JPH0252818B2 - - Google Patents
Info
- Publication number
- JPH0252818B2 JPH0252818B2 JP57072113A JP7211382A JPH0252818B2 JP H0252818 B2 JPH0252818 B2 JP H0252818B2 JP 57072113 A JP57072113 A JP 57072113A JP 7211382 A JP7211382 A JP 7211382A JP H0252818 B2 JPH0252818 B2 JP H0252818B2
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- liquid
- working electrode
- measurement cell
- enzyme reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000007788 liquid Substances 0.000 claims description 39
- 238000005259 measurement Methods 0.000 claims description 36
- 238000006911 enzymatic reaction Methods 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 239000005515 coenzyme Substances 0.000 claims description 5
- 239000005871 repellent Substances 0.000 claims description 4
- 210000004027 cell Anatomy 0.000 description 29
- 239000000126 substance Substances 0.000 description 16
- 229940088598 enzyme Drugs 0.000 description 14
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- 229910052697 platinum Inorganic materials 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 108010015776 Glucose oxidase Proteins 0.000 description 3
- 239000004366 Glucose oxidase Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 229940116332 glucose oxidase Drugs 0.000 description 3
- 235000019420 glucose oxidase Nutrition 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 2
- 229910021607 Silver chloride Inorganic materials 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 238000003487 electrochemical reaction Methods 0.000 description 1
- 235000012209 glucono delta-lactone Nutrition 0.000 description 1
- 229960003681 gluconolactone Drugs 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003754 machining Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 230000002940 repellent Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明は、酵素あるいは補酵素を固定化してな
る酵素電極を備え、基質の濃度あるいは酵素活性
を迅速かつ簡便に測定することのできる送液式の
酵素反応測定セルに関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a liquid-feed type enzyme reaction measurement cell that is equipped with an enzyme electrode on which an enzyme or coenzyme is immobilized and that can quickly and easily measure substrate concentration or enzyme activity. .
現在、酵素の有する特異的触媒作用の工業的利
用の一例として、酵素反応系と電気化学反応系を
結びつけたいわゆる酵素電極により、基質の濃度
や酵素活性を測定することが試みられている。例
えば、酵素反応にともなつて増減するH2O2、
NH3、CO2、O2などの物質について、各々の物
質に対応した特定物質検出用の電極を用いて電気
化学的に検出し、基質濃度や酵素活性を測定する
ことが出来る。その一例として、グルコース濃度
の測定について以下に説明する。グルコースオキ
シダーゼの作用により基質であるグルコースが酸
化されて、(1)式のごとくH2O2が生成する。次に
このH2O2を(2)式の様に白金電極を用いて酸化し、
この時得られる酸化電流値から試料中のグルコー
ス濃度を知ることができる。あるいはまた、(1)式
におけるO2の減少量を酸素濃度検出電極で測定
する方法もある。 Currently, as an example of industrial use of the specific catalytic action of enzymes, attempts are being made to measure substrate concentration and enzyme activity using a so-called enzyme electrode that combines an enzyme reaction system and an electrochemical reaction system. For example, H 2 O 2 increases and decreases with enzymatic reactions,
Substances such as NH 3 , CO 2 , and O 2 can be electrochemically detected using specific substance detection electrodes corresponding to each substance, and the substrate concentration and enzyme activity can be measured. As an example, measurement of glucose concentration will be described below. The substrate glucose is oxidized by the action of glucose oxidase, producing H 2 O 2 as shown in equation (1). Next, this H 2 O 2 is oxidized using a platinum electrode as shown in equation (2),
The glucose concentration in the sample can be determined from the oxidation current value obtained at this time. Alternatively, there is also a method of measuring the amount of decrease in O 2 in equation (1) using an oxygen concentration detection electrode.
グルコース＋O2グルコースオキシダーゼ
――――――――――――→
グルコノラクトン＋H2O2 ………(1)
H2O2→2H+＋2e＋O2 ………(2)
以上の様な測定を実用的に行うには、酵素の繰
り返し使用を可能にすることが必要であり、その
ために、酵素を固定化し、さらに特定物質検出用
の電極と一体化して適当な酵素反応測定セルに装
着する。測定セルの方式としては回分式と連続送
液式に大別されるが、臨床分析における血糖分析
の様に多数の試料について迅速・簡便な測定が要
求される場合には、連続送液式の測定セルが好都
合である。 Glucose + O 2 glucose oxidase――――――――――→ Gluconolactone + H 2 O 2 ………(1) H 2 O 2 →2H + +2e＋O 2 ………(2) As above In order to carry out practical measurements, it is necessary to be able to use the enzyme repeatedly, and for this purpose, the enzyme must be immobilized, integrated with an electrode for detecting a specific substance, and attached to an appropriate enzyme reaction measurement cell. do. Measuring cell methods can be roughly divided into batch type and continuous liquid feeding type, but when rapid and simple measurement of a large number of samples is required, such as blood sugar analysis in clinical analysis, the continuous liquid feeding type is used. A measuring cell is convenient.
従来、連続送液式の酵素反応測定セルとして
は、第１図あるいは第２図に概略を示す構造のも
のがある。第１図に示すセル構造について説明す
るとセル本体１の内部に管状の流路を備え、液入
口側に管３を、液出口側に管４をそれぞれ接続
し、矢印の方向に連続的に送液する（以下、同様
に矢印で送液方向を示す）。酵素反応に対応した
電気化学的な検出は、一例として示したが、本体
の流路２の内壁面に設けた特定物質検出用の電極
（作用極）５、参照極６、対極７からなる電極系
を用いて行なうことができる。この様に管内に電
極系を設けた場合、円滑な流れを得るためには各
電極の構造を円筒状とする必要がある。しかしな
がら、実用的見地からは、円筒構造とするための
工作上の難しさや、一般に広く用いられている酵
素固定化膜についてはこれを装着するのが大変困
難であるなどの欠点を有する。 BACKGROUND ART Conventionally, a continuous liquid feeding type enzyme reaction measurement cell has a structure schematically shown in FIG. 1 or 2. To explain the cell structure shown in Fig. 1, a tubular flow path is provided inside the cell body 1, a tube 3 is connected to the liquid inlet side, and a tube 4 is connected to the liquid outlet side, and the liquid is continuously fed in the direction of the arrow. liquid (hereinafter, the direction of liquid feeding is similarly indicated by an arrow). Electrochemical detection corresponding to an enzyme reaction is shown as an example, but an electrode consisting of an electrode (working electrode) 5, a reference electrode 6, and a counter electrode 7 for detecting a specific substance is provided on the inner wall surface of the channel 2 of the main body. This can be done using a system. When an electrode system is provided in a tube in this manner, each electrode must have a cylindrical structure in order to obtain smooth flow. However, from a practical point of view, it has drawbacks such as the difficulty in machining to form a cylindrical structure and the fact that it is very difficult to attach the generally widely used enzyme-immobilized membrane.
一方、第２図に示したセル構造は上記欠点が改
良されたものであり（第１図と共通する素子には
同一番号を付し一部説明を略す）、本体１の液流
入口側に接続された管３から本体内の管状流路２
を通つて測定室８に流入し、流路２′から管４へ
と流出する。この構造においては、特定物質検出
用の電極５として、例えば先述のグルコースの場
合の様にH2O2を検出する場合には、円板状の白
金電極上にグルコースオキシダーゼを固定化した
膜を重ね合せて装着し使用することは容易であ
る。また、その着脱も構造上容易である。しかし
ながら、第２図に示した構造においては測定室８
を必要とするため、第１図に示した構造の場合の
様な円滑な液の流れは得られない。すなわち、連
続的な液流に試料を注入し、これを測定室に流入
させた場合、試料を含む液は測定室に滞まり易く
なるため、測定室から完全に流出するまでに長時
間を要することになる。すなわち、電極の応答に
テーリングが生ずる。このことは、多数の試料に
ついて連続的にかつ迅速に測定を行う必要のある
場合には大変不利なことである。この様な欠点を
改良するために測定室の形状を半管状とするなど
も考えられるが、これは、特定物質検出用の電極
の有効面積（被検液に接する面の面積）の減少と
なり、応答感度の低下を招くことになる。また、
第１図に示した構造の場合と同様な円滑な流れは
得られない。 On the other hand, the cell structure shown in FIG. 2 has the above-mentioned drawbacks improved (elements common to those in FIG. 1 are given the same numbers and some explanations are omitted). From the connected pipe 3 to the tubular flow path 2 in the main body
It flows into the measuring chamber 8 through the channel 2' and out into the tube 4. In this structure, as the electrode 5 for detecting a specific substance, for example, when detecting H 2 O 2 as in the case of glucose mentioned above, a membrane in which glucose oxidase is immobilized on a disc-shaped platinum electrode is used. It is easy to attach and use them one on top of the other. Moreover, its structure makes it easy to attach and detach. However, in the structure shown in FIG.
Therefore, smooth liquid flow as in the case of the structure shown in FIG. 1 cannot be obtained. In other words, when a sample is injected into a continuous liquid stream and allowed to flow into the measurement chamber, the liquid containing the sample tends to stay in the measurement chamber, so it takes a long time for it to completely flow out of the measurement chamber. It turns out. That is, tailing occurs in the response of the electrode. This is a great disadvantage when it is necessary to carry out continuous and rapid measurements on a large number of samples. In order to improve this drawback, it is possible to make the measurement chamber semi-tubular, but this would reduce the effective area of the electrode for detecting a specific substance (the area of the surface in contact with the sample liquid). This results in a decrease in response sensitivity. Also,
The same smooth flow as in the structure shown in FIG. 1 is not achieved.
本発明は、以上に述べた諸点について種々改良
し、優れた特性を有する送液式の酵素反応測定セ
ルを提供するものである。 The present invention provides a liquid-feed type enzyme reaction measurement cell that has various improvements in the above-mentioned points and has excellent characteristics.
本発明の酵素反応測定セルの特徴は、特定物質
検出用の電極（作用極）を備え、この電極の近傍
に少くとも酵素又は補酵素のいずれかを固定化し
てなり、前記電極に対向して下方に配置したノズ
ル部を有し、ノズル部から電極面に被検液を下か
ら噴出することによりノズル部と電極面との間に
液柱を形成せしめる様にした点にある。 The enzyme reaction measurement cell of the present invention is characterized by comprising an electrode (working electrode) for detecting a specific substance, at least an enzyme or a coenzyme immobilized near the electrode, and a cell facing the electrode. The present invention has a nozzle portion disposed below, and a liquid column is formed between the nozzle portion and the electrode surface by jetting the test liquid from below onto the electrode surface from the nozzle portion.
第３図に本発明の一実施例の断面模式図を示
す。被検液は矢印で示すごとく、管１７より円筒
形状のノズル部９に設けた管状の流路１８を通
り、ノズル部の先端から円板状の特定物質検出用
の電極２０の中心部近傍に向けて噴出する。噴出
した被検液は、ノズル部外周面に沿つて流下し、
流路１８′から管１９へと流出する。定常状態に
おいては、ノズル先端部と特定物質検出用の電極
の間には破線１０で示す様な液柱が形成される。
さらには、先述の(1)、(2)式に示す反応で必要な酵
素の補給にも役立つものである。また、液柱の部
分は第２図に示した測定室８に相当するものであ
る。それ故、円滑な流れを得るためには、体積が
小さくかつ安定した液柱を形成する必要がある。
この様な観点から、特定物質検出用の電極を保持
する枠体１１の底面１２が撥水性である必要があ
る。すなわち、底面１２が親水性であると、液柱
は破線で示した様な形状とはならず底面にまで拡
大し、液柱部分の体積が増加する。このため円滑
な流れは損われ、テーリングを生じることにな
る。撥水性の枠体とするには、撥水性の樹脂など
を枠体の材料として使用するか、あるいは枠体の
底面にグリースを塗布するなど種々の方法が考え
られる。 FIG. 3 shows a schematic cross-sectional view of an embodiment of the present invention. As shown by the arrow, the test liquid passes from the tube 17 through the tubular flow path 18 provided in the cylindrical nozzle part 9, and from the tip of the nozzle part to the vicinity of the center of the disc-shaped specific substance detection electrode 20. Squirt towards. The spouted test liquid flows down along the outer circumferential surface of the nozzle,
It flows out from channel 18' into pipe 19. In a steady state, a liquid column as shown by the broken line 10 is formed between the nozzle tip and the specific substance detection electrode.
Furthermore, it is useful for replenishing enzymes necessary for the reactions shown in equations (1) and (2) above. Further, the liquid column portion corresponds to the measurement chamber 8 shown in FIG. Therefore, in order to obtain smooth flow, it is necessary to form a stable liquid column with a small volume.
From this point of view, the bottom surface 12 of the frame 11 that holds the electrode for detecting a specific substance needs to be water repellent. That is, when the bottom surface 12 is hydrophilic, the liquid column does not take the shape shown by the broken line, but expands to the bottom surface, and the volume of the liquid column portion increases. This impairs the smooth flow and causes tailing. In order to make the frame water-repellent, various methods can be considered, such as using a water-repellent resin or the like as a material for the frame, or applying grease to the bottom surface of the frame.
以上に述べた様にセルを構成することにより、
特定物質検出用の電極面に到達した試料液は速や
かに流出するため、迅速な応答が得られ、かつ従
来例に見られる様なテーリングは起こらない。 By configuring the cells as described above,
Since the sample liquid that reaches the electrode surface for detecting a specific substance quickly flows out, a rapid response can be obtained, and tailing as seen in the conventional example does not occur.
次に本発明の実施例について述べる。 Next, embodiments of the present invention will be described.
実施例 １
特定物質検出用の電極（作用極）として次のも
のを作製した。電極担体としてポリカーボネート
多孔質膜（膜厚10μｍ、孔径2000Å）を用い、こ
の膜の片側面に白金をスパツタリングしてH2O2
検出用の作用極を形成した。次に、この膜にグル
コースオキシダーゼ水溶液（100mg／ml）を20μ
／cm2の割合で展開した後、乾燥し、続いてグル
タルアルデヒドを用いて酵素相互間を架橋し、膜
面上および孔内に固定化した。Example 1 The following electrode (working electrode) for detecting a specific substance was produced. A polycarbonate porous membrane (film thickness 10 μm, pore diameter 2000 Å) was used as an electrode carrier, and platinum was sputtered on one side of the membrane to generate H 2 O 2 .
A working electrode for detection was formed. Next, 20μ of glucose oxidase aqueous solution (100mg/ml) was applied to this membrane.
After developing at a ratio of /cm 2 , it was dried, and then the enzymes were cross-linked using glutaraldehyde to be immobilized on the membrane surface and in the pores.
このようにして得られた電極を第３図に示す酵
素反応測定セルに組み込んだ。組み込まれた電極
２０の白金スパツタ面は、白金板からなるリード
１４に接し、内径５mmのゴムパツキン１３を介し
てフツ素樹脂製の枠体１１で保持した。枠体１１
とノズル部９は通気孔１６を有する樹脂製ホルダ
ー１５に装着されており、これにより、ノズル部
先端と電極２０の間隔を適切に保つている。通気
孔１６はセル内外の気圧を同一にすることによ
り、管１９からの液の流出を円滑にするためのも
のである。電極系は、電極（作用極）２０に加え
てノズル部先端に設けた白金対極２１、および枠
体１１に設けた多孔質セラミツク層を有する
Ag／AgCl参照極２２から構成される。流路１８
の内径は0.8mm、ノズル部先端と電極２０の間隔
を2.5mmとした。 The electrode thus obtained was incorporated into the enzyme reaction measurement cell shown in FIG. The platinum spattered surface of the assembled electrode 20 was in contact with a lead 14 made of a platinum plate, and was held by a frame 11 made of fluororesin via a rubber packing 13 having an inner diameter of 5 mm. Frame body 11
The nozzle part 9 is attached to a resin holder 15 having a ventilation hole 16, thereby maintaining an appropriate distance between the tip of the nozzle part and the electrode 20. The vent hole 16 is provided to make the air pressure inside and outside the cell the same, thereby facilitating the outflow of the liquid from the pipe 19. The electrode system includes an electrode (working electrode) 20, a platinum counter electrode 21 provided at the tip of the nozzle, and a porous ceramic layer provided on the frame 11.
It consists of an Ag/AgCl reference electrode 22. Channel 18
The inner diameter of the nozzle was 0.8 mm, and the distance between the tip of the nozzle and the electrode 20 was 2.5 mm.
測定は、作用極の電位を＋0.60Vvs.Ag／AgCl
に設定し、別に外部に備えた送液ポンプと試料注
入装置により、グルコース水溶液（５×10-3モ
ル／）の一定量をPH5.6のリン酸緩衝液の流れ
に注入し、管３より測定セル内に導入した。この
様にして得られた電極の応答について、応答電流
と時間の関係を第４図に実線Ａで示す。 For measurement, set the potential of the working electrode to +0.60V vs.Ag/AgCl
A fixed amount of glucose aqueous solution (5 × 10 -3 mol/) was injected into the flow of phosphate buffer at pH 5.6 using a separate external liquid pump and sample injection device, and was introduced into the measurement cell. Regarding the response of the electrode obtained in this manner, the relationship between response current and time is shown by solid line A in FIG.
比較のために、従来の測定セルとして第２図に
示す構造のものを用い、これに上記と全く同様の
作用極を装着してグルコースに対する応答を測定
した。第２図に示す流路２の内径は１mm、測定室
は直径５mm、高さ0.8mmの円柱状となる様に構成
した。これ以外の測定条件は上記と全く同様にし
て行なつた。この様にして得られた従来の測定セ
ルによる電極の応答を第４図のＢに破線で示す。
図より明らかなごとく、本発明の酵素反応測定セ
ルによる応答ＡにおいてはＢと比較して、H2O2
の酸化電流の立上りが速やかであり、テーリング
もほとんどなく約８秒後にもとの電流値のレベル
となるなど、高感度でかつ迅速な応答を有するこ
とが判る。これは、すでに述べた様に、本発明の
酵素反応測定セルにおいては、特定物質検出用の
電極の近傍における液流が円滑であることによる
ものである。 For comparison, a conventional measurement cell having the structure shown in FIG. 2 was used, and a working electrode similar to that described above was attached to the cell to measure the response to glucose. The inner diameter of the channel 2 shown in FIG. 2 was 1 mm, and the measurement chamber was configured to have a cylindrical shape with a diameter of 5 mm and a height of 0.8 mm. Other measurement conditions were the same as above. The response of the electrodes obtained in the conventional measurement cell thus obtained is shown by the broken line in FIG. 4B.
As is clear from the figure, in response A by the enzyme reaction measurement cell of the present invention, H 2 O 2
The rise of the oxidation current is rapid, and there is almost no tailing, and the current value returns to the original level after about 8 seconds, indicating a highly sensitive and quick response. This is because, as already mentioned, in the enzyme reaction measurement cell of the present invention, the liquid flow is smooth in the vicinity of the electrode for detecting a specific substance.
実施例 ２
アルコール脱水素酵素の活性測定を行うための
特定物質検出用の電極として以下のものを作製し
た。まず、導電性担体として高純度の黒鉛粉末を
成型して直径10mm、厚さ約１mmのデイスク状の電
極を作製し、この平面上に、補酵素としてニコチ
ンアミドアデニンジヌクレオチド（NAD）の水
溶液（200mg／ml）を15μ展開・乾燥して吸着
固定化し、次に洗浄して余分のNADを除去した。
この電極を実施例１と同様にして、第３図および
第２図に示す構造の酵素反応測定セルに装着し、
送液ポンプで0.01モル／のエタノールを含むPH
7.0のリン酸緩衝液を測定セルに連続的に流し、
この流れに試料としてアルコール脱水素酵素水溶
液を注入し、電極の応答を測定した。実施例１の
結果を同様に、NADHの酸化電流に基づく応答
は、本発明の測定セルにおいては従来の測定セル
に比較して迅速かつ高感度であつた。Example 2 The following electrode was prepared for detecting a specific substance for measuring the activity of alcohol dehydrogenase. First, a disc-shaped electrode with a diameter of 10 mm and a thickness of approximately 1 mm was created by molding high-purity graphite powder as a conductive carrier. On this flat surface, an aqueous solution of nicotinamide adenine dinucleotide (NAD) as a coenzyme ( 200mg/ml) was developed with 15μ, dried, adsorbed and immobilized, and then washed to remove excess NAD.
This electrode was attached to an enzyme reaction measurement cell having the structure shown in FIGS. 3 and 2 in the same manner as in Example 1, and
PH containing 0.01 mol/ethanol with liquid pump
7.0 phosphate buffer is continuously flowed into the measurement cell.
An alcohol dehydrogenase aqueous solution was injected as a sample into this flow, and the response of the electrode was measured. Similarly to the results of Example 1, the response based on the oxidation current of NADH was faster and more sensitive in the measurement cell of the present invention than in the conventional measurement cell.
以上、実施例に示した様に、本発明の送液式の
酵素反応測定セルを用いることにより、多数の試
料について基質濃度や酵素活性などを迅速に連続
的に測定することができる。 As shown in the Examples above, by using the liquid feeding type enzyme reaction measurement cell of the present invention, it is possible to rapidly and continuously measure the substrate concentration, enzyme activity, etc. of a large number of samples.
なお、上記実施例においてはノズル部からの被
検液は連続送液式としたが、１回の噴流によつて
液柱が作用極の表面にひろがる程度になつておれ
ば、間けつ的に噴出してもよい。また、特定物質
検出用の電極、対極および参照極などの電極系、
酵素や補酵素については実施例に示したものに限
定されることはなく、酵素反応系、使用条件や目
的等に応じたものを選択すればよい。さらに電極
の構成、装着方法、配置や枠体の材料、およびこ
れらに関連した測定セルの構造については実施例
に限定されることはない。第３図に示した通気孔
１６、流路１８′の位置、形状は、これに限定さ
れることはなく、本発明の目的に合致するもので
あれば良い。対極の配置については前記実施例に
示したようにノズル部の先端部に限定されること
はなく、作用極に対してノズル部側に設ければよ
い。また、実施例においては３電極系について示
したが、これに限定されることはなく、対極と参
照極を一つの電極で兼用した２電極系を適用する
こともできる。 In the above example, the test liquid was fed continuously from the nozzle part, but if the liquid column is spread over the surface of the working electrode by one jet, it can be fed intermittently. You may squirt. In addition, electrode systems such as electrodes for specific substance detection, counter electrodes, and reference electrodes,
Enzymes and coenzymes are not limited to those shown in the examples, and may be selected depending on the enzyme reaction system, usage conditions, purpose, etc. Furthermore, the configuration, mounting method, and arrangement of the electrodes, the material of the frame, and the structure of the measurement cell related thereto are not limited to the examples. The positions and shapes of the ventilation hole 16 and flow path 18' shown in FIG. 3 are not limited to these, and may be any suitable as long as they meet the purpose of the present invention. The arrangement of the counter electrode is not limited to the tip of the nozzle part as shown in the above embodiments, but may be provided on the nozzle part side with respect to the working electrode. Further, although a three-electrode system is shown in the embodiment, the present invention is not limited to this, and a two-electrode system in which one electrode serves as both a counter electrode and a reference electrode can also be applied.
以上のように本発明の酵素反応測定セルにおい
ては、被検液を作用電極に下から噴射させるもの
であるので、被検液が作用極部で滞留することが
なく速かに流れ、又噴流部は大気とよく接触する
ので酸素の補給も十分となる。したがつて被検液
は速やかにかつ円滑に流れるために、迅速な応答
が得られ、また被検液の入れ換えも容易になり、
正確かつ迅速な測定をすることができる。 As described above, in the enzyme reaction measurement cell of the present invention, the test liquid is injected onto the working electrode from below, so the test liquid does not stagnate at the working electrode and flows quickly. Since the parts are in good contact with the atmosphere, oxygen supply is sufficient. Therefore, the test liquid flows quickly and smoothly, providing a quick response and making it easy to replace the test liquid.
Accurate and quick measurements can be taken.
第１図および第２図は従来の異なる実施例の酵
素反応測定セルの断面模式図、第３図は本発明の
一実施例の酵素反応測定セルの断面模式図、第４
図は第３図の酵素反応測定セルにおけるグルコー
スに対する応答電流と時間の関係を示す図であ
る。
９……ノズル部、１０……液柱、１１……枠
体、１２……枠体底面、１３……ゴムパツキン、
１４……白金板のリード、１５……樹脂製ホルダ
ー、１６……通気孔、１７……管（液入口側）、
１８，１８′……流路、１９……管（液出口側）、
２０……作用極、２１……対極、２２……参照
極。
1 and 2 are schematic cross-sectional diagrams of enzyme reaction measurement cells of different conventional embodiments, FIG. 3 is a schematic cross-sectional diagram of an enzyme reaction measurement cell of one embodiment of the present invention, and FIG.
The figure shows the relationship between the response current to glucose and time in the enzyme reaction measurement cell of FIG. 3. 9... Nozzle part, 10... Liquid column, 11... Frame body, 12... Frame bottom surface, 13... Rubber gasket,
14...Platinum plate lead, 15...Resin holder, 16...Vent hole, 17...Pipe (liquid inlet side),
18, 18'...channel, 19...pipe (liquid outlet side),
20... Working electrode, 21... Counter electrode, 22... Reference electrode.
Claims (1)
し、被検液を噴流する室と、前記室内に下向に露
出させた前記作用極と、前記作用極に対向して配
置した被検液を下から噴出するノズル部と、前記
ノズル部側に配置してなる前記対極と、前記室に
外気を通ずる通気孔と、前記室内から被検液を流
出する流路を具備し、前記作用極の近傍に少なく
とも酵素または補酵素のいずれかを固定化してな
る酵素反応測定セル。 ２ 作用極は撥水性を有する枠体で保持されてい
る特許請求の範囲第１項に記載の酵素反応測定セ
ル。[Scope of Claims] 1. A chamber having an electrode system consisting of at least a working electrode and a counter electrode, into which a test liquid is jetted, the working electrode exposed downward in the chamber, and an electrode system facing the working electrode. A nozzle section for ejecting the disposed test liquid from below, the counter electrode disposed on the side of the nozzle section, a vent hole for passing outside air into the chamber, and a flow path for flowing out the test liquid from the chamber. and an enzyme reaction measurement cell, wherein at least either an enzyme or a coenzyme is immobilized near the working electrode. 2. The enzyme reaction measurement cell according to claim 1, wherein the working electrode is held by a water-repellent frame.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57072113A JPS58189549A (en) | 1982-04-28 | 1982-04-28 | Measuring cell of enzyme reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57072113A JPS58189549A (en) | 1982-04-28 | 1982-04-28 | Measuring cell of enzyme reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58189549A JPS58189549A (en) | 1983-11-05 |
| JPH0252818B2 true JPH0252818B2 (en) | 1990-11-14 |
Family
ID=13479991
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57072113A Granted JPS58189549A (en) | 1982-04-28 | 1982-04-28 | Measuring cell of enzyme reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58189549A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2611045B1 (en) * | 1987-02-13 | 1990-05-11 | Setric Genie Ind | METHOD AND DEVICE FOR ANALYSIS OR ASSAY IN A LIQUID MEDIUM |
| JP2600073B2 (en) * | 1987-12-03 | 1997-04-16 | 国立身体障害者リハビリテーションセンター総長 | Flow injection analysis system and analysis method using the same |
| JP2009058334A (en) * | 2007-08-31 | 2009-03-19 | Kanagawa Prefecture | Reactor for electrochemical measurement, and electrochemical measuring system |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5414297A (en) * | 1977-07-04 | 1979-02-02 | Hitachi Ltd | Distribution type ion responsive electrode device |
| JPS5491293A (en) * | 1977-12-28 | 1979-07-19 | Nikkiso Co Ltd | Electrode supporting apparatus for measuring sample |
-
1982
- 1982-04-28 JP JP57072113A patent/JPS58189549A/en active Granted
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5414297A (en) * | 1977-07-04 | 1979-02-02 | Hitachi Ltd | Distribution type ion responsive electrode device |
| JPS5491293A (en) * | 1977-12-28 | 1979-07-19 | Nikkiso Co Ltd | Electrode supporting apparatus for measuring sample |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58189549A (en) | 1983-11-05 |
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