JPH01248570A - Photosensor using avidin-biotin and manufacture thereof - Google Patents
Photosensor using avidin-biotin and manufacture thereofInfo
- Publication number
- JPH01248570A JPH01248570A JP63074605A JP7460588A JPH01248570A JP H01248570 A JPH01248570 A JP H01248570A JP 63074605 A JP63074605 A JP 63074605A JP 7460588 A JP7460588 A JP 7460588A JP H01248570 A JPH01248570 A JP H01248570A
- Authority
- JP
- Japan
- Prior art keywords
- avidin
- substrate
- membrane
- protein
- film
- 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
- 229960002685 biotin Drugs 0.000 title claims abstract description 10
- 239000011616 biotin Substances 0.000 title claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 239000000758 substrate Substances 0.000 claims abstract description 38
- 150000002632 lipids Chemical class 0.000 claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 28
- 108090001008 Avidin Proteins 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 16
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 12
- 235000020958 biotin Nutrition 0.000 claims abstract description 6
- 239000012528 membrane Substances 0.000 claims description 67
- 230000006287 biotinylation Effects 0.000 claims description 5
- 238000007413 biotinylation Methods 0.000 claims description 5
- 230000003993 interaction Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 108091006004 biotinylated proteins Proteins 0.000 claims description 2
- 210000003764 chromatophore Anatomy 0.000 abstract description 24
- 229920001220 nitrocellulos Polymers 0.000 abstract description 16
- 239000010409 thin film Substances 0.000 abstract description 16
- 239000000020 Nitrocellulose Substances 0.000 abstract description 15
- 239000010408 film Substances 0.000 abstract description 12
- 239000011521 glass Substances 0.000 abstract description 12
- 239000000126 substance Substances 0.000 abstract description 6
- XOLBLPGZBRYERU-UHFFFAOYSA-N tin dioxide Chemical compound O=[Sn]=O XOLBLPGZBRYERU-UHFFFAOYSA-N 0.000 abstract description 5
- 238000000151 deposition Methods 0.000 abstract description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 2
- 239000010931 gold Substances 0.000 abstract description 2
- 229910052737 gold Inorganic materials 0.000 abstract description 2
- 239000002504 physiological saline solution Substances 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 2
- -1 ITO Chemical compound 0.000 abstract 1
- 238000010521 absorption reaction Methods 0.000 abstract 1
- 230000008878 coupling Effects 0.000 abstract 1
- 238000010168 coupling process Methods 0.000 abstract 1
- 238000005859 coupling reaction Methods 0.000 abstract 1
- 230000002195 synergetic effect Effects 0.000 abstract 1
- 230000004044 response Effects 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 9
- 230000000243 photosynthetic effect Effects 0.000 description 8
- 210000002377 thylakoid Anatomy 0.000 description 8
- 230000000638 stimulation Effects 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000190932 Rhodopseudomonas Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000003100 immobilizing effect Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241000192700 Cyanobacteria Species 0.000 description 3
- 108010059332 Photosynthetic Reaction Center Complex Proteins Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229910001887 tin oxide Inorganic materials 0.000 description 2
- 238000007740 vapor deposition Methods 0.000 description 2
- UQZHJQWIISKTJN-YALINYFNSA-N 1-[6-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCNC(=O)CCCC[C@H]1[C@H]2NC(=O)N[C@H]2CS1 UQZHJQWIISKTJN-YALINYFNSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000191025 Rhodobacter Species 0.000 description 1
- 241000191043 Rhodobacter sphaeroides Species 0.000 description 1
- 241001441724 Tetraodontidae Species 0.000 description 1
- 229940072049 amyl acetate Drugs 0.000 description 1
- PGMYKACGEOXYJE-UHFFFAOYSA-N anhydrous amyl acetate Natural products CCCCCOC(C)=O PGMYKACGEOXYJE-UHFFFAOYSA-N 0.000 description 1
- XFLVBMBRLSCJAI-UHFFFAOYSA-N biotin amide Natural products N1C(=O)NC2C(CCCCC(=O)N)SCC21 XFLVBMBRLSCJAI-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000002788 crimping Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- MNWFXJYAOYHMED-UHFFFAOYSA-M heptanoate Chemical compound CCCCCCC([O-])=O MNWFXJYAOYHMED-UHFFFAOYSA-M 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- AMGQUBHHOARCQH-UHFFFAOYSA-N indium;oxotin Chemical group [In].[Sn]=O AMGQUBHHOARCQH-UHFFFAOYSA-N 0.000 description 1
- 238000003475 lamination Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000007738 vacuum evaporation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E10/00—Energy generation through renewable energy sources
- Y02E10/50—Photovoltaic [PV] energy
- Y02E10/549—Organic PV cells
Landscapes
- Light Receiving Elements (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は、光電変換機能を有する蛋白質を含む脂質膜を
利用した感光装置とその製造方法に関し、特に光電変換
機能を有する蛋白質を含む脂質膜を基板上に固定化した
感光装置とその製造方法に関する。Detailed Description of the Invention [Industrial Application Field] The present invention relates to a photosensitive device using a lipid membrane containing a protein having a photoelectric conversion function and a method for manufacturing the same, and particularly to a lipid membrane containing a protein having a photoelectric conversion function. The present invention relates to a photosensitive device in which is immobilized on a substrate and a method for manufacturing the same.
［従来の技術］
光電変換機能を有する蛋白質を含む脂質膜として、たと
えばチラコイド膜が知られている。植物、藍藻、光合成
細菌等の光合成能を有する細胞に含まれるチラコイド膜
は、光合成低能に関する蛋白質および脂質を含む生体膜
である。この種の膜は、方向性を持って配列した光合成
反応中心蛋白質複合体を有し、光を吸収した時、膜を挾
んで電位差を生じる能力を持つ、このため、光電変換素
子等の感光装置への光合成能を有する細胞の利用が期待
されている。[Prior Art] For example, a thylakoid membrane is known as a lipid membrane containing a protein having a photoelectric conversion function. Thylakoid membranes contained in cells with photosynthetic ability such as plants, blue-green algae, and photosynthetic bacteria are biological membranes containing proteins and lipids related to low photosynthetic ability. This type of membrane has a photosynthetic reaction center protein complex arranged in a directional manner, and when it absorbs light, it has the ability to sandwich the membrane and generate a potential difference.For this reason, it is used in photosensitive devices such as photoelectric conversion elements. It is expected that cells with photosynthetic ability will be used for this purpose.
従来の調製方法によると、チラコイド膜は細胞を破砕し
た後に微細な膜断片らしくは膜小胞として得られる場合
が多い。このような膜を挾んで生ずる電位差を直接外部
に取り出すことは困難であった。そこで測定の場合も、
螢光等を利用した間接的測定方法により、主として溶液
状態での光電変換機構等の研究が成されてきた（たとえ
ば、 Ｈｅｔｈｏｄｓ ｉｎ Ｅｎｚｙｍｏｌｏｏｙ
６９巻、４０９−７１５頁（１９８０年）Ａｃａｄｅｌ
ｌｉｃ Ｐｒｅｓｓ）。According to conventional preparation methods, thylakoid membranes are often obtained as minute membrane fragments or membrane vesicles after disrupting cells. It has been difficult to directly extract the potential difference generated by sandwiching such a membrane to the outside. Therefore, in the case of measurement,
Research on photoelectric conversion mechanisms, etc., mainly in solution state, has been carried out by indirect measurement methods using fluorescence, etc. (for example,
69, pp. 409-715 (1980) Acadel
lic Press).
「発明か解決しようとする問題点〕
光電変換機能を有する蛋白質を含む脂質膜を光電変換素
子として利用するには、膜の表裏を揃えて固定化し、膜
を挾んで生ずる電気的信号を取り出す技術の開発が望ま
れる。電気的信号は、たとえば、電位差ないしは電流で
ある。"Problem to be solved by the invention" In order to use a lipid membrane containing a protein with a photoelectric conversion function as a photoelectric conversion element, a technology is required to immobilize the membrane with the front and back sides aligned and extract the electrical signal generated by sandwiching the membrane. The electrical signal is, for example, a potential difference or a current.
本発明の目的は電極を備えた基板上に光電変換機能を有
する蛋白質を含む脂質膜を方向性をもって固定化した感
光装置を提供することである。An object of the present invention is to provide a photosensitive device in which a lipid film containing a protein having a photoelectric conversion function is directionally immobilized on a substrate provided with an electrode.
本発明の他の目的は基板上に光電変換機能を有する蛋白
質を含む脂質膜を方向性をもって固定化し、感光装置を
製造する方法を提供することである。Another object of the present invention is to provide a method for manufacturing a photosensitive device by directionally immobilizing a lipid film containing a protein having a photoelectric conversion function on a substrate.
「問題点を解決するための手段］ 電極を備えた基板上にアビジンを結合させる。“Means for solving problems” Bind avidin onto a substrate with electrodes.
一方、光電変換機能を有する蛋白質を含む脂質膜の特定
の部位を選択的にビオチン化する。On the other hand, specific sites of the lipid membrane containing proteins with photoelectric conversion function are selectively biotinylated.
アビジン化した基板表面にビオチン化した光電変換機能
を有する蛋白質を含む脂質膜を、アビジン−ビオチン相
互作用によって、結合させることにより、脂質膜を方向
性を持たせて固定化する。A lipid membrane containing a biotinylated protein having a photoelectric conversion function is bonded to the surface of an avidinized substrate through an avidin-biotin interaction, thereby immobilizing the lipid membrane with directionality.
光電変換機能を有する蛋白質を含む脂質膜として、たと
えば、植物の葉緑体、藍藻、光合成細菌等の細胞に含ま
れるチラコイド膜が用いられるが。As a lipid membrane containing a protein having a photoelectric conversion function, for example, thylakoid membranes contained in cells such as plant chloroplasts, blue-green algae, and photosynthetic bacteria are used.
チラコイド膜の中でもロドシュードモナス・ビリシス（
ＡＴＣＣＩ ９５６７）、ロドバクタ−スフェロイデス
（ＡＴＣＣ１７０２３＞等の細胞を破砕して得られる標
品であるクロマトホア膜は好適な材料である。Among the thylakoid membranes, Rhodopseudomonas viricis (
A chromatophore membrane, which is a standard product obtained by disrupting cells of Rhodobacter spheroides (ATCCI 9567), Rhodobacter sphaeroides (ATCC 17023), etc., is a suitable material.
電極には、インジウム−錫・酸化物（ＩＴＯ）。The electrode is indium-tin oxide (ITO).
酸化錫（Ｓｎ０２）、金属蒸着膜、半導体等を用いるこ
とができる。電極基板の作成は、たとえば。Tin oxide (Sn02), metal vapor deposition film, semiconductor, etc. can be used. For example, create an electrode substrate.
真空蒸着等の方法により、基板上に導電性膜を形成する
こと等により行うことかできる。This can be done by forming a conductive film on the substrate using a method such as vacuum evaporation.
電極基板へのアビジン分子の結合は、たとえば。The binding of avidin molecules to electrode substrates, e.g.
化学結合、吸着等で行うことかできる。This can be done by chemical bonding, adsorption, etc.
種々のビオチン化試薬で、光電変換機能を有する蛋白質
を化学修飾する場合９条件を選択することにより、特定
の部位を選択的にビオチン化することができる。When a protein having a photoelectric conversion function is chemically modified using various biotinylation reagents, a specific site can be selectively biotinylated by selecting nine conditions.
このようにしてビオチン化した脂質膜を、アビジン化し
た電極基板に取り付ける。The thus biotinylated lipid membrane is attached to an avidinized electrode substrate.
［作用］ アビジンとビオチンとは強い特異的相互作用を有する。[Effect] Avidin and biotin have a strong specific interaction.
光電変換機能を有する蛋白質を含む脂質膜の蛋白質複合
体の特定部位をビオチン化すると光電変換機能を有する
蛋白質を含む脂質膜がアビジン分子との結合性に関して
方向性を付与される。When a specific site of a protein complex of a lipid membrane containing a protein having a photoelectric conversion function is biotinylated, the lipid membrane containing a protein having a photoelectric conversion function is given directionality with respect to binding with an avidin molecule.
アビジン化した基板表面にはビオチンが結合するので、
基板上に方向性をもって光電変換機能を有する蛋白質を
含む脂質膜を固定化できる。Since biotin binds to the avidinized substrate surface,
A lipid film containing a protein having a photoelectric conversion function can be directionally immobilized on a substrate.
［実施例コ
光電変換機能を有する蛋白質を含む脂質膜の材料はたと
えばチラコイド膜であり、植物、藍藻、光合成細菌等の
細胞より調製することができる。[Example] The material of the lipid membrane containing a protein having a photoelectric conversion function is, for example, a thylakoid membrane, which can be prepared from cells of plants, blue-green algae, photosynthetic bacteria, etc.
中でも光合成細菌ロドシュードモナスピリシス（ＡＴＣ
Ｃ１９５６７） 、Ｑ ト／＜’；’ タース７工０イ
デス（ＡＴＣＣ１７０２３）等の細胞を破砕して得られ
るクロマトホア膜小胞は、好適な材料である。これらの
クロマトホア膜においては、光合成を行う反応中心の蛋
白質複合体のサブユニットにはり、Ｍ、Ｈ等のサブユニ
ットがある。タロマトホア膜懸濁液に、ビオチン基を有
する試薬を反応させ、蛋白質の化学修飾を行うと、Ｈサ
ブユニットを選択的にビオチン化する事ができる。この
ようにしてビオチン化を行ったタロマトホアは、アビジ
ンを介して基板上に方向性をもって固定化することが可
能である。Among them, the photosynthetic bacterium Rhodopseudomonas spirisis (ATC)
Chromatophore membrane vesicles obtained by disrupting cells such as C19567) and Q<'><';' Tase70oides (ATCC17023) are suitable materials. In these chromatophore membranes, there are subunits such as M and H among the subunits of the protein complex that is the reaction center for photosynthesis. When a talomatophore membrane suspension is reacted with a reagent having a biotin group to chemically modify the protein, the H subunit can be selectively biotinylated. Talomatophore biotinylated in this manner can be directionally immobilized on a substrate via avidin.
すなわち、基板表面に化学結合、吸着等によってアビジ
ン分子を結合させ、さらにビオチン化したタロマトホア
膜を作用させる。ビオチン−アビジンの特異的な相互作
用によって、クロマトホア膜が方向性を持って基板に結
合する。That is, avidin molecules are bonded to the substrate surface by chemical bonding, adsorption, etc., and then a biotinylated talomatophore membrane is applied. The specific biotin-avidin interaction causes the chromatophore membrane to directionally bind to the substrate.
このようにしてクロマトホア膜を固定化させ。In this way, the chromatophore membrane was immobilized.
電極を備えた感光装置について、光〜電位応答ないし光
−電流応答等の光電応答を検出利用する。For photosensitive devices equipped with electrodes, a photoelectric response such as a photo-potential response or a photo-current response is used for detection.
以下に１図面を参服して本発明をさらに詳細に説明する
。The present invention will be explained in more detail below with reference to one drawing.
［クロマトホア膜の調製］
光合成細菌であるロドシュードモナスビリシス（ＡＴＣ
Ｃ１９５６７）を、嫌気状態で、光照射下、３０°Ｃで
培養した。得られた菌体より以下の手順によってタロマ
トホア膜を調製した。すなわち、菌体をフレンチプレス
によって破砕し、庶稠密度勾配遠心等の遠心分画法によ
って膜画分を調製し、５０ｍＭ ノ炭酸ナトリウムＮａ
ＨＣ０３（１）８８．９）水溶液に透析しな（Ａｒｃｈ
、 Ｂｉｏｃｈｅｍ、 Ｂｉｏｐｈｙｓ、、２２３巻２
８２−２９０頁（１９７９年））。このようにして第１
図（Ａ）に示すようなタロマトホア膜を調製した。脂質
膜２に光合成反応中心の蛋白質複合体１が埋め込まれた
構造を有している。[Preparation of chromatophore membrane] Photosynthetic bacterium Rhodopseudomonas bilisis (ATC
C19567) was cultured under anaerobic conditions at 30°C under light irradiation. A talomatophore membrane was prepared from the obtained bacterial cells according to the following procedure. That is, the bacterial cells were disrupted using a French press, a membrane fraction was prepared by a centrifugal fractionation method such as concentrated density gradient centrifugation, and 50 mM sodium carbonate Na
HC03(1)88.9) Do not dialyze into aqueous solution (Arch
, Biochem, Biophys, Volume 223 2
82-290 (1979)). In this way the first
A talomatophore membrane as shown in Figure (A) was prepared. It has a structure in which a protein complex 1 serving as a photosynthetic reaction center is embedded in a lipid membrane 2.
［クロマトホア膜のビオチン化］
タロマトホア膜のＱｆｆｉ液（Ａ１０２０＝１０）に、
８ｍｑ／ｍ ｌスルホスクシニミジル６−（ビオチンア
ミド）ヘキサノエートを加え、３０℃で３０分処理し、
膜蛋白質のビオチン化修飾を行う、ビオチン化したクロ
マトホア膜は第１図（Ｂ）に示すように、方向性をもっ
て、ビオチン基３を備える。ビオチン化後のクロマトホ
ア膜は、リン酸緩衝液（ｐＨ７，４）を含む生理的食塩
水（ＰＢＳ）に恕濁する。[Biotinylation of chromatophore membrane] In Qffi solution (A1020=10) of talomatophore membrane,
Add 8 mq/ml sulfosuccinimidyl 6-(biotinamido)hexanoate and treat at 30°C for 30 minutes.
A biotinylated chromatophore membrane, in which membrane proteins are modified by biotinylation, is provided with biotin groups 3 in a directional manner, as shown in FIG. 1(B). The biotinylated chromatophore membrane is suspended in physiological saline (PBS) containing phosphate buffer (pH 7,4).
［アビジンの基板表面への固定化コ
固定化の基板としては、ガラス基板４に酸化錫（Ｓｎ０
２ ）、ＩＴＯ，金等の導電性物質を蒸着させて薄膜状
電極５としたものを用い、第１図（Ｃ）に示すように、
その表面に蛋白質吸着能力の高いニトロセルロース等の
物質でさらに薄膜６をつくり、第１図（Ｄ）に示すよう
にアビジン７を吸着させる方法を用いる。このような薄
膜の作成については、ニトロセルロースの０．００２％
酢酸アミル溶液（コロジオン溶液）５０マイクロリツト
ルを２平方センチメートルの電極面状に載せて乾燻同化
させる方法、及び２％コロジオン溶液２０マイクロリツ
トルを水面状に展開して１溶媒を蒸発させて固化した薄
膜を電極面に圧着する方法等か用いられる。これらの電
極を２０μｇ／ｍ１アビジンを含むＰＢＳで３０℃で２
０分処理を行い、アビジン分子７を吸着させる。上述の
ニトロセルロース薄膜６を介した固定化法は各種材料か
らなる基板へのアビジン固定化に広く用いることができ
る。[As a substrate for co-immobilization of avidin on the substrate surface, tin oxide (Sn0
2), using a thin film electrode 5 made by depositing a conductive substance such as ITO or gold, as shown in FIG. 1(C),
A method is used in which a thin film 6 is further formed on the surface of a substance such as nitrocellulose having a high protein adsorption capacity, and avidin 7 is adsorbed as shown in FIG. 1(D). For the creation of such thin films, 0.002% of nitrocellulose
A method of placing 50 microliters of amyl acetate solution (collodion solution) on a 2 square centimeter electrode surface and dry-smoking assimilation, and a thin film solidified by spreading 20 microliters of 2% collodion solution on a water surface and evaporating one solvent. A method such as crimping the electrode onto the electrode surface is used. These electrodes were incubated with PBS containing 20 μg/ml avidin at 30°C for 2 hours.
Treatment is performed for 0 minutes to adsorb avidin molecule 7. The above-described immobilization method via the nitrocellulose thin film 6 can be widely used for immobilizing avidin on substrates made of various materials.
電極の素材によっては、ホルムアルデヒド、グルタルア
ルデヒド等の架橋試薬を用いて、アビジン分子を電極平
面に化学的に結合させることも可能である。Depending on the material of the electrode, it is also possible to chemically bond avidin molecules to the electrode plane using a crosslinking reagent such as formaldehyde or glutaraldehyde.
ニトロセルロース薄膜は非共有結合でアビジン分子を吸
着し、架橋試薬の残基をつけた場合はアビジン分子と共
有結合すると考えられる。It is thought that the nitrocellulose thin film adsorbs avidin molecules non-covalently, and when attached with residues of a cross-linking reagent, covalently bonds with the avidin molecules.
［ビオチン化りロマトホア膜のアビジン化基板表面への
固定化］
アビジン化ガラス基板をビオチン化クロマ１ヘホア膜（
Ａ１０２０＝５．０）で３０°Ｃで２０分処理し、反応
ｆ＆ Ｐ Ｂ Ｓで洗浄して未結合のクロマトホア膜を
除く０以上の操作により、第１図（Ｅ）に示すように、
クロマトホア膜８を固定化することができる。[Immobilization of a biotinylated chromatophor membrane onto the surface of an avidinized substrate] An avidinized glass substrate was immobilized with a biotinylated chromatophor membrane (
A1020 = 5.0) at 30 °C for 20 minutes, and washed with reaction f&PBS to remove unbound chromatophore membranes. As shown in Figure 1 (E),
The chromatophore membrane 8 can be immobilized.
このｔ＊１．７％ホルムアルデヒドを含むＰＢＳで３０
℃で３０分処理を行い、ＰＢＳで洗浄してから第１図（
Ｆ）に示すようにアビジンをを含むＰＢＳで処理する段
階に戻って、クロマトホア膜１０の固定化を行う。この
ように複数回の固定化を行えば、クロマトホア膜の多層
積層を行うことができる。第１図（Ｅ）は、ガラス基板
４上の５ｎ０２電８ｉ！５の表面にニトロセルロース薄
膜６を形成し、その上にアビジン７を吸着させ夕ロマト
ホア膜８を１回固定化した場合を示し、第１図（Ｆ）は
、同様に２回固定化積層した場合を示す。This t*30 in PBS containing 1.7% formaldehyde.
After treatment at ℃ for 30 minutes and washing with PBS, Figure 1 (
As shown in F), returning to the step of treatment with PBS containing avidin, the chromatophore membrane 10 is immobilized. By performing immobilization multiple times in this manner, multilayer stacking of chromatophore membranes can be performed. FIG. 1(E) shows the 5n02 electric 8i! on the glass substrate 4! Figure 1 (F) shows a case in which a nitrocellulose thin film 6 is formed on the surface of the nitrocellulose film 6, on which avidin 7 is adsorbed and a chromatophore membrane 8 is immobilized once. Indicate the case.
また、積層の方法としては固定化クロマトホア膜の上に
コロジオン薄膜を重ね、その上に改めて固定化を行う方
法も用いられる。Furthermore, as a method of lamination, a method of stacking a collodion thin film on an immobilized chromatophore membrane and then immobilizing it again is also used.
このような方法で、５ｎ０２電極５を備えたガラス基板
４上にロドシュードモナスビリシスのクロマトホア膜８
．１０を１０回積層した感光装置の吸収スペクトルを第
２図に示す、特徴的な光吸収が行われていることが判る
。In this way, a chromatophore film 8 of Rhodopseudomonas bilisis was formed on a glass substrate 4 equipped with a 5n02 electrode 5.
．． FIG. 2 shows the absorption spectrum of a photosensitive device in which 10 layers were stacked 10 times, and it can be seen that characteristic light absorption occurs.
上述の固定化方法により、チラコイド膜を方向性をもっ
て固定化できる。By the above immobilization method, the thylakoid membrane can be immobilized with directionality.
積層することもできる。チラコイド膜のみに限らず、蛋
白質を含む脂質膜を方向性を持って固定化できるものと
考えられる。It can also be laminated. It is thought that it is possible to immobilize not only thylakoid membranes but also lipid membranes containing proteins with directionality.
タロマトホア膜を固定化した基板を１％牛血清アルブミ
ン（ＢＳＡ）水溶液に浸した後、乾燥させる。The substrate on which the talomatophore membrane is immobilized is immersed in a 1% bovine serum albumin (BSA) aqueous solution and then dried.
［光電応答の検出コ
基板上に固定化した脂質膜上に、対極となる電極を取り
付けて、光刺激を与え、光な応答による両ｅ ｋ間の電
気信号を取り出すことができる。たとえば、電位、電流
の変化を測定する。対極は。[Detection of Photoelectric Response A counter electrode is attached to the lipid membrane immobilized on the co-substrate, optical stimulation is applied, and an electrical signal between both e and k due to the optical response can be extracted. For example, measuring changes in potential and current. The opposite is true.
感光装置の表面に水銀玉を載せる。金属薄膜を圧着する
。金属薄膜を蒸着する等の方法で形成できる。光刺激の
光源は、太陽光等自然のものでもよいし、ストロボラン
プ、発光ダイオード（ＬＥＤ）、レーザ、アーク燈等で
もよい。A mercury ball is placed on the surface of the photosensitive device. Press the metal thin film. It can be formed by a method such as vapor deposition of a metal thin film. The light source for optical stimulation may be a natural source such as sunlight, or may be a strobe lamp, a light emitting diode (LED), a laser, an arc lamp, or the like.
第３図に感光装置の光電応答検出系の概略を示す、ガラ
ス基板１５上の５ｎ０２電極１４上にニトロセルロース
薄１Ｂ５１！１３を介してクロマトホア膜層１２を固定
化積層した。その上に、水銀玉をのせる等の方法で対極
１１を形成した。FIG. 3 schematically shows a photoelectric response detection system of a photosensitive device. A chromatophore membrane layer 12 was fixed and laminated on a 5N02 electrode 14 on a glass substrate 15 via a thin nitrocellulose 1B51!13. On top of that, a counter electrode 11 was formed by placing a mercury ball or the like.
図中下側より、ストロボランプ、ＬＥＤ等の光源２０か
ら光刺激をガラス基板１５．５ｎ０２電＠１４、ニトロ
セルロース薄［１３を介して、クロマトホア膜層１２へ
与える。光刺激によって画電極１１．１４間に生じる電
位変化を導線１６．１つで取り出し、差動アンプ１７を
介してオシロスコープ１８で観察した。From the bottom of the figure, light stimulation is applied from a light source 20 such as a strobe lamp or LED to the chromatophore membrane layer 12 via a glass substrate 15.5n02 electrode@14 and a nitrocellulose thin layer 13. A potential change occurring between the picture electrodes 11.14 due to optical stimulation was taken out using a conductive wire 16.1 and observed with an oscilloscope 18 via a differential amplifier 17.
第４図に光刺激に対する感光装置の電位応答の例を示す
、ストロボランプ光の刺激に対して、ミリ秒以下の速い
応答の立ち上がりがみられた６本例においては、１０回
固定化を行った感光装置を用いた。Figure 4 shows an example of the potential response of a photosensitive device to light stimulation. In the 6 cases in which a rapid response of less than a millisecond was observed in response to strobe lamp light stimulation, the immobilization was performed 10 times. A photosensitive device was used.
［発明の効果］
光な変換機能を有する蛋白質を含む脂質膜を方向性をも
って固定化できる。[Effects of the Invention] A lipid membrane containing a protein having a light conversion function can be directionally immobilized.
これにより、感光装置が実現できる。Thereby, a photosensitive device can be realized.
第１図はタロマトホア膜の固定化を示す概略図であり、
（Ａ＞は未処理のタロマトホア膜、
（Ｂ）はビオチン化したタロマトホア膜、（Ｃ）はニト
ロセルロース膜を張った５ｎ０２電極を備えたガラス基
板、
（Ｄ）はニトロセルロース膜を張り、アビジンを吸着さ
せた５ｎ０２電極を備えたガラス基板、（Ｅ）はガラス
基板上の５ｎ０２電極上にニトロセルロース薄膜を張り
、アビジンを吸着させタロマトホア膜を固定化した場合
。
（Ｆ）は（Ｅ）と同様の固定化を２回行い１積層した場
合を示す。
第２図は５ｎ０２電極上にニトロセルロース薄膜を張り
、タロマトホア膜を１０回固定化積層した７ｉ、ＰＢＳ
中にて測定した吸収スペクトルである。
第３図は感光装置の光電応答検出系の概略図である。
第４図はストロボランプ光の光刺激に対する感光装置の
電位応答を示す図である。タロマトホア膜を１０回固定
化積層させた感光装置を用いて測定した。
符号の説明
１ 光合成反応中心蛋白質複合体
２ 脂質膜
３ ビオチン化試薬ニスルホスクシニミジル６−（ビオ
チンアミド）ヘキサノエート
４ ガラス基板
５ ５ｎ０２電極
６ ニトロセルロース薄膜
７ ニトロセルロース薄膜に吸着されたアビジン
８ 固定化１層目の脂質膜
９ 固定化クロマトホア膜上に結合したアビジン
１０ 固定化２層目の脂質膜
１１ 対極となる電極
１２ 固定化、積層されたクロマトホア膜層１３ ニト
ロセルロース薄膜
１４ ５ｎ０２電極
１５ ガラス基板
１６ 対極に接続された導線
１７ 差動アンプ
１８ オシロスコープ
１９ ５ｎ０２電５に接続された導線
２０ 光源
指定代理人 工業技術院微生物工業技術研究所長鈴木智
雄
復代理人 弁理士 高橋敬四部Figure 1 is a schematic diagram showing the immobilization of talomatophore membranes, (A> is an untreated talomatophore membrane, (B) is a biotinylated talomatophore membrane, and (C) is a 5n02 electrode covered with a nitrocellulose membrane. (D) is a glass substrate with a 5n02 electrode on which a nitrocellulose membrane is attached and adsorbed avidin; (E) is a glass substrate with a nitrocellulose thin film attached to the 5n02 electrode on the glass substrate to which avidin is adsorbed and talomatophore is attached. When the membrane is immobilized. (F) shows the case where the same immobilization as in (E) is performed twice and one layer is stacked. Figure 2 shows a nitrocellulose thin film stretched on the 5n02 electrode, and a talomatophore membrane is immobilized 10 times. layered 7i, PBS
This is an absorption spectrum measured inside. FIG. 3 is a schematic diagram of a photoelectric response detection system of a photosensitive device. FIG. 4 is a diagram showing the potential response of the photosensitive device to optical stimulation of strobe lamp light. The measurement was performed using a photosensitive device in which talomatophore membranes were fixed and laminated 10 times. Explanation of symbols 1 Photosynthetic reaction center protein complex 2 Lipid membrane 3 Biotinylation reagent nisulfosuccinimidyl 6-(biotinamide) hexanoate 4 Glass substrate 5 5n02 electrode 6 Nitrocellulose thin film 7 Avidin 8 adsorbed on the nitrocellulose thin film Immobilization First layer lipid membrane 9 Avidin 10 bound on the immobilized chromatophore membrane Second immobilized lipid membrane 11 Counter electrode 12 Immobilized and stacked chromatophore membrane layer 13 Nitrocellulose thin film 14 5n02 electrode 15 Glass Board 16 Leading wire 17 connected to the counter electrode Differential amplifier 18 Oscilloscope 19 Leading wire 20 connected to 5n02 electric wire 5 Light source Designated agent Tomoo Suzuki Director, Institute of Microbial Technology, Agency of Industrial Science and Technology Patent attorney Keiji Takahashi
Claims (2)
された蛋白質を含む脂質膜と、 アビジンを固定化した電極基板と を含む感光装置。(1) A photosensitive device that has a photoelectric conversion function and includes a lipid membrane containing a protein biotinylated at a specific site and an electrode substrate on which avidin is immobilized.
アビジン化する工程と、 光電変換機能を有する蛋白質を含む脂質膜をビオチンを
含む処理剤で処理し、蛋白質のビオチン化修飾を行う工
程と、 表面をアビジン化した基板にビオチン化修飾した光電変
換機能を有する蛋白質を含む脂質膜を作用させ、アビジ
ン−ビオチンの相互作用を利用して光電変換機能を有す
る蛋白質を含む脂質膜を基板上に固定化する工程と、 を含む感光装置の製造方法。(2) The step of treating the substrate with a treatment agent containing avidin to avidinize the surface, and treating the lipid membrane containing a protein with a photoelectric conversion function with a treatment agent containing biotin to modify the protein with biotinylation. A lipid membrane containing a biotinylated protein with a photoelectric conversion function is applied to a substrate whose surface has been converted to avidin, and the lipid membrane containing a protein with a photoelectric conversion function is applied to the substrate using the avidin-biotin interaction. A method for manufacturing a photosensitive device, comprising:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63074605A JPH0719927B2 (en) | 1988-03-30 | 1988-03-30 | Photoelectric conversion device using avidin-biotin system and manufacturing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63074605A JPH0719927B2 (en) | 1988-03-30 | 1988-03-30 | Photoelectric conversion device using avidin-biotin system and manufacturing method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01248570A true JPH01248570A (en) | 1989-10-04 |
| JPH0719927B2 JPH0719927B2 (en) | 1995-03-06 |
Family
ID=13551965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63074605A Expired - Lifetime JPH0719927B2 (en) | 1988-03-30 | 1988-03-30 | Photoelectric conversion device using avidin-biotin system and manufacturing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0719927B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05183150A (en) * | 1991-12-27 | 1993-07-23 | Oki Electric Ind Co Ltd | Arrangement of ultrafine particle |
| WO2002042411A1 (en) * | 2000-11-22 | 2002-05-30 | Japan Science And Technology Corporation | Apparatus for microscopic observation of long-term culture of single cell |
| JP2005274141A (en) * | 2004-03-22 | 2005-10-06 | Sanyo Electric Co Ltd | Membrane protein fixing substrate and membrane protein fixing method |
| JP2014081365A (en) * | 2012-09-26 | 2014-05-08 | Fujita Gakuen | Method for measuring antibody with protein on cell surface as antigen |
| CN113874728A (en) * | 2019-01-30 | 2021-12-31 | 代表亚利桑那大学的亚利桑那校董事会 | Bioelectronic circuits, systems, and methods of making and using the same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63295600A (en) * | 1986-11-20 | 1988-12-01 | ハンス オー．リビ | Lipid-protein composition and product and preparation of them |
-
1988
- 1988-03-30 JP JP63074605A patent/JPH0719927B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63295600A (en) * | 1986-11-20 | 1988-12-01 | ハンス オー．リビ | Lipid-protein composition and product and preparation of them |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05183150A (en) * | 1991-12-27 | 1993-07-23 | Oki Electric Ind Co Ltd | Arrangement of ultrafine particle |
| WO2002042411A1 (en) * | 2000-11-22 | 2002-05-30 | Japan Science And Technology Corporation | Apparatus for microscopic observation of long-term culture of single cell |
| US7092154B2 (en) | 2000-11-22 | 2006-08-15 | Japan Science And Technology Corporation | Apparatus for microscopic observation of long-term culture of single cell |
| JP2005274141A (en) * | 2004-03-22 | 2005-10-06 | Sanyo Electric Co Ltd | Membrane protein fixing substrate and membrane protein fixing method |
| JP2014081365A (en) * | 2012-09-26 | 2014-05-08 | Fujita Gakuen | Method for measuring antibody with protein on cell surface as antigen |
| CN113874728A (en) * | 2019-01-30 | 2021-12-31 | 代表亚利桑那大学的亚利桑那校董事会 | Bioelectronic circuits, systems, and methods of making and using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0719927B2 (en) | 1995-03-06 |
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