JPH0548419B2 - - Google Patents
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- Publication number
- JPH0548419B2 JPH0548419B2 JP59209166A JP20916684A JPH0548419B2 JP H0548419 B2 JPH0548419 B2 JP H0548419B2 JP 59209166 A JP59209166 A JP 59209166A JP 20916684 A JP20916684 A JP 20916684A JP H0548419 B2 JPH0548419 B2 JP H0548419B2
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- Prior art keywords
- enzyme
- immobilized
- film
- photoresist
- semiconductor
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-
- 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
-
- 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
- G01N27/403—Cells and electrode assemblies
- G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
- G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
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- Proteomics, Peptides & Aminoacids (AREA)
- Microelectronics & Electronic Packaging (AREA)
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- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Spectroscopy & Molecular Physics (AREA)
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- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は半導体マルチバイオセンサの製造方法
に関し、特に表面に酵素固定化膜が設けられた半
導体電界効果型イオンセンサを集積化してなる半
導体マルチイオンセンサの製造方法に関するもの
である。Detailed Description of the Invention (Industrial Application Field) The present invention relates to a method for manufacturing a semiconductor multi-biosensor, and particularly to a semiconductor multi-biosensor that is formed by integrating semiconductor field-effect ion sensors each having an enzyme-immobilized membrane on its surface. The present invention relates to a method for manufacturing an ion sensor.
（従来技術）
従来、溶液中の特定の有機物の濃度を測定する
半導体バイオセンサの一種に半導体電界効果型イ
オンセンサ（Ｉon Ｓensitive Ｆield Ｅffect
Ｔransistor、以下ISFETと略す）の表面に酵
素を固定化した膜が設けられたものが知られてい
る。（宮原裕二、塩川祥子、森泉豊栄、松岡英明、
軽部征夫、鈴木周一：「半導体技術を用いたバイ
オセンサ」、電子通信学会、電子部品・材料研究
会資料CPM81−93、61（1981））、このISFETバ
イオセンサは、溶液中の特定の有機物が酵素固定
化膜中で酵素の触媒作用により分解された時に生
ずる膜中の水素イオン濃度の変化をISFETで検
出するこにより、特定の有機物の濃度を測定する
ものである。この選択性をもつ酵素固定化膜の例
として、たとえば尿素検出用としてウレアーゼ固
定化膜、グルコース検出用としてグルコースオキ
シダーゼ固定化膜などが知られている。また、酵
素固定化膜が設けられたISFETと設けられてい
ないISFETの出力の差を測定することにより、
溶液の電位変化の影響を打ち消すことができ、プ
ラチナや金などの金属電極を参照電極に使用する
ことが近年報告されている。（Y.Hanzato and
S.Shiono：Bioelectrode Vsing Two Hydrogen
Ion Sensitive Tran−sistor and ａ Platinam
Wire Pseudo Reference Electrode、Proc.of
the Inter−national Meeting on Chemical
Sensors、P.513（1983））
（従来技術の問題点）
しかしながら、溶液中の多成分の有機物を同時
に測定できるマルチバイオセンサを実現するため
には複数の酵素固定膜をそれぞれ所定のISFET
表面に設けることが必要となるが、従来は１つの
半導体チツプ上で行うことができず、酵素固定化
膜をもつ個々のISFETを基板にはりつけること
によりマルチ化が行なわれた。（花里、中子、塩
野、『複合型酵素センサーの試作、第44回応用物
理学会講演予稿集P.606（1983））そのため、
ISFETの特徴であるIC製造技術が適用できず、
大量生産化や微小化が困難であるという欠点が生
じた。(Prior Art) Conventionally, a semiconductor field-effect ion sensor is a type of semiconductor biosensor that measures the concentration of a specific organic substance in a solution .
It is known that a transsistor (hereinafter abbreviated as ISFET) is provided with a membrane on which an enzyme is immobilized on the surface. (Yuji Miyahara, Shoko Shiokawa, Toyosaka Morizumi, Hideaki Matsuoka,
Yukio Karube, Shuichi Suzuki: "Biosensor using semiconductor technology", Institute of Electronics and Communication Engineers, Electronic Components and Materials Study Group Materials CPM81-93, 61 (1981)). The concentration of a specific organic substance is measured by using ISFET to detect changes in the hydrogen ion concentration in the enzyme-immobilized membrane when it is decomposed by the catalytic action of the enzyme. Known examples of enzyme-immobilized membranes having this selectivity include urease-immobilized membranes for urea detection and glucose oxidase-immobilized membranes for glucose detection. In addition, by measuring the difference in output between ISFETs with and without enzyme-immobilized membranes,
In recent years, it has been reported that a metal electrode such as platinum or gold can be used as a reference electrode to cancel the effects of potential changes in the solution. (Y.Hanzato and
S.Shiono：Bioelectrode Vsing Two Hydrogen
Ion Sensitive Tran-sistor and a Platinum
Wire Pseudo Reference Electrode, Proc.of
the International Meeting on Chemical
Sensors, p. 513 (1983)) (Problems with conventional technology) However, in order to realize a multi-biosensor that can simultaneously measure multiple organic substances in a solution, it is necessary to connect multiple enzyme-immobilized membranes to each predetermined ISFET.
Although it is necessary to provide it on the surface, conventionally this could not be done on a single semiconductor chip, and multiplication was achieved by attaching individual ISFETs each having an enzyme-immobilized film to a substrate. (Hanazato, Nakako, Shiono, “Prototype production of a composite enzyme sensor,” Proceedings of the 44th Japan Society of Applied Physics Conference, p. 606 (1983)) Therefore,
The IC manufacturing technology that characterizes ISFET cannot be applied,
The drawback is that mass production and miniaturization are difficult.
（発明の目的）
本発明の目的は、この様な従来の欠点を除去
し、互いに異なる酵素固定化膜を同一チツプ上に
つくられたISFETのそれぞれの表面の所定の位
置にウエーハの段階で形成することができる大量
生産に適し、かつ一チツプ化された微小なマルチ
バイオセンサの製造方法を提供することにある。(Objective of the Invention) The object of the present invention is to eliminate such conventional drawbacks and form different enzyme-immobilized films at predetermined positions on the surfaces of ISFETs fabricated on the same chip at the wafer stage. It is an object of the present invention to provide a method for manufacturing a minute multi-biosensor that is suitable for mass production and that is integrated into a single chip.
（発明の構成）
本発明によれば、複数個の、異なる酵素固定化
膜を表面にもつ半導体電界効果型イオンセンサが
集積化されてなる半導体マルチバイオセンサの製
造方法において、半導体電界効果型イオンセンサ
が形成された半導体ウエーハ上にフオトレジスト
を塗布し、この後フオトリソグラフイー法により
酵素固定化膜が設けられる所定の半導体電界効果
型イオンセンサの表面のフオトレジストを除く工
程と、酵素と加橋剤を含む蛋白質溶液を塗布し酵
素固定化膜を形成する工程と、フオトレジストを
溶かし所定の半導体電界効果型イオンセンサの表
面以外に存在する酵素固定化膜を除去する工程と
を備え複数個の異なる酵素固定化膜をそれぞれ所
定の半導体電界効果型イオンセンサの表面に設け
ることを特徴とする半導体マルチバイオセンサの
製造方法が得られる。(Structure of the Invention) According to the present invention, in a method for manufacturing a semiconductor multi-biosensor in which a plurality of semiconductor field-effect ion sensors having different enzyme-immobilized membranes on the surface are integrated, semiconductor field-effect ion A process of coating a photoresist on a semiconductor wafer on which a sensor is formed, and then removing the photoresist from the surface of a given semiconductor field-effect ion sensor on which an enzyme-immobilized film is provided by photolithography, and a process of adding an enzyme A step of applying a protein solution containing a bridging agent to form an enzyme-immobilized film, and a step of dissolving the photoresist and removing the enzyme-immobilized film existing on areas other than the surface of a predetermined semiconductor field-effect ion sensor. A method for manufacturing a semiconductor multi-biosensor is obtained, which is characterized in that enzyme-immobilized membranes of different types are provided on the surface of a predetermined semiconductor field-effect ion sensor.
（実施例）
以下本発明の一実施例について図面を参照して
詳細に説明する。(Example) An example of the present invention will be described in detail below with reference to the drawings.
第１図〜第６図はサフアイア基板上に設けられ
た島状シリコン層を用いて形成された複数の
ISFET上に２種類の酵素固定化膜をそれぞれ設
ける場合について示している。なお、金属参照電
極がサフアイア基板１の裏面に蒸着されており、
溶液の電位を定める仮の参照電極として働く。第
１図〜第６図において、１はサフアイア基板、２
は高不純物濃度ｎ形シリコン領域、３はｐ形シリ
コン領域、４は酸化シリコン膜、５は窒化シリコ
ン膜、６はアセトンに可溶なフオトレジスト膜、
７は第１の酵素固化膜であるウレアーゼ固定化
膜、８はアセトンに可溶なフオトレジスト膜、９
は第２の酵素固定化膜でたとえばグルコースオキ
シダーゼ固定化膜、１０は金電極である。 Figures 1 to 6 show a plurality of silicon layers formed using island-like silicon layers provided on a sapphire substrate.
The case where two types of enzyme-immobilized membranes are respectively provided on the ISFET is shown. Note that a metal reference electrode is deposited on the back surface of the sapphire substrate 1,
It acts as a temporary reference electrode to determine the potential of the solution. In Figures 1 to 6, 1 is a sapphire substrate, 2
3 is a high impurity concentration n-type silicon region, 3 is a p-type silicon region, 4 is a silicon oxide film, 5 is a silicon nitride film, 6 is an acetone-soluble photoresist film,
7 is a urease immobilized membrane which is the first enzyme solidified membrane; 8 is an acetone-soluble photoresist membrane; 9
1 is a second enzyme-immobilized membrane, for example, a glucose oxidase-immobilized membrane, and 10 is a gold electrode.
次に構造工程を順を追つて説明する。サフアイ
ア基板１表面の島状シリコン層を用いてISFET
形成したサフアイア基板１裏面に金１０を蒸着し
たウエーハの表面にアセトンに可溶性のフオトレ
ジスト膜６としてシツプレー社（Shipley Co.）
製AZ1450Jを3000r.p.mの回転数でスピン塗布し
た後、フオトマスクを用いたフオトリソグラフイ
ーにより第１の酵素固定化膜たとえばウレアーゼ
固定化膜が設けられるISFETの表面のフオトレ
ジスト膜を除去する（第１図）。次に第１の酵素
と架橋剤を含む蛋白質溶液として15％牛血清アル
ブミンを含む0.2M、PH8.5のトリス・塩酸緩衝液
250μに、同じく緩衝液で調製した100mg／dlウ
レアーゼ（ペーリンガー・マンハイム社製、約
50V／mg）溶液250μを加え、0.75％グルタール
アルデヒド水溶液500μと撹拌混合した溶液を
3000r.p.mの回転数でスピン塗布する（第２図）。
この工程によりウレアーゼ固定化膜７を5000Å以
下の厚さでほぼ均一に形成することができた。ウ
レアーゼ固定化膜の窒化シリコン膜への密着性も
良好であつたが、さらに密着性を向上させるため
第１の酵素固定化膜のスピン塗布の前に窒化シチ
コン膜の表面をプライマー処理することも可能で
ある。その後、ウエーハをアセトンに浸しフオト
レジスト６を溶かし、同時にフオトレジスト上に
塗布されていたウレアーゼ固定化膜を除去する。
ウレアーゼ固定化膜中のウレアーゼはアセトンに
よつて失活されないので、第１のISFETの表面
だけに活性なウレアーゼ固定化膜７を形成するこ
とができた（第３図）。 Next, the structural steps will be explained step by step. ISFET using the island-like silicon layer on the surface of the sapphire substrate 1
On the surface of the wafer with gold 10 deposited on the back surface of the formed sapphire substrate 1, an acetone-soluble photoresist film 6 was applied by Shipley Co.
After spin-coating AZ1450J manufactured by AZ1450J at a rotational speed of 3000 rpm, the photoresist film on the surface of the ISFET on which the first enzyme-immobilized film, such as the urease-immobilized film, is provided is removed by photolithography using a photomask (the first Figure 1). Next, a 0.2M Tris/HCl buffer containing 15% bovine serum albumin at pH 8.5 is used as a protein solution containing the first enzyme and crosslinking agent.
250μ, 100mg/dl urease (manufactured by Pähringer Mannheim, approx.
Add 250μ of 50V/mg) solution and stir and mix with 500μ of 0.75% glutaraldehyde aqueous solution.
Spin coating at a rotation speed of 3000 rpm (Figure 2).
Through this process, the urease-immobilized film 7 could be formed almost uniformly with a thickness of 5000 Å or less. The adhesion of the urease-immobilized film to the silicon nitride film was also good, but in order to further improve the adhesion, the surface of the nitride nitride film may be treated with a primer before spin-coating the first enzyme-immobilized film. It is possible. Thereafter, the wafer is immersed in acetone to dissolve the photoresist 6, and at the same time, the urease immobilized film coated on the photoresist is removed.
Since urease in the urease-immobilized membrane was not inactivated by acetone, an active urease-immobilized membrane 7 could be formed only on the surface of the first ISFET (FIG. 3).
次に、再びAZ1450Jを3000r.p.mの回転数でス
ピン塗布した後、室温、減圧下でフオトレジスト
を乾燥しフオトマスクを用いたフオトリソグラフ
イーにより第２の酵素固定化膜としてグルコース
固定化膜が設けられるISFETの表面のフオトレ
ジストの膜を除去する（第４図）。この時、室温
でレジストを乾燥するのは、高温（60℃以上）で
は第１の酵素が失活するためである。次に、第２
の酵素と架橋剤を含む蛋白質溶液として15％牛血
清アルブミン液50μに、２mgのグルコースオキ
シダーゼ（ベーリンガー・マインハイム社製、約
250V／mg）水溶液450μを加え、0.75％グルタ
ールアルデヒド水溶液500μと撹拌混合した溶
液を3000r.p.mの回転数でスピン塗布する（第５
図）。この工程によりグルコースオキシダーゼ固
定化膜９を5000Å以下の厚さでほぼ均一に形成す
ることができた。グルコースオキシダーゼ固定化
膜の窒化シリコン膜への密着性も良好であつた
が、さらに密着剤を向上させるため第２の酵素固
定化のスピン塗布の前に窒化シリコン膜の表面を
プライマー処理することも可能である。この後ウ
エーハをアセトンに浸しフオトレジスト８を溶か
し、同時にフオトレジスト上に塗布されていたウ
レアーゼ固化膜を除去する。グルコースオキシダ
ーゼ固定化膜中のグルコースはアセトンにより失
活されないので、第2ISFETの表面だけに活性な
グルコース固定化膜９を形成することができた
（第６図）。 Next, after spin-coating AZ1450J again at a rotational speed of 3000 rpm, the photoresist was dried at room temperature under reduced pressure, and a glucose-immobilized film was formed as a second enzyme-immobilized film by photolithography using a photomask. Remove the photoresist film on the surface of the ISFET (Figure 4). At this time, the resist is dried at room temperature because the first enzyme is inactivated at high temperatures (60° C. or higher). Next, the second
Add 2 mg of glucose oxidase (manufactured by Boehringer Meinheim, approx.
Add 450μ of an aqueous solution (250V/mg) and stir and mix the solution with 500μ of a 0.75% glutaraldehyde aqueous solution at a rotation speed of 3000rpm (5th
figure). Through this step, the glucose oxidase immobilized membrane 9 could be formed almost uniformly with a thickness of 5000 Å or less. The adhesion of the glucose oxidase immobilization membrane to the silicon nitride membrane was also good, but in order to further improve the adhesion, the surface of the silicon nitride membrane may be treated with a primer before spin coating for the second enzyme immobilization. It is possible. Thereafter, the wafer is immersed in acetone to dissolve the photoresist 8, and at the same time, the urease solidified film coated on the photoresist is removed. Since glucose in the glucose oxidase-immobilized membrane was not deactivated by acetone, an active glucose-immobilized membrane 9 could be formed only on the surface of the second ISFET (FIG. 6).
（発明の効果）
本発明により同一チツプ上に異なる種類の素素
固定化膜がそれぞれ設けられた複数のISFETを
容易に形成することができ、マルチバイオセンサ
を容易に大量生産し、微小化することができた。(Effects of the Invention) According to the present invention, multiple ISFETs each having a different type of element immobilization film can be easily formed on the same chip, and multi-biosensors can be easily mass-produced and miniaturized. I was able to do that.
本発明の工程を繰り返すことにより３種類以上
の酵素固定化膜がそれぞれ表面に形成された
ISFETを同一チツプ上に設けることも可能であ
ることは明らかであり、さらに、酵素固定化膜が
設けられいない同一チツプ上のISFETの出力と
比較することにより金属電極を参照電極に使用す
ることによる溶液の電位変動の影響を除去できる
ことも明らかである。 By repeating the process of the present invention, three or more types of enzyme-immobilized films were formed on each surface.
It is clear that it is possible to provide an ISFET on the same chip, and furthermore, it is possible to use a metal electrode as a reference electrode by comparing the output of an ISFET on the same chip without an enzyme-immobilized membrane. It is also clear that the effects of potential fluctuations in the solution can be eliminated.
第１図〜第６図は本発明による半導体マルチバ
イオセンサの製造方法の一実施例を説明するため
の図。
同図において、１はサフアイア基板、２は高不
純物濃度ｎ形シリコン領域、３はｐ形シリコン領
域、４は酸化シリコン膜、５は窒化シリコン膜、
６はアセトンに可溶なフオトレジスト膜、７は第
１の酵素固定化膜、８はアセトンに可溶なフオト
レジスト膜、９は第２の酵素固定化膜、１０は金
電極である。
1 to 6 are diagrams for explaining an embodiment of the method for manufacturing a semiconductor multi-biosensor according to the present invention. In the figure, 1 is a sapphire substrate, 2 is a high impurity concentration n-type silicon region, 3 is a p-type silicon region, 4 is a silicon oxide film, 5 is a silicon nitride film,
6 is an acetone-soluble photoresist film, 7 is a first enzyme-immobilized film, 8 is an acetone-soluble photoresist film, 9 is a second enzyme-immobilized film, and 10 is a gold electrode.
Claims (1)
導体電界効果型イオンセンサが集積化されてなる
半導体マルチバイオセンサの製造方法において、
半導体電界効果型イオンセンサが形成された半導
体ウエーハ上にフオトレジストを塗布し、この後
フオトリソグラフイー法により酵素固定化膜が設
けられる所定の半導体電界効果型イオンセンサの
表面のフオトレジストを除く工程と、酵素と加橋
剤を含む蛋白質溶液を塗布し酵素固定化膜を形成
する工程と、フオトレジストを溶かし所定の半導
体電界効果型イオンセンサの表面以外に存在する
酵素固定化膜を除去する工程とを備え複数個の異
なる酵素固定化膜を所定の半導体電界効果型イオ
ンセンサの表面に設けることを特徴とする半導体
マルチバイオセンサの製造方法。1. In a method for manufacturing a semiconductor multi-biosensor in which semiconductor field-effect ion sensors having a plurality of different enzyme-immobilized membranes on the surface are integrated,
A step of applying a photoresist on a semiconductor wafer on which a semiconductor field-effect ion sensor is formed, and then removing the photoresist on the surface of a given semiconductor field-effect ion sensor on which an enzyme-immobilized film is provided using a photolithography method. , a step of applying a protein solution containing an enzyme and a cross-linking agent to form an enzyme-immobilized film, and a step of dissolving the photoresist and removing the enzyme-immobilized film existing on areas other than the surface of the specified semiconductor field-effect ion sensor. 1. A method for manufacturing a semiconductor multi-biosensor, comprising: providing a plurality of different enzyme-immobilized membranes on the surface of a predetermined semiconductor field-effect ion sensor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59209166A JPS6188136A (en) | 1984-10-05 | 1984-10-05 | Production of semiconductor multibiosensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59209166A JPS6188136A (en) | 1984-10-05 | 1984-10-05 | Production of semiconductor multibiosensor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6188136A JPS6188136A (en) | 1986-05-06 |
| JPH0548419B2 true JPH0548419B2 (en) | 1993-07-21 |
Family
ID=16568419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59209166A Granted JPS6188136A (en) | 1984-10-05 | 1984-10-05 | Production of semiconductor multibiosensor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6188136A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0717162Y2 (en) * | 1988-05-13 | 1995-04-19 | ミノルタ株式会社 | Flexible printed circuit board |
| JP4277777B2 (en) | 2004-09-28 | 2009-06-10 | セイコーエプソン株式会社 | Mounting structure, mounting substrate, electro-optical device, and electronic apparatus |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59209165A (en) * | 1983-05-13 | 1984-11-27 | Yokogawa Hokushin Electric Corp | Recorder |
-
1984
- 1984-10-05 JP JP59209166A patent/JPS6188136A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6188136A (en) | 1986-05-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |