不便であった。 Moreover, the use of silver-silver chloride counter electrode potential becomes stable, which is inconvenient in terms of labor to manufacture and integration.

参照極を設置して3電極系にすることによって電位が安定し、測定極,対極,参照極が同面積でも精度よく測定することが可能となった。 The reference electrode and ground potential is stabilized by the three-electrode system, the measuring electrode, counter electrode, the reference electrode became possible to measure accurately even in the same area. これにより、小型化が可能となった。 As a result, it became possible to miniaturize.

第4図は塩化ビニル樹脂よりなる絶縁性基板17の上に白金をスパッタ法あるいは蒸着法により測定極18と対極19および参照極20を薄膜状に形成した例を示す。 Figure 4 shows an example of forming a measuring electrode 18 and the counter electrode 19 and reference electrode 20 by sputtering or vapor deposition of platinum on an insulating substrate 17 made of vinyl chloride resin into a thin film.
スパッタすることにより電極面積を自由に調節でき、特に同一の電極を大量に製造する時、効果が大であった。 Electrode area by sputtering can be adjusted freely, especially when producing large quantities of the same electrode, the effect was large.
この上に点線で示すように酵素と酸化型色素を保持したナイロン不織布をのせ試料を添加すると、第3図の電極と同様に良い応答が得られたため、電極毎交換することも可能となった。 The addition of sample loaded nylon nonwoven fabric, which holds the enzyme and oxidized dye as shown by the dotted lines thereon, for equally good response and the electrode of Figure 3 has been obtained, has become possible to each electrode exchange .

酸化型色素としては、上記に用いたフェリシアン化カリウムが安定に反応するので適しているが、P−ベンゾキノンを使えば、反応速度が早いので高速化に適している。 The oxidized dye is potassium ferricyanide used in the above are suitable because reacts stable With P- benzoquinone, the reaction rate is suitable for quick because speed. 又、2,6−ジクロロフェノールインドフェノール,メチレンブルー,フェナジンメトサルフェート,β Further, 2,6-dichlorophenol indophenol, methylene blue, phenazine methosulfate, beta
−ナフトキノン4−スルホン酸カリウムなども使用できる。 - potassium naphthoquinone 4-sulfonic acid may also be used.

酸化型色素および酵素を含む多孔体は、試料液をすみやかに吸収して酵素反応をおこなわせることができるように、親水性の多孔体膜であることが望ましい。 Porous body containing an oxidizing dye and enzyme, as can be the sample liquid rapidly absorbed to thereby perform the enzymatic reaction, it is desirable that the hydrophilic porous membrane. たとえば、ろ紙やパルプの不織布,ガラスの多孔体,セラミック多孔体などを用いると、試料液が均一にすばやく浸透し再現性も良好であった。 For example, non-woven fabric of filter paper or pulp, porous glass, the use of a ceramic porous body, the sample solution was also good uniform and quickly penetrated reproducibility. さらにナイロン不織布において、界面活性剤で処理したものは、処理しなかったものよりすみやかに液が浸透し、再現性が向上した。 In addition nylon nonwoven, those treated with surfactant, rapidly liquid penetrates than those not treated, with improved reproducibility.

酸化型色素と酵素を細かく粉砕後加圧して成形体として電極上に設置することもできる。 It may be installed to pressurize after finely pulverizing the oxidized dye and enzyme electrode on a shaped body. この加圧成形体に血液を添加すると、すみやかに浸透し迅速に反応した。 The addition of blood to the pressed compact was rapidly penetrate quickly react. なお、酸化型色素と酵素を加圧成形する際、SiO のような結着剤を少量混合すると、成形体の強度が増すので取り扱いが簡易となる。 It should be noted that in the pressure molding oxidation dye and an enzyme, when mixing a small amount of a binder such as SiO 2, the handling is simplified because the strength of the molded body increases. 結着剤としては、酵素反応及び電極反応に無関係で親水性のものが適している。 As the binder, those unrelated hydrophilic enzyme reactions and electrode reactions are suitable.

酸化型色素および酵素は、なるべく血液の液体成分に早く溶ける状態におくことが望ましい。 Oxidation dye and enzymes are desirably placed in a state as much as possible dissolves quickly in the liquid component of the blood. そこで、色素の溶液をナイロン不織布に浸漬後、ドライヤーにより熱風乾燥すると、真空乾燥したものより非常に細かい結晶となり、液体にとけやすくなった。 Therefore, after the dipping solution of the dye in the nylon nonwoven fabric, when dried with hot air by dryer, it becomes very fine crystals than those dried under vacuum to easily soluble in liquid. 又、色素の溶液を浸漬したナイロン不織布を、エタノールのような水にとける有機溶媒中に浸漬後、真空乾燥すると、さらに細かい結晶を担持することができた。 Further, a nylon nonwoven fabric was immersed solution of the dye, after immersion in an organic solvent soluble in water, such as ethanol, and dried in vacuo, it was possible to carry the finer crystals. 酵素は熱などにより活性が失活するので、浸漬後真空乾燥を行なった。 Since the enzyme is active is deactivated by thermal and subjected to immersion after vacuum drying.

第5図は、第3図と同じ電極系の上に、グルコースオキシダーゼ15を含浸後真空乾燥により担持したナイロン不織布21を、さらにその上部にフェリシアン化カリウム16を含浸後エタノールに浸漬し乾燥して担持したナイロン不織布22を設置した例を示す。 Figure 5 is on the same electrode system and the third figure, nylon nonwoven fabric 21 which is carried by vacuum drying after the impregnation of the glucose oxidase 15, and then immersed potassium ferricyanide 16 in its upper part after impregnation ethanol dried carrier It shows an example where the nylon nonwoven fabric 22 described. 血液を添加すると、フェリシアン化カリウムがすみやかに溶け、グルコースオキシダーゼの層に浸透するため、反応時間が約1 The addition of blood, dissolve the potassium ferricyanide rapidly, in order to penetrate the layers of glucose oxidase, the reaction time of about 1
分間と短く、再現性も良好であった。 Short and minutes between, it was also good reproducibility. グルコースオキシダーゼの層とフェリシアン化カリウムの層を逆に設置しても同様に迅速に反応した。 It was rapidly react similarly be provided a layer of layers with potassium ferricyanide glucose oxidase reversed.

本発明のセンサは、グルコースに限らず、アルコールセンサや、鮮度に関係するイノシンセンサなど酸化還元酵素の関与する系に用いることができる。 Sensor of the present invention is not limited to glucose, can be used and the alcohol sensor, the involved systems oxidoreductase such as inosine sensor associated with freshness. 酵素は固定して担持してもよく、固定化することにより、酵素の活性を長期間安定に保持することができる。 Enzyme may be supported and fixed, by immobilizing, can be long-term stably retain the activity of the enzyme.

発明の効果 測定極と対極および参照極からなる電極系に酸化還元酵素と酸化還元酵素と共役する色素を含んだ親水性の多孔体を設置し、直接試料液を添加して測定することにより、微量の試料液を感度よく測定できるようになった。 The effect measurement electrode and a counter electrode and a redox enzyme to an electrode system consisting of a reference electrode and containing an oxidoreductase and conjugated to a dye hydrophilic porous body of the invention was placed, by measuring directly adding the sample liquid, the sample solution traces can now be sensitively measured.

【図面の簡単な説明】 BRIEF DESCRIPTION OF THE DRAWINGS

第1図及び第2図は従来のグルコースセンサの構成を示す略図、第3図は本発明の一実施例のグルコースセンサの断面模式図、第4図は他の例の要部の平面図、第5図はさらに他の例の断面模式図である。 FIGS. 1 and 2 is schematic diagram illustrating the configuration of a conventional glucose sensor, FIG. 3 is cross-sectional schematic view of a glucose sensor of one embodiment of the present invention, Figure 4 is a plan view of a main part of another embodiment, FIG. 5 is a schematic sectional view of still another example. 10……基板、11……測定極、12……対極、13… 10 ...... substrate, 11 ...... measuring electrode, 12 ...... counter electrode, 13 ...
…参照極、14……多孔体、15……酵素、16……色素。 ... reference electrode, 14 ...... porous body, 15 ...... enzyme, 16 ...... dye.

Claims (2)

    【特許請求の範囲】 [The claims]
  1. 【請求項1】絶縁性の基板上に測定極と対極および参照極からなる電極系を設け、この電極系を酸化還元酵素および酸化還元酵素と共役する酸化型色素を含有する多孔体を乾燥状態で被覆したバイオセンサ。 1. A provided with an electrode system consisting of measuring electrode and a counter electrode and a reference electrode on an insulating substrate, a porous body dry state containing an oxidizing dye conjugate to a redox enzyme and an oxidoreductase this electrode system in coated biosensor.
  2. 【請求項2】多孔体が親水性多孔体膜である特許請求の範囲第1項記載のバイオセンサ。 2. A biosensor ranging first claim of the porous body is a hydrophilic porous membrane claims.
JP59030544A 1984-02-20 1984-02-20 Biosensor Expired - Lifetime JPH0640086B2 (en)

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