JPH0332743B2 - - Google Patents
Info
- Publication number
- JPH0332743B2 JPH0332743B2 JP57177243A JP17724382A JPH0332743B2 JP H0332743 B2 JPH0332743 B2 JP H0332743B2 JP 57177243 A JP57177243 A JP 57177243A JP 17724382 A JP17724382 A JP 17724382A JP H0332743 B2 JPH0332743 B2 JP H0332743B2
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- glucose
- platinum
- platinum layer
- wire mesh
- 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
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 12
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 229910052751 metal Inorganic materials 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 239000011810 insulating material Substances 0.000 claims 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 42
- 229910052697 platinum Inorganic materials 0.000 description 21
- 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 11
- 239000008103 glucose Substances 0.000 description 11
- 239000010409 thin film Substances 0.000 description 10
- 108010015776 Glucose oxidase Proteins 0.000 description 7
- 239000004366 Glucose oxidase Substances 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 229940116332 glucose oxidase Drugs 0.000 description 7
- 235000019420 glucose oxidase Nutrition 0.000 description 7
- 229910001220 stainless steel Inorganic materials 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 108010093096 Immobilized Enzymes Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000010935 stainless steel Substances 0.000 description 3
- 238000003466 welding Methods 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- 229910021607 Silver chloride Inorganic materials 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 2
- 108010089254 Cholesterol oxidase Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005868 electrolysis reaction Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920006254 polymer film Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000007665 sagging Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000005476 soldering Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (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】
産業上の利用分野
本発明は、固定化酵素電極を利用したバイオセ
ンサの改良に関するものである。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an improvement in a biosensor using an immobilized enzyme electrode.
従来例の構成とその問題点
従来のバイオセンサの中で、もつとも応答が迅
速で、高性能なのは、固定化酵素を利用した酵素
電極である。酵素電極の１つとして、グルコース
センサに例をとると、白金層を有する多孔質性薄
膜（第１の電極）と、白金層を有し、かつグルコ
ースオキシダーゼを固定化した多孔質性薄膜（第
２の電極）から構成されていた。Configuration of conventional examples and their problems Among conventional biosensors, the one with the quickest response and highest performance is an enzyme electrode that uses immobilized enzymes. Taking a glucose sensor as an example of an enzyme electrode, a porous thin film (first electrode) having a platinum layer and a porous thin film (first electrode) having a platinum layer and immobilized glucose oxidase are used. It consisted of two electrodes).
その電極の拡大断面模式図を第１図に示した。
同図において、１は第１の電極で、高分子フイル
ムなどの多孔質性薄膜２の片面に白金層３を設け
たものである。４は第２の電極で、多孔質性薄膜
５の片面に白金層６を設け、反対側及び細孔内に
グルコースオキシダーゼ７を固定化している。 An enlarged schematic cross-sectional view of the electrode is shown in FIG.
In the figure, reference numeral 1 denotes a first electrode, which has a platinum layer 3 provided on one side of a porous thin film 2 such as a polymer film. Reference numeral 4 denotes a second electrode, in which a platinum layer 6 is provided on one side of a porous thin film 5, and glucose oxidase 7 is immobilized on the other side and within the pores.
グルコースオキシダーゼにより被検液中のグル
コースが酸化され、生成したH2O2は白金層６で
さらに酸化され、その酸化電流値によりグルコー
スの濃度を検知する。 Glucose in the test liquid is oxidized by glucose oxidase, and the generated H 2 O 2 is further oxidized in the platinum layer 6, and the concentration of glucose is detected based on the oxidation current value.
その際、被検液中に含まれ白金層で酸化される
アスコルビン酸や尿酸を第１の電極の白金層３で
除去するしくみになつている。しかし、多孔質性
薄膜は、被検液を早く拡散させる必要があるた
め、非常に薄いものが用いられる。 At this time, ascorbic acid and uric acid contained in the test liquid and oxidized by the platinum layer are removed by the platinum layer 3 of the first electrode. However, since the porous thin film needs to quickly diffuse the test liquid, a very thin porous film is used.
そのため強度が弱く、取扱いに注意を要し、製
造上困難な点が多々生じていた。又、各白金層
３，６からリードを取る場合、白金板に圧着した
り、銀ペーストによる接続が試みられているが、
接触が不安定で、確実な応答を得ることが困難で
あつた。これらの問題点はグルコースセンサに限
らずコレステロールやイノシンなど生体関連物質
を検知するセンサにもあてはまり、改良が望まれ
ている。 As a result, it has low strength, requires careful handling, and has many manufacturing difficulties. Furthermore, when taking leads from each of the platinum layers 3 and 6, attempts have been made to bond them to a platinum plate or connect them using silver paste.
Contact was unstable and it was difficult to obtain a reliable response. These problems apply not only to glucose sensors but also to sensors that detect biological substances such as cholesterol and inosine, and improvements are desired.
発明の目的
本発明は、上記多孔質性薄膜による製造上の難
点を克服し、簡易にかつ確実に応答の得られる高
性能のバイオセンサを提供することを目的とす
る。OBJECTS OF THE INVENTION It is an object of the present invention to overcome the manufacturing difficulties of the above-mentioned porous thin film and to provide a high-performance biosensor that can easily and reliably obtain a response.
発明の構成
本発明は、上記の目的を達成するため、多孔質
性薄膜のかわりに導電性の多孔性金属電極、たと
えば金網を用いることを特徴とする。さらに具体
的には、従来の妨害物質除去用の第１の電極のか
わりに白金、ステンレス鋼などのように耐食性を
有する導電性の多孔性金属電極を用い、生体関連
物質、たとえばグルコースに応答する第２の電極
のかわりに、測定対象の生体関連物質に特異的に
働き過酸化水素が反応系に関与する酵素、たとえ
ばグルコースオキシダーゼを固定化した過酸化水
素分解能を有する導電性の多孔性電極を用いる。
各々の電極からは、スポツト溶接やハンダ付けに
よりリードをとる。Structure of the Invention In order to achieve the above object, the present invention is characterized in that a conductive porous metal electrode, such as a wire mesh, is used in place of the porous thin film. More specifically, instead of the conventional first electrode for removing interfering substances, a conductive porous metal electrode such as platinum or stainless steel is used to respond to biologically related substances, such as glucose. Instead of the second electrode, an electrically conductive porous electrode with hydrogen peroxide decomposition ability immobilized with an enzyme, such as glucose oxidase, which acts specifically on the biological substance to be measured and in which hydrogen peroxide participates in the reaction system, is used. use
Leads are taken from each electrode by spot welding or soldering.
ここに用いる金網などの導電性の多孔性電極
は、多孔質薄膜に比べて強度も充分であるから、
製造が簡単になり、かつリードも確実にとること
ができる。又、多孔質性薄膜では白金層上で数Ω
の抵抗があつたが、金網などでは導電性が良く、
かつ反応面積が増加するため、感度を向上させる
ことができる。 The conductive porous electrodes used here, such as wire mesh, have sufficient strength compared to porous thin films.
Manufacture is simplified and leads can be taken reliably. In addition, in porous thin films, the resistance of several Ω on the platinum layer
There was some resistance, but wire mesh has good conductivity,
In addition, since the reaction area increases, sensitivity can be improved.
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
について説明する。Description of Examples A glucose sensor will be described as one of the biosensors.
第１の電極および第２の電極の担体として、厚
さ80ミクロン、直径10mmで目の粗さ2000メツシユ
のステンレス鋼製の円板状の金網を用いる。この
金網の両面にスパツタリング法により、厚さ数百
〜数千オングストロームの白金層を形成し、第１
電極とした。第２の電極は、上記と同様の電極に
濃度100mg／c.c.のグルコースオキシダーゼ水溶液
を展開し、グルタルアルデヒド蒸気中で固定化し
たものを用いた。 A stainless steel disc-shaped wire mesh having a thickness of 80 microns, a diameter of 10 mm, and a mesh size of 2000 is used as a carrier for the first electrode and the second electrode. A platinum layer with a thickness of several hundred to several thousand angstroms is formed on both sides of this wire mesh by a sputtering method.
It was used as an electrode. The second electrode used was one in which an aqueous glucose oxidase solution with a concentration of 100 mg/cc was developed on the same electrode as above and fixed in glutaraldehyde vapor.
第２図に上記の第１及び第２の電極を用いた酵
素電極全体の拡大断面模式図を示した。８が第１
の電極で、金網を構成するステンレス鋼線９の表
面に白金層１０を有している。１１は第２の電極
で、ステンレス鋼線１２表面の白金層１３の上に
グルコースオキシダーゼ１４が固定化されてい
る。第１の電極と第２の電極の間には、樹脂片か
らなるスペーサー１５を設けて２つの電極を絶縁
した。 FIG. 2 shows an enlarged schematic cross-sectional view of the entire enzyme electrode using the above-mentioned first and second electrodes. 8 is the first
This electrode has a platinum layer 10 on the surface of a stainless steel wire 9 constituting a wire mesh. 11 is a second electrode, and glucose oxidase 14 is immobilized on a platinum layer 13 on the surface of a stainless steel wire 12. A spacer 15 made of a resin piece was provided between the first electrode and the second electrode to insulate the two electrodes.
上記の電極を円筒形の電極ホルダーに装着した
断面と電極系について第３図に模式図で示した。
図中、１６は上記の電極であり、第２の電極１１
がホルダーの内側になるように外とう管１７で本
体１８に装着されている。第２の電極１１の白金
層１３は白金リード１９に、第１の電極８の白金
層１０は白金リード２０にそれぞれスポツト溶接
によりつながれている。第１の電極に対する
Ag／AgCl参照極２１と対極２２は電極ホルダー
の外側に、第２電極に対するAg／AgCl参照極２
３と対極２４は電極ホルダー内部に配して電極系
を構成している。又電極ホルダー内は電解液２５
で満たされている。 FIG. 3 schematically shows the cross section and electrode system of the above electrode mounted on a cylindrical electrode holder.
In the figure, 16 is the above-mentioned electrode, and the second electrode 11
It is attached to the main body 18 with the outer tube 17 so that it is inside the holder. The platinum layer 13 of the second electrode 11 is connected to a platinum lead 19, and the platinum layer 10 of the first electrode 8 is connected to a platinum lead 20 by spot welding. for the first electrode
An Ag/AgCl reference electrode 21 and a counter electrode 22 are placed on the outside of the electrode holder.
3 and the counter electrode 24 are arranged inside the electrode holder to constitute an electrode system. Also, the electrolyte 25 is inside the electrode holder.
filled with.
第１図に示した従来例のグルコースセンサＢと
本発明による上記グルコースセンサＡをそれぞれ
PH5.6のリン酸緩衡液に浸漬し、それぞれ＋
0.6VVSAg／AgClに設定し、濃度10-3モル／の
グルコース溶液を30μ添加して、応答性能を比
較した。第４図は、横軸にグルコースを添加して
からの時間を表し、縦軸にグルコースに対する電
流増加を表している。センサＡはＢの２倍以上の
応答電流が流れ、応答速度も迅速であつた。これ
は、導電性のよいステンレス鋼金網を使用したた
めと考えられる。又妨害物質の除去効果も良好で
あつた。さらに、製造上、多孔質性薄膜をたるみ
なく均一にセルに装着し、リードをとるのは非常
に難しかつたが、金網は扱いやすく、スポツト溶
接により確実にリードをとることができた。 The conventional glucose sensor B shown in FIG. 1 and the glucose sensor A according to the present invention are respectively
Immerse in phosphate buffer with pH 5.6 and
The response performance was compared by setting 0.6V VS Ag/AgCl and adding 30μ of a glucose solution with a concentration of 10 -3 mol/. In FIG. 4, the horizontal axis represents the time since glucose was added, and the vertical axis represents the increase in current relative to glucose. Sensor A had a response current that was more than twice that of sensor B, and its response speed was quick. This is thought to be due to the use of stainless steel wire mesh with good conductivity. The effect of removing interfering substances was also good. Furthermore, during manufacturing, it was very difficult to uniformly attach the porous thin film to the cell without sagging and to take the lead, but wire mesh was easy to handle and we were able to securely take the lead by spot welding.
上記の例では、導電性の多孔性電極としてステ
ンレス鋼性金網を用いたが、ステンレス鋼に限ら
ず、白金やニツケルなどでも利用でき、又金網に
限らず、カーボン電極なども使用でき、良好な応
答が得られた。又、過酸化水素分解能をもつもの
として白金を利用したが、銀や二酸化マンガンな
ども利用できる。酵素としては、グルコースオキ
シダーゼに限らず、ウリカーゼ、コレステロール
オキシダーゼ、キサンチンオキシダーゼ等、過酸
化水素が反応系に関与するものは用いることがで
きる。 In the above example, a stainless steel wire mesh was used as the conductive porous electrode, but it is not limited to stainless steel, but platinum, nickel, etc. can also be used, and not only wire mesh, but carbon electrodes can also be used, and they are good. I got a response. Furthermore, although platinum was used as a material capable of decomposing hydrogen peroxide, silver, manganese dioxide, etc. can also be used. As the enzyme, not only glucose oxidase but also enzymes that involve hydrogen peroxide in the reaction system, such as uricase, cholesterol oxidase, and xanthine oxidase, can be used.
発明の効果
以上のように、本発明によれば、前電解用の耐
食性を有する導電性の多孔性電極と酵素を固定化
した過酸化水素分解能を有する導電性の多孔性電
極を使用することにより、製造が簡単になり、確
実に応答がとれるようになり、さらに、応答特性
も高めることができる。Effects of the Invention As described above, according to the present invention, by using a conductive porous electrode having corrosion resistance for pre-electrolysis and a conductive porous electrode having hydrogen peroxide decomposition ability on which an enzyme is immobilized. This simplifies manufacturing, ensures reliable response, and improves response characteristics.
第１図は従来の電極の拡大断面模式図、第２図
は本発明の一実施例の電極の拡大断面模式図、第
３図は電極およびセルの断面図、第４図は電極の
応答を示す図である。
８……第１の電極、１１……第２の電極、９，
１２……多孔性電極、１０，１３……白金層、１
４……固定化酵素。
Fig. 1 is an enlarged schematic cross-sectional view of a conventional electrode, Fig. 2 is an enlarged schematic cross-sectional view of an electrode according to an embodiment of the present invention, Fig. 3 is a cross-sectional view of the electrode and cell, and Fig. 4 shows the response of the electrode. FIG. 8...first electrode, 11...second electrode, 9,
12... Porous electrode, 10, 13... Platinum layer, 1
4...immobilized enzyme.
Claims (1)
を固定化した多孔性金属電極からなる第２の電極
から構成され、前記第１および第２の電極は絶縁
材を介して隣接され、前記第２の電極が過酸化水
素を電解酸化することを特徴とするバイオセン
サ。1 Consisting of a first electrode made of a porous metal electrode and a second electrode made of a porous metal electrode on which an enzyme is immobilized, the first and second electrodes are adjacent to each other via an insulating material, and the A biosensor characterized in that the second electrode electrolytically oxidizes hydrogen peroxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57177243A JPS5965753A (en) | 1982-10-07 | 1982-10-07 | Biosensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57177243A JPS5965753A (en) | 1982-10-07 | 1982-10-07 | Biosensor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5965753A JPS5965753A (en) | 1984-04-14 |
| JPH0332743B2 true JPH0332743B2 (en) | 1991-05-14 |
Family
ID=16027650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57177243A Granted JPS5965753A (en) | 1982-10-07 | 1982-10-07 | Biosensor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5965753A (en) |
-
1982
- 1982-10-07 JP JP57177243A patent/JPS5965753A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5965753A (en) | 1984-04-14 |
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