JPH02193054A - Enzyme electrode with flow cell - Google Patents
Enzyme electrode with flow cellInfo
- Publication number
- JPH02193054A JPH02193054A JP1012273A JP1227389A JPH02193054A JP H02193054 A JPH02193054 A JP H02193054A JP 1012273 A JP1012273 A JP 1012273A JP 1227389 A JP1227389 A JP 1227389A JP H02193054 A JPH02193054 A JP H02193054A
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- enzyme
- contact
- flow cell
- enzyme electrode
- 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.)
- Pending
Links
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 77
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 77
- 239000012528 membrane Substances 0.000 claims abstract description 36
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 34
- 239000000758 substrate Substances 0.000 claims description 9
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 abstract description 10
- 229920001296 polysiloxane Polymers 0.000 abstract description 4
- 230000006641 stabilisation Effects 0.000 abstract 1
- 238000011105 stabilization Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 66
- 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 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 239000008103 glucose Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- WABPQHHGFIMREM-UHFFFAOYSA-N lead(0) Chemical compound [Pb] WABPQHHGFIMREM-UHFFFAOYSA-N 0.000 description 5
- 238000011088 calibration curve Methods 0.000 description 4
- 239000007772 electrode material Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- SMEGJBVQLJJKKX-HOTMZDKISA-N [(2R,3S,4S,5R,6R)-5-acetyloxy-3,4,6-trihydroxyoxan-2-yl]methyl acetate Chemical compound CC(=O)OC[C@@H]1[C@H]([C@@H]([C@H]([C@@H](O1)O)OC(=O)C)O)O SMEGJBVQLJJKKX-HOTMZDKISA-N 0.000 description 2
- 229940081735 acetylcellulose Drugs 0.000 description 2
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 2
- 229920002301 cellulose acetate Polymers 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000010408 film Substances 0.000 description 2
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 102000020006 aldose 1-epimerase Human genes 0.000 description 1
- 108091022872 aldose 1-epimerase Proteins 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000206 photolithography Methods 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 238000001771 vacuum deposition Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
【発明の詳細な説明】 （イ）産業上の利用分野 この発明は、フローセルを備えた酵素電極に関する。[Detailed description of the invention] (b) Industrial application fields The present invention relates to an enzyme electrode equipped with a flow cell.
（ロ）従来の技術
従来のフローセル付き酵素電極を、第１０図に示す。こ
の酵素電極は、外ケース４１、フローセル４４、キャッ
プ４日とにより構成される。外ケース４１は、作用電極
４２及び対照電極４３とを備えており、作用電極４２に
は丸棒状（例えば直径０．７ｍｍ）の白金、対照電極４
３には、円筒状（例えば内径２ＩＩｌＩ１１、外径６ｍ
ｍ）の銀がそれぞれ使用され、これらが同軸状に配され
ている。これら作用電極４２及び対照電極４３は、それ
ぞれの端面が外ケース４１の凸面４１ａに露出している
。(b) Prior Art A conventional enzyme electrode with a flow cell is shown in FIG. This enzyme electrode is composed of an outer case 41, a flow cell 44, and a cap. The outer case 41 is equipped with a working electrode 42 and a reference electrode 43.
3 has a cylindrical shape (for example, inner diameter 2IIlI11, outer diameter 6m)
m) are used, and these are arranged coaxially. The end surfaces of the working electrode 42 and the control electrode 43 are exposed on the convex surface 41a of the outer case 41.
一方、フローセル４４には、試料が流れる試料流路４５
が形成されている。この試料流路４５は、フローセル４
４の凹面４４ａに露出している。フローセル４４には、
凹面４４ａを覆うように固定化酵素膜４６が装着され、
０リング４７により固定されている。On the other hand, the flow cell 44 includes a sample channel 45 through which the sample flows.
is formed. This sample channel 45 is connected to the flow cell 4
It is exposed on the concave surface 44a of No. 4. In the flow cell 44,
An immobilized enzyme membrane 46 is attached to cover the concave surface 44a,
It is fixed by an O ring 47.
フローセル４４は、キャップ４８により外ケース４１に
取付けられる。この時、外ケース凸面４１ａがフローセ
ル凹面４４ａに嵌まり、固定化酵素膜４６が作用電極４
２及び対照電極４３に密着する。この状態で試料を試料
流路４５内に流すと共に、作用電極４２、対照電極４３
間に所定の電圧を印加すれば、試料中の基質濃度に比例
した電流（電極出力）が得られる。Flow cell 44 is attached to outer case 41 by cap 48 . At this time, the outer case convex surface 41a fits into the flow cell concave surface 44a, and the immobilized enzyme membrane 46 is attached to the working electrode 4.
2 and the reference electrode 43. In this state, the sample is allowed to flow into the sample channel 45, and the working electrode 42 and the reference electrode 43
By applying a predetermined voltage between them, a current (electrode output) proportional to the substrate concentration in the sample can be obtained.
（ハ）発明が解決しようとする課題
上記従来のフローセル付き酵素電極では、以下に列挙す
る問題点を有している。(c) Problems to be Solved by the Invention The above-mentioned conventional enzyme electrode with a flow cell has the following problems.
■固定化酵素膜４６をＯリング４７で固定するため、固
定化酵素膜４６の脱着が煩雑で、交換時に固定化酵素膜
４６が破損してしまう。この問題点を解決するために、
固定化酵素膜４６が予め装着されたフロ−セル４４全体
を交換することも考えられるが、フローセル４４はＦＪ
、雑な構造の部材であるから、ツーセルの使い捨ては大
幅なコスト上昇を生じさせる。(2) Since the immobilized enzyme membrane 46 is fixed with an O-ring 47, it is complicated to attach and detach the immobilized enzyme membrane 46, and the immobilized enzyme membrane 46 will be damaged during replacement. In order to solve this problem,
It is also conceivable to replace the entire flow cell 44 on which the immobilized enzyme membrane 46 is attached in advance, but the flow cell 44 is
Since it is a member with a crude structure, disposable two cells will cause a significant increase in cost.
■外ケース４１とフローセル４４の脱着を繰返すと、や
はり固定化酵素１ｆ１６の破損を生じる。(2) If the outer case 41 and the flow cell 44 are repeatedly attached and detached, the immobilized enzyme 1f16 will be damaged.
■フローセル凹面４４ａよりもさらに大きな固定化酵素
膜４６（例えば直径１５＋Ｍ以上）を必要とするが、実
際に酵素反応に必要な部分は試料流路４５に対応する部
分だけであり、効率が悪く固定化酵素膜４６が高価なも
のとなる。■Although it requires an immobilized enzyme membrane 46 that is larger than the flow cell concave surface 44a (for example, diameter 15+M or more), the only part actually required for the enzyme reaction is the part corresponding to the sample flow path 45, and the immobilization is inefficient. The converting enzyme membrane 46 becomes expensive.
■固定化酵素膜４６と電極４２・４３との密着性が悪く
両者の間に間隙が生じやすい。このため電極出力にノイ
ズが見られ、測定精度が低下してしまう。また、この密
着性は装着のたびごとに異なるから、電極出力が変動し
再現性が低下してしまう。(2) The adhesion between the immobilized enzyme membrane 46 and the electrodes 42 and 43 is poor, and gaps are likely to form between them. For this reason, noise is seen in the electrode output, reducing measurement accuracy. Furthermore, since this adhesion differs each time it is worn, the electrode output fluctuates and reproducibility decreases.
■固定化酵素膜４６とフローセル４４との間で試料流路
４５より試料が流出することがあり、測定精度が低下す
る。(2) The sample may flow out from the sample channel 45 between the immobilized enzyme membrane 46 and the flow cell 44, resulting in a decrease in measurement accuracy.
■外ケース凸面４ｔａとフローセル凹面４４ａが一部す
るように製造するためには、高度な技？、テををし、加
工費が増大する。■In order to manufacture the outer case convex surface 4ta and the flow cell concave surface 44a partially, it is necessary to use advanced techniques. , and the processing cost increases.
■複数項目の同時測定が困難である。■It is difficult to measure multiple items simultaneously.
この発明は上記に鑑みなされたもので、■〜■の問題点
を解決できるフローセル付き酵素電極の提供を目的とし
ている。This invention was made in view of the above, and aims to provide an enzyme electrode with a flow cell that can solve the problems (1) to (3).
（ニ）課題を解決するための手段及び作用上記課題を解
決するため、この発明のフローセル付き酵素電極は、絶
縁基板上に電極と、この電極を被覆する固定化酵素膜と
を形成し、電極の一部を露出させて接点部としてなる酵
素電極と、この酵素電極を支持する支持部材と
この支持部材上に支持された酵素電極に密着し、この酵
素電極の固定化酵素膜に接する試料流路と、酵素電極の
接点部に接触する接点端子とを備えてなるフローセル部
材と、
このフローセル部材と前記支持部材の密着状態を保持す
る密着手段とよりなるものである。(d) Means and action for solving the problems In order to solve the above problems, the enzyme electrode with a flow cell of the present invention has an electrode formed on an insulating substrate and an immobilized enzyme membrane covering the electrode. An enzyme electrode with a part exposed to serve as a contact part, a support member that supports this enzyme electrode, and a sample flow that is in close contact with the enzyme electrode supported on this support member and in contact with the immobilized enzyme membrane of this enzyme electrode. and a flow cell member comprising a contact terminal that contacts a contact portion of an enzyme electrode; and a contact means for maintaining the flow cell member and the support member in close contact.
この発明では、酵素電極が平板状で固定化酵素膜が一体
的に形成されている。このため固定化酵素膜の装着の手
間が不要となり、固定化酵素膜を破損する危険性が少な
い。また、酵素電極自体が小型で、固定化酵素膜も電極
を被覆する大きさだけがあればよく、酵素の効率がよく
安価であり、使い捨ても可能となる。さらに、固定化酵
素膜と電極との密着が安定しており、電極出力の安定及
び低ノイズ化を図ることができる。In this invention, the enzyme electrode is flat and the immobilized enzyme membrane is integrally formed. This eliminates the need to attach the immobilized enzyme membrane and reduces the risk of damaging the immobilized enzyme membrane. In addition, the enzyme electrode itself is small, and the immobilized enzyme membrane only needs to be large enough to cover the electrode, making the enzyme efficient, inexpensive, and disposable. Furthermore, the adhesion between the immobilized enzyme membrane and the electrode is stable, making it possible to stabilize the electrode output and reduce noise.
酵素電極は上にも述べたように平板状であり、フローセ
ル部材と支持部材の密着面は共に平面とすることができ
るから、酵素電極のフローセル部材への密着を完全にし
試料流路よりの試料の漏れが防止できると共に、フロー
セル部材、支持部材の加工が容易となる。また、上記密
着面に複数の酵素電極が配置できるから、複数項目の測
定も容易となる。As mentioned above, the enzyme electrode has a flat plate shape, and the contact surfaces of the flow cell member and the support member can both be flat, so that the enzyme electrode can be completely contacted with the flow cell member and the sample from the sample flow path can be made flat. In addition to preventing leakage, processing of the flow cell member and support member becomes easy. Furthermore, since a plurality of enzyme electrodes can be arranged on the contact surface, measurement of a plurality of items becomes easy.
（ホ）実施例
〈実施例１〉
この発明の第１の実施例を第１図乃至第５図に基づいて
以下に説明する。(e) Example (Example 1) A first example of the present invention will be described below based on FIGS. 1 to 5.
まず、第５図を参照しながら酵素電極１０を説明する。First, the enzyme electrode 10 will be explained with reference to FIG.
２は、絶縁基板であり、例えば厚さ０．１２５ｍｍのプ
ラスチックフィルムが使用される。絶縁基板２表面には
、電極材３．４がスパッタリングや真空蒸着等により薄
膜形成されており、この実施例では電極材３．４として
白金（Ｐｔ）を使用している。2 is an insulating substrate, for example, a plastic film with a thickness of 0.125 mm is used. A thin film of electrode material 3.4 is formed on the surface of the insulating substrate 2 by sputtering, vacuum deposition, etc., and in this embodiment, platinum (Pt) is used as the electrode material 3.4.
絶縁基板２表面には、電極材３．４を被覆するように絶
縁性保３１ｒｆｉ、５が形成される。この絶縁性保護膜
５には、感光性樹脂が使用されホトリソグラフィを適用
してバターニングし、電極材３．４の２つの部分をそれ
ぞれ露出させて作用電極３ａ、対照電極４ａ、接点部３
ｂ、４ｂとする。An insulating layer 31rfi, 5 is formed on the surface of the insulating substrate 2 so as to cover the electrode material 3.4. This insulating protective film 5 is made of a photosensitive resin, which is patterned using photolithography to expose the two parts of the electrode material 3.4, the working electrode 3a, the reference electrode 4a, and the contact part 3.
b, 4b.
絶縁基板２表面には、さらに固定化酵素膜６が形成され
、作用電極３ａ、対照電極４ａが被覆される。固定化酵
素膜６は、まずアセチルセルロース膜６ａをスピン法で
形成し、作用電極３ａ上・に酵素溶液６ｂを滴下して風
乾させ、さらにアセチルセルロース膜６ｃをスピン法で
形成してなるものである。酵素溶液は、例えばグルコー
スオキシダーゼ（ＣＯＤ）の場合には、ＣＯＤ ２ ｍ
ｇを０．１Ｍリン酸援衝液（ｐＨ７，０）１００ａｆに
溶解したものを、同じ緩衝液で調製した０、５％グルタ
ルアルデヒド溶液とを混合したものを用いる。An immobilized enzyme film 6 is further formed on the surface of the insulating substrate 2, covering the working electrode 3a and the control electrode 4a. The immobilized enzyme membrane 6 is formed by first forming an acetylcellulose membrane 6a by a spin method, dropping an enzyme solution 6b onto the working electrode 3a and air drying it, and then forming an acetylcellulose membrane 6c by a spin method. be. For example, in the case of glucose oxidase (COD), the enzyme solution is COD 2 m
g dissolved in 100af of 0.1M phosphate buffer (pH 7,0) and mixed with 0.5% glutaraldehyde solution prepared in the same buffer.
さて、第１図は、この第１の実施例に係るフローセル付
き酵素電極１の分解斜視図である。Now, FIG. 1 is an exploded perspective view of an enzyme electrode 1 with a flow cell according to the first embodiment.
１１は、アクリル板（支持部材）であり、この第１図で
は裏返した状態で示されている。蓋板１１の下面１１ａ
には、シリコンシート〔例えば厚さ０．１３ｍｍの５Ｉ
ＬＡＳＴＩＣ（登録商標）シー）ティング・ダウ、コー
ニング社製が使用される）１２を介して、上記酵素電極
１が装着される。シリコンシート１２は弾性を有してお
り、アクリル板１１と酵素電極１０との密着性を高める
。Reference numeral 11 denotes an acrylic plate (supporting member), which is shown upside down in FIG. Lower surface 11a of cover plate 11
For example, a silicone sheet [for example, 5I with a thickness of 0.13 mm] is used.
The enzyme electrode 1 is attached via a LASTIC (registered trademark) sheeting dow (made by Corning Inc.) 12. The silicone sheet 12 has elasticity and enhances the adhesion between the acrylic plate 11 and the enzyme electrode 10.
一方、フローセル１３には、試料流路１４が形成されて
おり、その両端部にはチューブライン１７ａ、１７ｂが
それぞれ接続される。フローセル上面１３ａには、この
試料流路１４が露出すると共に、接点金具（接点端子）
１５．１５が設けられている。接点金具１５．１５から
は、リード線１６が引出されている。１８．１８は、そ
れぞれ密着具であり、その凹溝１８ａ、１８ａ内には板
バネＩ９、！９がそれぞれ設けられている。On the other hand, a sample channel 14 is formed in the flow cell 13, and tube lines 17a and 17b are connected to both ends of the sample channel 14, respectively. The sample channel 14 is exposed on the upper surface 13a of the flow cell, and a contact fitting (contact terminal)
15.15 is provided. A lead wire 16 is drawn out from the contact fitting 15.15. 18. 18 is a contact tool, and inside the grooves 18a, 18a are plate springs I9, ! 9 are provided respectively.
第２図は、この実施例フローセル付き酵素電極１を組立
てた状態の外観斜視図、第３図は、この状態での縦断面
図を示している。この状態では、アクリル板１１とフロ
ーセル１３とが合わされ、両者側縁１１ｂ、１３ｂに密
着具１８が装着されている。密着具１８の板バネ１９の
力により、アクリル板１１とフローセル１３が密着させ
られる。FIG. 2 is a perspective view of the assembled state of the flow cell-equipped enzyme electrode 1 of this embodiment, and FIG. 3 is a longitudinal cross-sectional view of this state. In this state, the acrylic plate 11 and the flow cell 13 are put together, and the contact tool 18 is attached to the side edges 11b and 13b of both. The acrylic plate 11 and the flow cell 13 are brought into close contact with each other by the force of the leaf spring 19 of the contact tool 18.
この時、酵素電極ＩＯがフローセル上面１３ａに密着し
、試料流路１４の露出部が固定化酵素膜６に接する。一
方、酵素電極１０の接点部３ｂ、４ｂには、接点端子１
５．１５が圧接し、リード線１６と、作用電極３ａ、対
照電極４ａとの導道がとられる。At this time, the enzyme electrode IO is in close contact with the upper surface 13a of the flow cell, and the exposed portion of the sample channel 14 is in contact with the immobilized enzyme membrane 6. On the other hand, contact terminals 1
5.15 are brought into pressure contact, and a conductive path is established between the lead wire 16, the working electrode 3a, and the reference electrode 4a.
第４図は、実施例フローセル付き酵素電極ｒを用いた測
定システムを示している。リード線１６は、測定機器２
１に接続され、作用電極３ａ、対照電極４ａ間に所定の
電圧（この実施例の場合には０．５５Ｖ〜０．８Ｖ）が
印加されて、電極出力が測定される。FIG. 4 shows a measurement system using an enzyme electrode r with an embodiment flow cell. The lead wire 16 is connected to the measuring device 2
1, a predetermined voltage (0.55V to 0.8V in this example) is applied between the working electrode 3a and the reference electrode 4a, and the electrode output is measured.
チューブライン１７ａは、リン酸緩衝液（０，１Ｍ、ｐ
Ｈ７，０）容器２２又は試料容器２３に接続できる。一
方、チューブライン１７ｂは、ミニポンプ２４に接続さ
れ、リン酸緩衝液又は試料がフローセル１３の試料流路
１４を通って、このミニポンプ２４に吸引される。ミニ
ポンプ２４より排出されたリン酸緩衝液又は試料は、廃
液ボトル２５へ導かれる。The tube line 17a contains phosphate buffer (0.1M, p
H7,0) Can be connected to container 22 or sample container 23. On the other hand, the tube line 17b is connected to a mini-pump 24, and the phosphate buffer or sample passes through the sample channel 14 of the flow cell 13 and is sucked into the mini-pump 24. The phosphate buffer or sample discharged from the mini pump 24 is guided to a waste liquid bottle 25.
試料が試料流路１４内を流れる時、固定化酵素膜６に接
触する。固定化酵素膜６内では、の反応が生じ、生成し
た１１２０□濃度に応じた電極出力が得られる。この電
極出力に後述の第９図に示す検量線を適用し、試料中の
グルコース濃度を知ることができる。When the sample flows through the sample channel 14, it comes into contact with the immobilized enzyme membrane 6. A reaction occurs within the immobilized enzyme membrane 6, and an electrode output corresponding to the generated 1120□ concentration is obtained. By applying a calibration curve shown in FIG. 9, which will be described later, to this electrode output, the glucose concentration in the sample can be determined.
〈実施例２〉
この発明の第２の実施例を、第６図乃至第９図に基づい
て以下に説明する。<Example 2> A second example of the present invention will be described below based on FIGS. 6 to 9.
この第２の実施例に係るフローセル付き酵素電極３０は
、グルコースとシェフロース（蔗ＩＮ）の２つの項目の
測定に適用されるものであり、前述のＣＯＤを用いた酵
素電極ｌＯと、インベルターゼ、ムタロターゼ及びＣＯ
Ｄの三種の酵素を用いた同様の酵素電極１０’を使用す
る。The enzyme electrode 30 with a flow cell according to the second embodiment is applied to the measurement of two items, glucose and chefulose (potato IN), and includes the enzyme electrode IO using the above-mentioned COD, invertase, Mutarotase and CO
A similar enzyme electrode 10' using three types of enzymes in D is used.
第６図は、実施例フローセル付き酵素電極３０の分解斜
視図であり、アクリル板（支持部材）３１の上下を裏返
して示している。アクリル板下面３１ａには、シリコン
シート３２を介して酵素電極１０．１０°が並べて装着
される。FIG. 6 is an exploded perspective view of the enzyme electrode 30 with a flow cell according to the embodiment, and shows the acrylic plate (supporting member) 31 upside down. Enzyme electrodes 10.10° are attached to the lower surface 31a of the acrylic plate with a silicone sheet 32 interposed therebetween.
一方、フローセル３３には、アクリル板３１が嵌合する
凹部３３ａが形成される。この凹部３３ａの面３３ｂ内
には、試料流路３４が露出すると共に、２対の接点金具
３５、・・・ ３５が設けられ、リード線３６が接続さ
れる。試料流路３４は、フローセル側面３３ｃ、３３ｃ
において、チューブライン３７ａ、３７ｂが接続される
。また、接点金具３５、・・・、３５には、リード線３
６が電気的に接続している。フローセル上面３３ｄには
、固定具（密着手段）３９取り付は用ねじ３８．３８が
螺入されている。On the other hand, the flow cell 33 is formed with a recess 33a into which the acrylic plate 31 is fitted. In the surface 33b of the recess 33a, the sample flow path 34 is exposed, and two pairs of contact fittings 35, . . . , 35 are provided, to which a lead wire 36 is connected. The sample channel 34 is connected to the flow cell side surfaces 33c, 33c.
At this point, tube lines 37a and 37b are connected. In addition, the contact fittings 35, . . . , 35 have lead wires 3
6 is electrically connected. Screws 38 and 38 for attaching a fixture (closing means) 39 are screwed into the upper surface 33d of the flow cell.
第７図は、実施例フローセル付き酵素電極３０を組立て
た状態での外観斜視図、第８図は、その縦断面図である
。アクリル板３１は、フローセル凹部３３ａに嵌合し、
固定具３９で押さえ付けられ、フローセル３３に固定さ
れる。この時、第８図に示すように酵素電極１０．１０
”が面３３ｂに密着し、酵素電極１０、ｌＯｏのそれぞ
れの固定化酵素膜６．６”が試料流Ｂ３４の露出部を覆
う。また、接点金具３５、・・・ ３５が酵素電極１０
、ｌＯ”の接点部３ｂ、４ｂ、３° ｂ、４° ｂにそ
れぞれ接触し、酵素電極１０．１０゛がリード線３６に
導通する。FIG. 7 is a perspective view of the assembled enzyme electrode 30 with a flow cell according to the embodiment, and FIG. 8 is a longitudinal sectional view thereof. The acrylic plate 31 fits into the flow cell recess 33a,
It is pressed down with a fixture 39 and fixed to the flow cell 33. At this time, as shown in FIG.
" is in close contact with the surface 33b, and each of the immobilized enzyme membranes 6.6" of the enzyme electrode 10 and lOo covers the exposed portion of the sample flow B34. In addition, the contact fittings 35, . . . 35 are the enzyme electrodes 10.
, lO'' contact portions 3b, 4b, 3°b, and 4°b, respectively, and the enzyme electrodes 10 and 10'' are electrically connected to the lead wire 36.
このフローセル付き酵素膜ｉ３０も、第４図に示すもの
と全く同様の測定システムを適用することができる。両
酵素電極１０．１０″には、それぞれ０．５５〜０．８
ｖの電圧が印加される。なお、試料流路中央部３４ａに
おいて、酵素電極１０．１０“のいずれにも覆われない
開放した部分があり、この部分により試料が漏れるおそ
れがあるように第８図では見られるが、実際には酵素電
極１０．１０″は極めて薄いものであり（第３図、第５
図、第８図では、酵素電極の厚さが誇張して描かれてい
る）。また、試料流路３４内の試料は、ミニポンプ２４
に引かれて流動するので、試料が漏れることはまずない
。A measurement system completely similar to that shown in FIG. 4 can be applied to this enzyme membrane i30 with a flow cell. Both enzyme electrodes 10.10″ each have 0.55 to 0.8
A voltage of v is applied. In addition, in the center part 34a of the sample flow path, there is an open part that is not covered by any of the enzyme electrodes 10, 10'', and although it can be seen in FIG. 8 that there is a risk of the sample leaking due to this part, in reality The enzyme electrode 10.10'' is extremely thin (Fig. 3, 5).
In FIG. 8, the thickness of the enzyme electrode is exaggerated). In addition, the sample in the sample flow path 34 is transferred to the mini pump 24.
The sample is unlikely to leak because it is attracted by the fluid and flows.
さて、酵素電極１０の固定化酵素膜６内では、前述の（
１）式で表される反応が起こり、電極出力に第９図の検
量線を適用して、試料のグルコース濃度を知ることがで
きる。Now, within the immobilized enzyme membrane 6 of the enzyme electrode 10, the above-mentioned (
1) The reaction expressed by the formula occurs, and by applying the calibration curve of FIG. 9 to the electrode output, the glucose concentration of the sample can be determined.
一方、酵素電極１０°の固定化酵素膜６°内では、以下
の（２）〜（４）式で表される反応が生じる。On the other hand, within 6 degrees of the immobilized enzyme membrane of 10 degrees of the enzyme electrode, reactions expressed by the following formulas (2) to (4) occur.
α−Ｄ−グルコース＋フルクトース ・・・（２）α−
Ｄ−グルコース
ムクロターゼ
β−Ｄ−グルコース ・・・（３）
β−Ｄ−グルコース
ＯＤ
グルコン酸＋Ｈ２０□ ・・・（４）（４）式で生成
するｔｉ ２ｏ□の濃度に応じた電極出力が酵素電極１
０°で得られる。しかし、（４）式の反応に関与するグ
ルコースには、（２）（３）式の反応で生成したものだ
けではなく、試料中に初めから含まれているものも含ま
れる。従って、酵素電極１０’の電極出力より、酵素電
極１０の電極出力を減じた電極出力差が、試料中のシュ
クロース濃度に対応する。第９図中には、電極出力差に
対するシュクロース濃度の検量線が示されている。α-D-glucose + fructose ... (2) α-
D-glucose mucrotase β-D-glucose ... (3) β-D-glucose OD gluconic acid + H20□ ... (4) The electrode output according to the concentration of ti2o□ generated by the formula (4) is Enzyme electrode 1
Obtained at 0°. However, the glucose involved in the reaction of formula (4) includes not only that produced in the reaction of formulas (2) and (3), but also glucose that is originally contained in the sample. Therefore, the electrode output difference obtained by subtracting the electrode output of the enzyme electrode 10 from the electrode output of the enzyme electrode 10' corresponds to the sucrose concentration in the sample. FIG. 9 shows a calibration curve of sucrose concentration versus electrode output difference.
（へ）発明の詳細
な説明したように、この発明のフローセル付き酵素電極
は、絶！！基板上に電極と、この電極を被覆する固定化
酵素膜とを形成し、電極の一部を露出させて接点部とし
てなる酵素電極と、この酵素電極を支持する支持部材と
この支持部材上に支持された酵素電極に密着し、この酵
素電極の固定化酵素膜に接する試料流路と、酵素電極の
接点部に接触する接点端子とを備えてなるフローセル部
材と、このフローセル部材と前記支持部材の密着を保持
する密着手段とよりなるものである。(to) As explained in detail, the enzyme electrode with a flow cell of this invention is absolutely perfect! ! An electrode and an immobilized enzyme membrane covering this electrode are formed on a substrate, and a part of the electrode is exposed to serve as a contact part, an enzyme electrode, a support member that supports this enzyme electrode, and a support member that is placed on this support member. A flow cell member comprising a sample channel that closely contacts a supported enzyme electrode and an immobilized enzyme membrane of the enzyme electrode, and a contact terminal that contacts a contact portion of the enzyme electrode; the flow cell member and the support member; and an adhesion means for maintaining adhesion.
よって、酵素電極のコストを低減し、その使い捨てを可
能とすると共に、固定化酵素膜の脱着の必要がなくその
破損を防止することができる。また、電極と固定化酵素
膜との密着性が向上かつ安定化し、電極出力の低ノイズ
化及び安定化が図られ、測定精度及び再現性を向上させ
ることが可能となる。さらに、フローセル部材に特殊な
加工が要求されず、加工費の増大がおさえられると共に
、試料の漏れも防止される。加えて、複数項目の測定も
容易に行うことができる。Therefore, the cost of the enzyme electrode can be reduced, it can be made disposable, and there is no need to detach the immobilized enzyme membrane, thereby preventing its damage. In addition, the adhesion between the electrode and the immobilized enzyme membrane is improved and stabilized, the noise of the electrode output is reduced and stabilized, and measurement accuracy and reproducibility can be improved. Furthermore, no special processing is required for the flow cell member, which reduces processing costs and prevents sample leakage. In addition, multiple items can be easily measured.
第１図は、この発明の第１の実施例に係るフローセル付
き酵素電極の分解斜視図、第２図は、同フローセル付き
酵素電極の外観斜視図、第３図は、同フローセル付き酵
素電極の縦断面図、第４図は、同フローセル付き酵素電
極の適用される測定システムを説明する図、第５図（ａ
）は、同フローセル付き酵素電極に適用される酵素電極
の平面図、第５図（ｂ）及び第５図（Ｃ）は、それぞれ
同酵素電極の第５図（ａ）中Ｂ−Ｂ線、Ｃ−Ｃ線におけ
る断面図、第６図は、この発明の第２の実施例に係るフ
ローセル付き酵素電極の分解斜視図、第７図は、同フロ
ーセル付き酵素電極の外観斜視図、第８図は、同フロー
セル付き酵素電極の縦断面図、第９図は、同フローセル
付き酵素電極のグルコース及びシュクロースの検量線を
説明する図、第１０図は、従来のフローセル付き酵素電
極を説明する図である。
２：絶縁基板、 ３ａ：作用電極、４ａ：対照電極
、
３ｂ・４ｂ・３’ ｂ・４° ｂ：接点部、６・６°
：固定化酵素膜、
１０・１０゛ ：酵素電極、
１１・３１ニアクリル板、
１３・３３：フローセル、
１４・３４：試料流量、１５・３５：接点金具、１８：
密着具、 ３９：固定具。FIG. 1 is an exploded perspective view of an enzyme electrode with a flow cell according to the first embodiment of the present invention, FIG. 2 is an external perspective view of the enzyme electrode with a flow cell, and FIG. 3 is an exploded perspective view of the enzyme electrode with a flow cell. A vertical cross-sectional view, FIG. 4 is a diagram illustrating a measurement system to which the enzyme electrode with flow cell is applied, and FIG.
) is a plan view of the enzyme electrode applied to the enzyme electrode with flow cell, and FIG. 5(b) and FIG. 5(C) are the BB line in FIG. 5(a) of the enzyme electrode, respectively. 6 is an exploded perspective view of an enzyme electrode with a flow cell according to a second embodiment of the present invention; FIG. 7 is an external perspective view of the enzyme electrode with a flow cell; FIG. 9 is a diagram illustrating the calibration curves of glucose and sucrose for the enzyme electrode equipped with a flow cell, and FIG. 10 is a diagram illustrating a conventional enzyme electrode equipped with a flow cell. It is. 2: Insulating substrate, 3a: Working electrode, 4a: Control electrode, 3b, 4b, 3' b, 4° b: Contact part, 6, 6°
: Immobilized enzyme membrane, 10/10゛: Enzyme electrode, 11/31 Niacrylic plate, 13/33: Flow cell, 14/34: Sample flow rate, 15/35: Contact fitting, 18:
Adhering tool, 39: Fixing tool.
Claims (1)
酵素膜とを形成し、電極の一部を露出させて接点部とし
てなる酵素電極と、 この酵素電極を支持する支持部材と この支持部材上に支持された酵素電極に密着し、この酵
素電極の固定化酵素膜に接する試料流路と、酵素電極の
接点部に接触する接点端子とを備えてなるフローセル部
材と、 このフローセル部材と前記支持部材の密着状態を保持す
る密着手段とよりなるフローセル付き酵素電極。(1) An enzyme electrode formed by forming an electrode and an immobilized enzyme membrane covering the electrode on an insulating substrate and exposing a part of the electrode to serve as a contact part; a support member supporting the enzyme electrode; A flow cell member comprising: a sample channel that is in close contact with an enzyme electrode supported on a support member and in contact with an immobilized enzyme membrane of the enzyme electrode; and a contact terminal that is in contact with a contact portion of the enzyme electrode; and a contact means for maintaining the contact state of the support member.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1012273A JPH02193054A (en) | 1989-01-21 | 1989-01-21 | Enzyme electrode with flow cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1012273A JPH02193054A (en) | 1989-01-21 | 1989-01-21 | Enzyme electrode with flow cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02193054A true JPH02193054A (en) | 1990-07-30 |
Family
ID=11800759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1012273A Pending JPH02193054A (en) | 1989-01-21 | 1989-01-21 | Enzyme electrode with flow cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02193054A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07270377A (en) * | 1994-03-31 | 1995-10-20 | Toto Ltd | Polarograph analyser equipped with disposable flow cell |
| US5730149A (en) * | 1993-12-30 | 1998-03-24 | Toto Ltd. | Toilet-bowl-mounted urinalysis unit |
| KR100741270B1 (en) * | 2005-07-05 | 2007-07-19 | 학교법인 포항공과대학교 | Lab-on-a-chip for electrochemical analysis having dual fluid flow channel |
-
1989
- 1989-01-21 JP JP1012273A patent/JPH02193054A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5730149A (en) * | 1993-12-30 | 1998-03-24 | Toto Ltd. | Toilet-bowl-mounted urinalysis unit |
| JPH07270377A (en) * | 1994-03-31 | 1995-10-20 | Toto Ltd | Polarograph analyser equipped with disposable flow cell |
| KR100741270B1 (en) * | 2005-07-05 | 2007-07-19 | 학교법인 포항공과대학교 | Lab-on-a-chip for electrochemical analysis having dual fluid flow channel |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5707502A (en) | Sensors for measuring analyte concentrations and methods of making same | |
| US7455756B2 (en) | Biosensor | |
| AU6482198A (en) | Electrochemical biosensor | |
| JP2002090331A (en) | Biosensor provided with porous thin film having chromatography function | |
| US5507936A (en) | Member for the formation of at least one electrode and/or one sensor | |
| WO2003023389A9 (en) | Solid electrochemical sensor | |
| CN107957440B (en) | Planar ammonia selective sensing electrode and method for fabricating the same | |
| Yang et al. | Biosensors and their Applications | |
| Urban et al. | Performance of integrated glucose and lactate thin-film microbiosensors for clinical analysers | |
| Hintsche et al. | Microbiosensors using electrodes made in Si-technology | |
| CN112292446A (en) | Glycated protein sensor, measurement method, program, and method for producing sensor | |
| JPH02193054A (en) | Enzyme electrode with flow cell | |
| US20010024804A1 (en) | Disposable plate electrode with biologicalactive film and manufacture method thereof | |
| JPS6034061B2 (en) | electrode device | |
| AU2002366139A1 (en) | Measurement device for measuring electric signal emitted by biological sample | |
| JPH06109687A (en) | Flow cell for electrochemical measurement | |
| JPS59206756A (en) | Fet chemical sensor combined with reference electrode | |
| US5573649A (en) | Miniaturized oxygen electrode and process of producing same | |
| JPH03160358A (en) | Enzyme sensor | |
| Monroe | Potentiometric (bioselective electrodes) assay systems: Utility and limitations | |
| JPS6258154A (en) | Enzymatic function electrode | |
| CS274231B1 (en) | Regenerable enzyme sensor with exchangeable membrane system | |
| JPH04194661A (en) | Enzyme electrode | |
| JPH01203959A (en) | Enzyme-immobilized fet sensor | |
| JPS6457159A (en) | Biosensor |