JPS62225941A - Method of forming semiconductor biosensor enzyme immobilized membrane - Google Patents
Method of forming semiconductor biosensor enzyme immobilized membraneInfo
- Publication number
- JPS62225941A JPS62225941A JP61070149A JP7014986A JPS62225941A JP S62225941 A JPS62225941 A JP S62225941A JP 61070149 A JP61070149 A JP 61070149A JP 7014986 A JP7014986 A JP 7014986A JP S62225941 A JPS62225941 A JP S62225941A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- immobilized
- semiconductor
- film
- membrane
- 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.)
- Granted
Links
- 239000004065 semiconductor Substances 0.000 title claims abstract description 46
- 239000012528 membrane Substances 0.000 title claims abstract description 35
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims description 24
- 229920002120 photoresistant polymer Polymers 0.000 claims abstract description 18
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- 239000012460 protein solution Substances 0.000 claims abstract description 8
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 7
- 238000004528 spin coating Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 230000005669 field effect Effects 0.000 claims description 13
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 238000000206 photolithography Methods 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- 230000003100 immobilizing effect Effects 0.000 claims 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 abstract description 9
- 229910052710 silicon Inorganic materials 0.000 abstract description 9
- 239000010703 silicon Substances 0.000 abstract description 9
- 229910052594 sapphire Inorganic materials 0.000 abstract description 8
- 239000010980 sapphire Substances 0.000 abstract description 8
- 239000000758 substrate Substances 0.000 abstract description 8
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 5
- 239000010931 gold Substances 0.000 abstract description 5
- 229910052737 gold Inorganic materials 0.000 abstract description 5
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 abstract description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract 2
- 229910052760 oxygen Inorganic materials 0.000 abstract 2
- 239000001301 oxygen Substances 0.000 abstract 2
- 229940088598 enzyme Drugs 0.000 description 17
- 150000002500 ions Chemical class 0.000 description 8
- 239000000126 substance Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 229910052581 Si3N4 Inorganic materials 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- HQVNEWCFYHHQES-UHFFFAOYSA-N silicon nitride Chemical compound N12[Si]34N5[Si]62N3[Si]51N64 HQVNEWCFYHHQES-UHFFFAOYSA-N 0.000 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920002284 Cellulose triacetate Polymers 0.000 description 1
- 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 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical group [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- NNLVGZFZQQXQNW-ADJNRHBOSA-N [(2r,3r,4s,5r,6s)-4,5-diacetyloxy-3-[(2s,3r,4s,5r,6r)-3,4,5-triacetyloxy-6-(acetyloxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6s)-4,5,6-triacetyloxy-2-(acetyloxymethyl)oxan-3-yl]oxyoxan-2-yl]methyl acetate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](OC(C)=O)[C@H]1OC(C)=O)O[C@H]1[C@@H]([C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H](COC(C)=O)O1)OC(C)=O)COC(=O)C)[C@@H]1[C@@H](COC(C)=O)O[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@H]1OC(C)=O NNLVGZFZQQXQNW-ADJNRHBOSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 229910052814 silicon oxide Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は半導体電界効果型イオンセンサの表面に酵素固
定化膜が設けられてなる集積化された半導体バイオセン
サにおける酵素固定化膜の形成方法に関するものである
。Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for forming an enzyme-immobilized membrane in an integrated semiconductor biosensor in which an enzyme-immobilized membrane is provided on the surface of a semiconductor field-effect ion sensor. It is related to.
（従来の技術）
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（以
下ｒＩＳＦＥＴｊという）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。このバイオセンサは
、溶液中の特定の有機物が酵素固定化膜中で酵素の触媒
作用により化学反応をした時に生じる水素イオン濃度の
変化をＩＳＦ師で検出することにより、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、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膜な
どが知られている（センサーズ・アンド・アク千ユエイ
ターズ（５ｅｎｓｏｒｓ ａｎｄ Ａｃｔｕ−ａｔｏｒ
ｓ ）第７巻１頁〜１０頁（１９８５乃。また、サファ
イア基板上に設けられた島状シリコンを用いて酵素固定
化膜か設けられたＩ ５ＦＥＴと失活した酵素固定化膜
が設けられたＩ ５ＦＥＴを同一チップ上に形成し、裏
面に参照電極として金電極を設けることにより、ワンチ
ップ化されたバイオセンサも開発されている（第１６回
、１９８４インタナシコナル・カンファレンス・オン・
ソリッド中ステート・デバイシズ・アンド・マテリアル
ズ、レイト・ニューズ・アプストラクツ（１９８４Ｉｎ
ｔｅｒｎａｔｉｏｎａｌＣｏｎｆｅｒｅｎｃｅ ｏｎ
５ｏｌｉｄ ５ｔａｔｅ Ｄｅｖｉｃｅｓ
ａｎｄＭａｔｅｒｉａｌｓ、 Ｌａｔｅ Ｎｅｗｓ Ａ
ｂｓｔｒａｃｔｓ） ６６頁〜６７頁（１９８４））。(Prior Art) Conventionally, one type of semiconductor biosensor for measuring the concentration of a specific organic substance in a solution is a semiconductor field-effect ion sensor (hereinafter referred to as rISFET) in which a membrane on which an enzyme is immobilized is provided on the surface. Are known. This biosensor measures the concentration of specific organic substances by using an ISF device to detect changes in hydrogen ion concentration that occur when specific organic substances in a solution undergo a chemical reaction due to the catalytic action of enzymes in an enzyme-immobilized membrane. It is something to do. Examples of enzyme-immobilized membranes with this selectivity include urease-immobilized membranes for urea detection and glucose oxidase membranes for glucose detection (Sensors and Actuators).
s) Vol. 7, pp. 1-10 (1985). Also, an I5FET was provided with an enzyme immobilization film using island-shaped silicon provided on a sapphire substrate, and an I5FET was provided with an enzyme immobilization film inactivated. A single-chip biosensor has also been developed by forming two I5FETs on the same chip and providing a gold electrode as a reference electrode on the back side (16th, 1984 International Conference on
Solid State Devices and Materials, Late News Abstracts (1984In
InternationalConference on
5solid 5tate Devices
andMaterials, Late News A
bstructs) pp. 66-67 (1984)).
この様なバイオセンサを製造するにあたり、所定のｌ５
ＦＥＴ上に酵素固定化膜を形成する必要がある。酵素固
定化膜の形成法にはいくつかの方法が知られているが、
ウェハの段階で酵素固定化膜を形成できバイオセンサの
大量生産を可能ならしめるものとしては、例えば前記引
用文献のように三酢酸セルロースを担体とした酵素固定
化膜をウェハ全面に形成した後ホトマスクを介して紫外
線を照射、所定のｌ５ＦＥＴ上以外の酵素固定化膜中の
酵素を失活させるという方法がある。In manufacturing such a biosensor, a predetermined l5
It is necessary to form an enzyme-immobilized membrane on the FET. Several methods are known for forming enzyme-immobilized membranes, but
An example of a method that enables mass production of biosensors by forming an enzyme-immobilized film at the wafer stage is to form an enzyme-immobilized film using cellulose triacetate as a carrier on the entire surface of the wafer, and then use a photomask, as shown in the above cited document. There is a method in which the enzymes in the enzyme-immobilized membrane other than those on the predetermined 15FET are inactivated by irradiating ultraviolet rays through the 15FET.
（発明が解決しようとする問題点）
しかし、上記の方法は酵素固定化を形成するのに１日以
上を要する上、酵素固定化膜の性状、例えば厚さ、酵素
含有量などバイオセンサの出力に重大な影響を及ぼす要
因の変更が等しいという問題点を有していた。これに対
し、短時間で酵素固定化膜を得られ酵素固定化膜中の酵
素含有量の変更も容易であるリフトオフ法が提案されて
いる（特願昭５９−２０９１６５）。しかし、このリフ
トオフ法では、酵素固定化膜が有機溶剤と接触するため
。(Problems to be Solved by the Invention) However, the above method requires more than one day to form the enzyme immobilization, and the properties of the enzyme immobilization membrane, such as thickness, enzyme content, etc. The problem was that changes in factors that had a significant impact on both were equally important. In contrast, a lift-off method has been proposed (Japanese Patent Application No. 59-209165), which allows an enzyme-immobilized membrane to be obtained in a short time and allows for easy changes in the enzyme content in the enzyme-immobilized membrane. However, in this lift-off method, the enzyme-immobilized membrane comes into contact with the organic solvent.
有機溶剤で失活を受けやすい酵素を利用した半導体バイ
オセンサ酵素固定化膜の形成にはリフトオフ法を採用す
ることができなかった。The lift-off method could not be used to form a semiconductor biosensor enzyme-immobilized membrane using enzymes that are easily inactivated by organic solvents.
（問題点を解決するための手段）
本発明は半導体電界効果型イオンセンサの表面に酵素固
定化膜が設けられてなる半導体バイオセンサの酵素固定
化膜形成方法において。(Means for Solving the Problems) The present invention relates to a method for forming an enzyme-immobilized membrane for a semiconductor biosensor, in which an enzyme-immobilized membrane is provided on the surface of a semiconductor field-effect ion sensor.
（ａ） 半導体電界効果型イオンセンサが形成された
半導体ウェハ上に７オトレジストを塗布した後、フォト
リソグラフィー法により酵素固定化膜が設けられるべき
所定の半導体電界効果型イオンセンサの表面のフォトレ
ジストを除く工程と、（ｂ） 前記半導体ウェハ表面
に、親水性プライマ溶液をスピン塗布し、前記所定の半
導体電界効果型イオンセンサの表面を親水性プライマ処
理する工（ｃ） 前記工程を経た半導体ウェハ上に残
ったフォトレジストを剥離する工程と。(a) After coating the semiconductor wafer on which the semiconductor field-effect ion sensor is formed with a photoresist, the photoresist is applied to the surface of the predetermined semiconductor field-effect ion sensor on which the enzyme-immobilized film is to be provided by photolithography. (b) spin-coating a hydrophilic primer solution on the surface of the semiconductor wafer and treating the surface of the predetermined semiconductor field-effect ion sensor with a hydrophilic primer; and the process of peeling off the remaining photoresist.
（ｄ） さらｌこ前記工程を経た半導体ウェハ表面に
酵素と架橋剤を含む蛋白質溶液をスピン塗布して前記半
導体ウェハ表面上に酵素固定化膜を形成した後、該半導
体ウェハを所定のｐＨ範囲の水溶液に浸漬し、超音波処
理を行なうことにより、前記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し
、前記所定の半導体電界効果型イオンセンサの表面に酵
素固定化膜を形成することを特徴とする半導体バイオセ
ンサ酵素固定化膜の形成方法である。(d) After forming an enzyme-immobilized film on the surface of the semiconductor wafer by spin-coating a protein solution containing an enzyme and a crosslinking agent on the surface of the semiconductor wafer that has gone through the above steps, the semiconductor wafer is heated to a predetermined pH range. By immersing the sensor in an aqueous solution and performing ultrasonic treatment, the enzyme-immobilized film existing on areas other than the surface of the predetermined semiconductor field-effect ion sensor is removed, and the enzyme is immobilized on the surface of the predetermined semiconductor field-effect ion sensor. This is a method for forming a semiconductor biosensor enzyme-immobilized membrane, the method comprising forming an immobilized membrane.
（作用）
本発明の方法によれば、半導体ウェハ上にフォトレジス
トを塗布した後、フォトリソグラフィー法により酵素固
定化膜が設けられるべき所定のｌ５ＦＥＴの表面のフォ
トレジストを除き、次にフォトレジストが除かれたｌ５
ＦＥＴの表面を親水性プライマで処理するため、親水性
プライマをスピン塗布する。この工程により所定のｌ５
ＦＥＴの表（６）。(Function) According to the method of the present invention, after coating a photoresist on a semiconductor wafer, the photoresist on the surface of a predetermined 15FET on which an enzyme immobilization film is to be provided is removed by photolithography, and then the photoresist is coated on a semiconductor wafer. removed l5
To treat the surface of the FET with a hydrophilic primer, the hydrophilic primer is spin-coated. Through this process, the predetermined l5
FET table (6).
面に親水性プライマの反応基が導入され、蛋白質溶液中
に含まれる架橋剤との反応に供される。親水性プライマ
処理工程を経た半導体ウェハ上に残存するフォトレジス
トは有機溶剤などで剥離する。A reactive group of a hydrophilic primer is introduced onto the surface and subjected to reaction with a crosslinking agent contained in the protein solution. The photoresist remaining on the semiconductor wafer that has undergone the hydrophilic primer treatment step is removed using an organic solvent or the like.
フォトレジスト剥離に使用する薬品は親水性プライマの
反応基を損うものであってはならない。以上の工程を経
た半導体ウェハ上に酵素上架橋剤を含む蛋白質溶液をス
ピン塗布する。架橋剤による架橋反応が完了した後、こ
の半導体ウェハを所定のｐＨ以上の水溶液に浸漬する。The chemicals used to strip the photoresist must not damage the reactive groups of the hydrophilic primer. A protein solution containing an enzymatic crosslinking agent is spin-coated onto the semiconductor wafer that has undergone the above steps. After the crosslinking reaction by the crosslinking agent is completed, the semiconductor wafer is immersed in an aqueous solution having a predetermined pH or higher.
所定のｐＨ以上の水溶液中では酵素固定化膜、半導体ウ
ェハ双方とも負の電荷を帯び、電気的反撥力により親水
性プライマの反応基との化学結合によって保持されてい
る所定のｌ５ＦＥＴ表面上の酵素固定化膜以外は、半導
体ウェハ表面から浮き上がり、この状態で超音波を作用
させると、浮き上がった酵素固定化膜は力学的に除去さ
れ、所定のｌ５ＦＥＴ表面上にのみ酵素固定化膜が形成
される。酵素固定化膜の材質や他の条件にもよるが望ま
しくはｐＨ８以上の水溶液がよい。In an aqueous solution with a predetermined pH or higher, both the enzyme-immobilized membrane and the semiconductor wafer are negatively charged, and the enzyme on the surface of the predetermined 15FET is held by chemical bonds with the reactive groups of the hydrophilic primer due to electrical repulsion. Everything other than the immobilized film rises from the surface of the semiconductor wafer, and when ultrasonic waves are applied in this state, the lifted enzyme-immobilized film is mechanically removed and the enzyme-immobilized film is formed only on the predetermined 15FET surface. . Although it depends on the material of the enzyme-immobilized membrane and other conditions, an aqueous solution with a pH of 8 or higher is preferable.
（実施例） 以下本発明の実施例について図面を参照して説明する。(Example) Embodiments of the present invention will be described below with reference to the drawings.
第１図は本発明による半導体バイオセンサ酵素固定化膜
の形成方法の一実施例の工程説明図で。FIG. 1 is a process explanatory diagram of one embodiment of the method for forming a semiconductor biosensor enzyme-immobilized membrane according to the present invention.
サファイア基板上に形成された２つ１組のｌ５ＦＥＴの
一方にのみ酵素固定化膜を形成する場合について示しで
ある。第１図（ａ）〜（ｅ）において、ｌはサファイア
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は酸化ンリコン膜、５は窒化シリコン膜
、６はフォトレジスト膜、７は金電極である。次に形成
工程を順を追って説明する。サファイア基板１の表面の
島状ンリコン層を用いてｌ５ＦＥＴを形成し、サファイ
ア基板１の裏面の島状シリコン層を用いてｌ５ＦＥＴを
形成し、サファイア裏面に金７を蒸着したウェハ表面に
フォトレジスト膜６、例えばンップレー社製マイクロポ
ジット１３００−２７をスピン塗布する（第１図（ａ）
）。次に、フォトマスクを用い露光、現像により酵素固
定化膜が設けられるｌ５ＦＥＴの表面８のフォトレジス
ト膜を除去する（第１図（ｂ））。その後親水性プライ
マ、例えばγ−アミノプロピルトリエトキシシランの１
％水溶液をウェハ上にスピン塗布し、１１０℃で５分間
熱処理を行ない、酵素固定化膜が設けられるｌ５ＦＥＴ
の表面にγ−アミノプロピルトリエトキンシランを結合
させる。This figure shows a case where an enzyme-immobilized film is formed only on one of a pair of 15FETs formed on a sapphire substrate. In FIGS. 1(a) to (e), l is a sapphire substrate, 2 is a high impurity concentration n-type silicon region, 3 is a p-type silicon region, 4 is an oxide silicon film, 5 is a silicon nitride film, and 6 is a photoresist. The membrane 7 is a gold electrode. Next, the formation process will be explained step by step. An 15FET is formed using an island silicon layer on the front surface of the sapphire substrate 1, an 15FET is formed using an island silicon layer on the back surface of the sapphire substrate 1, and a photoresist film is formed on the wafer surface with gold 7 deposited on the back surface of the sapphire substrate. 6. For example, spin-coat Microposit 1300-27 manufactured by Nppley (Fig. 1(a)).
). Next, the photoresist film on the surface 8 of the 15FET on which the enzyme-immobilized film is provided is removed by exposure and development using a photomask (FIG. 1(b)). Then a hydrophilic primer, e.g. 1 of γ-aminopropyltriethoxysilane
% aqueous solution onto a wafer and heat-treated at 110°C for 5 minutes to form an 15FET with an enzyme-immobilized film.
γ-Aminopropyltriethquin silane is bonded to the surface of.
このウェハを５チグルタルアルデヒド水溶液に１５分間
浸漬し、ｌ５ＦＥＴの表面上に結合しているγ−アミノ
プロピルトリエトキシンランのアミン基とグルタルアル
デヒドのアルデヒド基を反応させ、酵素固定化膜が設け
られるｌ５ＦＥＴの表面をアルデヒド基で修飾する。次
にこの半導体ウェハをアセトン中に浸漬してフォトレジ
ストを除去する（第１図（Ｃ））。水洗、乾燥の後、酵
素と架橋剤を含む蛋白質溶液、例えは３００ｍｇ牛血清
アルブミンを含む０．１ Ｍピペラジン−Ｎ 、 Ｎ’
−ヒス（２−エタンスルフォン酸）−水酸化ナトリウム
（ｐＨ６，８）２体積部に５０ｍｇ／−のウレアーゼ（
マイルスラボラトリー・ズ製）水溶液１体積部を加え、
さらに２重量％グルタルアルデヒド水溶液１体積部を加
えた後よく混合した溶液９００μｌをスピン塗布する。This wafer is immersed in an aqueous solution of 5tiglutaraldehyde for 15 minutes to cause the amine group of γ-aminopropyltriethoxine bonded on the surface of the 15FET to react with the aldehyde group of glutaraldehyde, thereby forming an enzyme-immobilized membrane. The surface of the 15FET is modified with aldehyde groups. Next, this semiconductor wafer is immersed in acetone to remove the photoresist (FIG. 1(C)). After washing with water and drying, a protein solution containing an enzyme and a cross-linking agent, for example 0.1 M piperazine-N, N' containing 300 mg bovine serum albumin, is added.
- His(2-ethanesulfonic acid) - 50 mg/- of urease in 2 parts by volume of sodium hydroxide (pH 6,8)
Add 1 part by volume of aqueous solution (manufactured by Miles Laboratories),
Further, 1 part by volume of a 2% by weight aqueous glutaraldehyde solution was added, and 900 μl of the well-mixed solution was spin-coated.
１時間常温で放置してグルタルアルデヒドによる架橋反
応を完了させて酵素固定化膜を形成する（第１図（ｄ）
）。このようにして酵素固定化膜９が形成された半導体
ウェハをｐＨ８以上の水溶液、例えば０．１ Ｍ ｌ−
リス（ヒドロキンメチル）アミノメタン−塩酸緩衝液に
浸漬し、親水性プライマ処理したｌ５ＦＥＴ上以外の酵
素固定化膜が半導体ウェハ表面から浮き上がるのをまっ
て超音波処理を行ない、浮き上がった酵素固定化膜を除
去する悄１図（ｅ））。The enzyme-immobilized membrane is formed by leaving it at room temperature for 1 hour to complete the crosslinking reaction with glutaraldehyde (Fig. 1(d)).
). The semiconductor wafer on which the enzyme-immobilized film 9 has been formed in this way is treated with an aqueous solution having a pH of 8 or higher, for example, 0.1 M l-
The enzyme-immobilized film other than that on the 15FET, which had been immersed in a lith(hydroquine-methyl)aminomethane-hydrochloric acid buffer and treated with a hydrophilic primer, was lifted from the semiconductor wafer surface, and then subjected to ultrasonication to immobilize the enzyme. Figure 1 (e)).
以上の工程により所定のｌ５ＦＥＴの表面にだけ酵素固
定化膜を形成することができた。Through the above steps, it was possible to form an enzyme-immobilized membrane only on the surface of a predetermined 15FET.
（発明の効果）
本発明の方法によれば、酵素固定化膜は蛋白質溶液をス
ピン塗布するために、酵素固定化膜の厚さはウェハ上の
各ｌ５ＦＥＴ上で均一であり、形成の各工程は酵素活性
に影響を与える要因を含まず、ｌ５ＦＥＴを用いたバイ
オセンサの大量生産が可能である。(Effects of the Invention) According to the method of the present invention, since the enzyme-immobilized film is formed by spin-coating a protein solution, the thickness of the enzyme-immobilized film is uniform on each 15FET on the wafer, and the thickness of the enzyme-immobilized film is uniform at each step of formation. does not contain any factors that affect enzyme activity and allows mass production of biosensors using 15FET.
第１図（ａ）〜（ｅ）は本発明の方法を半導体バイオセ
ンサの製造工程に適用した際の工程説明図。
図において、１はサファイア基板、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は酸化ンリ
コン膜、５は窒化シリコン膜、６はフォトレジスト膜、
７は金電極、８は酵素固定化膜が設けられるｌ５ＦＥＴ
表面、９は酵素固定化膜。
亭 １ 回FIGS. 1(a) to 1(e) are process explanatory diagrams when the method of the present invention is applied to the manufacturing process of a semiconductor biosensor. In the figure, 1 is a sapphire substrate, 2 is a high impurity concentration n
3 is a p-type silicon region, 4 is a silicon oxide film, 5 is a silicon nitride film, 6 is a photoresist film,
7 is a gold electrode, and 8 is an 15FET provided with an enzyme-immobilized membrane.
On the surface, 9 is an enzyme-immobilized membrane. Tei 1 time
Claims (1)
設けられてなる半導体バイオセンサの酵素固定化膜形成
方法において、 （ａ）半導体電界効果型イオンセンサが形成された半導
体ウェハ上にフォトレジストを塗布した後、フォトリソ
グラフィー法により酵素固定化膜が設けられるべき所定
の半導体電界効果型イオンセンサの表面のフォトレジス
トを除く工程と、 （ｂ）前記半導体ウェハ表面に、親水性プライマ溶液を
スピン塗布し、前記所定の半導体電界効果型イオンセン
サの表面を親水性プライマ処理する工程と、 （ｃ）前記工程を経た半導体ウェハ上に残ったフォトレ
ジストを剥離する工程と、 （ｄ）さらに前記工程を経た半導体ウェハ表面に酵素と
架橋剤を含む蛋白質溶液をスピン塗布して前記半導体ウ
ェハ表面上に酵素固定化膜を型成した後、該半導体ウェ
ハを所定のｐＨ範囲の水溶液に浸漬し、超音波処理を行
なうことにより、前記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し、前記
所定の半導体電界効果型イオンセンサの表面に酵素固定
化膜を形成することを特徴とする半導体バイオセンサ酵
素固定化膜の形成方法。[Scope of Claims] A method for forming an enzyme-immobilized film of a semiconductor biosensor in which an enzyme-immobilized film is provided on the surface of a semiconductor field-effect ion sensor, comprising: (a) a semiconductor on which a semiconductor field-effect ion sensor is formed; After coating a photoresist on the wafer, removing the photoresist from the surface of a predetermined semiconductor field effect ion sensor on which an enzyme immobilization film is to be provided by photolithography; (b) applying a hydrophilic coating to the surface of the semiconductor wafer; (c) peeling off the photoresist remaining on the semiconductor wafer after the above process; d) Furthermore, after forming an enzyme-immobilized film on the surface of the semiconductor wafer by spin-coating a protein solution containing an enzyme and a crosslinking agent on the surface of the semiconductor wafer that has undergone the above steps, the semiconductor wafer is coated in an aqueous solution with a predetermined pH range. The enzyme-immobilized film existing on areas other than the surface of the predetermined semiconductor field-effect ion sensor is removed by immersing the sensor in water and performing ultrasonic treatment, thereby immobilizing the enzyme on the surface of the predetermined semiconductor field-effect ion sensor. A method for forming a semiconductor biosensor enzyme-immobilized membrane, characterized by forming a membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61070149A JPS62225941A (en) | 1986-03-27 | 1986-03-27 | Method of forming semiconductor biosensor enzyme immobilized membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61070149A JPS62225941A (en) | 1986-03-27 | 1986-03-27 | Method of forming semiconductor biosensor enzyme immobilized membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62225941A true JPS62225941A (en) | 1987-10-03 |
| JPH0481739B2 JPH0481739B2 (en) | 1992-12-24 |
Family
ID=13423228
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61070149A Granted JPS62225941A (en) | 1986-03-27 | 1986-03-27 | Method of forming semiconductor biosensor enzyme immobilized membrane |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62225941A (en) |
-
1986
- 1986-03-27 JP JP61070149A patent/JPS62225941A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0481739B2 (en) | 1992-12-24 |
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