JP2573429B2 - Bio element and method for discriminating and quantifying chemical substance using the same - Google Patents
Bio element and method for discriminating and quantifying chemical substance using the sameInfo
- Publication number
- JP2573429B2 JP2573429B2 JP9699791A JP9699791A JP2573429B2 JP 2573429 B2 JP2573429 B2 JP 2573429B2 JP 9699791 A JP9699791 A JP 9699791A JP 9699791 A JP9699791 A JP 9699791A JP 2573429 B2 JP2573429 B2 JP 2573429B2
- Authority
- JP
- Japan
- Prior art keywords
- liposome
- membrane potential
- chemical substance
- change
- fluorescence intensity
- 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 - Fee Related
Links
- 239000000126 substance Substances 0.000 title claims description 46
- 238000000034 method Methods 0.000 title claims description 12
- 239000002502 liposome Substances 0.000 claims description 42
- 239000012528 membrane Substances 0.000 claims description 39
- 230000008859 change Effects 0.000 claims description 32
- 239000007864 aqueous solution Substances 0.000 claims description 21
- 102000004310 Ion Channels Human genes 0.000 claims description 14
- 239000007850 fluorescent dye Substances 0.000 claims description 13
- 239000000975 dye Substances 0.000 claims description 3
- 108010009551 Alamethicin Proteins 0.000 description 11
- LGHSQOCGTJHDIL-UTXLBGCNSA-N alamethicin Chemical compound N([C@@H](C)C(=O)NC(C)(C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)NC(C)(C)C(=O)N[C@H](C(=O)NC(C)(C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NC(C)(C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NC(C)(C)C(=O)NC(C)(C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](CO)CC=1C=CC=CC=1)C(C)C)C(=O)C(C)(C)NC(=O)[C@@H]1CCCN1C(=O)C(C)(C)NC(C)=O LGHSQOCGTJHDIL-UTXLBGCNSA-N 0.000 description 11
- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 235000001258 Cinchona calisaya Nutrition 0.000 description 4
- 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 4
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 4
- 230000010365 information processing Effects 0.000 description 4
- 229960000948 quinine Drugs 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 239000003012 bilayer membrane Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 229960004793 sucrose Drugs 0.000 description 3
- BTUZNIQVZBANAX-SWQYCTROSA-N (13Z,17Z,21Z,29Z)-48-(8-aminooctan-2-yl)-8,10,16,20,24,26,28,32,36,38,40,42,44,46-tetradecahydroxy-9,15,17,19,21,25,31,33,39,41,47-undecamethyl-23-[(2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-1-oxacyclooctatetraconta-13,17,21,29-tetraen-2-one Chemical compound CC1CCC(CC(C(C(C(C(CC(CC(C(C(OC(=O)CCCCCC(C(C(CC/C=C\C(C(/C(=C\C(C(/C(=C\C(C(C(C(CC(/C=C\C(C1O)C)O)O)C)O)O[C@@H]2[C@H]([C@H]([C@@H]([C@H](O2)CO)O)O)O)/C)O)C)/C)O)C)O)C)O)C(C)CCCCCCN)C)O)O)O)C)O)C)O)O BTUZNIQVZBANAX-SWQYCTROSA-N 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 235000013681 dietary sucrose Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003909 pattern recognition Methods 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- PRYIJAGAEJZDBO-ZEQHCUNVSA-N 5,6alpha-epoxy-5alpha-cholestan-3beta-ol Chemical compound C([C@]12O[C@H]1C1)[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 PRYIJAGAEJZDBO-ZEQHCUNVSA-N 0.000 description 1
- 101100301837 Arabidopsis thaliana RH46 gene Proteins 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006386 memory function Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000608 photoreceptor cell Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000004382 visual function Effects 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION
【０００１】[0001]
【産業上の利用分野】この発明は、生物の情報処理機能
を模倣したバイオ素子及びこれを使用して化学物質の判
別及び定量の双方又は一方を行う方法に関するものであ
る。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bioelement imitating the information processing function of living organisms and a method for discriminating and / or quantifying chemical substances using the same.
【０００２】[0002]
【従来の技術】従来のコンピューターは、シリコン半導
体素子等によって構成されており、フォン・ノイマン
（Ｖｏｎ Ｎｅｕｍａｎｎ）方式によって直列型の論理
演算を実行するもの（以下、「ノイマン型コンピュータ
ー」と称する。）であった。しかし、この方式は、迅速
な論理演算を行うことは出来るが、多数の情報処理を同
時に並行して行うことが本質的に困難であるという欠点
を有していた。2. Description of the Related Art A conventional computer is constituted by a silicon semiconductor device or the like, and executes a serial logic operation by a Von Neumann system (hereinafter, referred to as a "Neumann computer"). Met. However, this method has a drawback that although it is possible to perform a quick logical operation, it is inherently difficult to perform a large number of information processes simultaneously in parallel.
【０００３】これに対し高等生物は、周知の通り、パタ
ーン認識等を容易に行う。従って、脳に見られるような
パターン認識や学習・記憶機能がどのような原理に基い
て実行されているのかを解明しそれを模倣すれば、ノイ
マン型コンピューターでは満足し得なかった様々な機能
を持つコンピュータ例えばバイオコンピュータの実現が
可能になると期待される。そこで、近年、生体機能の模
倣に関する研究が活発化している。On the other hand, higher organisms easily perform pattern recognition and the like, as is well known. Therefore, by elucidating the principles on which pattern recognition and learning and memory functions such as those found in the brain are performed and imitating them, various functions that could not be satisfied with Neumann-type computers can be achieved. It is expected that the realization of a computer having, for example, a biocomputer will be possible. Therefore, in recent years, research on imitation of biological functions has been activated.
【０００４】例えばこの出願人に係る特開昭６３−１１
１４２８号公報には、バイオコンピューターのセンサー
としての使用が期待出来るバイオ素子が開示されてい
る。[0004] For example, Japanese Patent Application Laid-Open No.
Japanese Patent No. 1428 discloses a bioelement that can be expected to be used as a sensor in a biocomputer.
【０００５】このバイオ素子は、生体の視覚機能を模倣
したものであり、生体類似物質を用いて形成したリポソ
ームであって入射した光をイオン又は化学物質に変換す
るリポソームを基板上に二次元配列して構成されてい
た。これにより、生体の網膜上での視細胞の電位変化の
場と同様な場を得ようとしている。[0005] This bio-element simulates the visual function of a living body, and a liposome formed by using a bio-analogous substance, which converts incident light into ions or chemical substances, is two-dimensionally arranged on a substrate. Was configured. This seeks to obtain a field similar to the potential change field of the photoreceptor cells on the retina of the living body.
【０００６】[0006]
【発明が解決しようとする課題】しかしながら、従来の
バイオ素子では、入射した光をイオン又は化学物質に変
換することはできたが、化学物質による刺激には応答で
きなかった。However, in the conventional biodevice, incident light can be converted into ions or chemical substances, but it cannot respond to stimulation by the chemical substances.
【０００７】化学物質による刺激に対して応答するバイ
オ素子が実現出来れば、これは化学物質の判別や定量に
利用できるので有用である。また、例えばバイオコンピ
ューターが実現された場合にはその機能向上に寄与する
ので有用と考えられる。If a biodevice that responds to a stimulus by a chemical substance can be realized, it is useful because it can be used for discrimination and quantification of a chemical substance. Further, for example, when a biocomputer is realized, it is considered useful because it contributes to the improvement of its function.
【０００８】この発明はこのような点に鑑みなされたも
のであり、従ってこの発明の目的は、化学物質に応答す
るバイオ素子であってその応答が蛍光強度変化として現
われるバイオ素子を提供すること、及び、このバイオ素
子を使用した化学物質の判別定量方法を提供することに
ある。SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and it is therefore an object of the present invention to provide a biodevice which responds to a chemical substance, wherein the response appears as a change in fluorescence intensity. Another object of the present invention is to provide a method for discriminating and quantifying a chemical substance using the bioelement.
【０００９】[0009]
【課題を解決するための手段】この目的の達成を図るた
め、この出願の第一発明であるバイオ素子によれば、膜
電位配向性イオンチャンネルを組み込んだリポソーム
と、該リポソームの膜電位に応答する膜電位感受性蛍光
色素とを水溶液中に含んで成ることを特徴とする。In order to achieve this object, according to the bioelement of the first invention of the present application, a liposome incorporating a membrane potential-oriented ion channel, and a liposome which responds to the membrane potential of the liposome. And a membrane potential-sensitive fluorescent dye.
【００１０】また、この出願の第二発明の化学物質の判
別定量方法によれば、膜電位配向性イオンチャンネルを
組み込んだリポソームと、該リポソームの膜電位に応答
する膜電位感受性蛍光色素と、化学物質とを水溶液中に
混在させ、該化学物質の種類及び濃度並びに前述のイオ
ンチャンネルの開放及び閉鎖に起因する前述のリポソー
ムの電位変化に応じて前述の膜電位感受性色素で生じる
蛍光強度変化を測定し、該蛍光強度変化に基いて前述の
化学物質の判別及び定量の双方又は一方を行うことを特
徴とする。Further, according to the method for discriminating and quantifying a chemical substance according to the second invention of this application, a liposome incorporating a membrane potential-oriented ion channel, a membrane potential-sensitive fluorescent dye responding to the membrane potential of the liposome, A substance is mixed in an aqueous solution, and the type and concentration of the chemical substance and the change in fluorescence intensity generated by the membrane potential-sensitive dye according to the change in potential of the liposome caused by opening and closing of the ion channel are measured. In addition, the present invention is characterized in that both or one of the above-described determination and quantification of the chemical substance is performed based on the change in the fluorescence intensity.
【００１１】なお、第一及び第二発明の実施に当たり、
膜電位配向性イオンチャンネルとしては公知の種々のも
のを用いることができる。例えば、アラメチシン、モナ
ゾマイシン等を挙げることが出来る。In implementing the first and second inventions,
Various known membrane potential orientation ion channels can be used. For example, alamethicin, monazomycin and the like can be mentioned.
【００１２】また、第二発明の実施に当たり、蛍光強度
変化に基いて判別・定量を行う場合の基いてとは、例え
ば蛍光強度変化の周期を測定しこの周期を種々の化学物
質の予め測定しておいた同様な周期と照合すること、或
は、蛍光強度変化の振幅を種々の化学物質の予め測定し
ておいた同様な振幅と照合すること等であることが出来
る。もちろんこれ以外の方法でも良い。In the second embodiment of the present invention, the determination and quantification based on the change in the fluorescence intensity is based on, for example, measuring the cycle of the change in the fluorescence intensity and measuring this cycle in advance for various chemical substances. It can be compared with the same cycle as described above, or the amplitude of the change in fluorescence intensity can be compared with the previously measured similar amplitude of various chemical substances. Of course, other methods may be used.
【００１３】また、ここでいう化学物質は、例えば匂い
物質、にがみ物質、酸味物質、甘み物質など種々のもの
であることが出来る。The chemical substance mentioned here can be various substances such as, for example, odorous substances, bite substances, sour substances, and sweet substances.
【００１４】[0014]
【作用】この出願の第一発明のバイオ素子の構成によれ
ば以下のような作用が得られる。According to the structure of the bioelement of the first invention of the present application, the following effects can be obtained.
【００１５】このバイオ素子に具わるリポソームでは化
学物質の刺激によって膜電位が上昇或は下降する。ま
た、このリポソームに組み込まれている膜電位配向性イ
オンチャンネルは、リポソームの膜電位が所定電位にな
ると閉鎖状態から開放状態へ（イオンチャンネルの種類
によっては開放状態から閉鎖状態へ）と変化する。イオ
ンチャンネルでこのような状態変化が起きるとリポソー
ムの膜電位は無刺激時の電位（以下、ここでは「ベース
電位」という。）に復帰する。この復帰によりチャンネ
ルは再び閉じる。そして、このリポソームでは、ベース
電位と前記所定電位との間において膜電位変化が繰り返
えされる。このベース電位はリポソームに刺激を与える
化学物質の種類・濃度によって異なる。このため、化学
物質の種類、濃度がリポソームのベース電位を低い値に
するものの場合と、リポソームのベース電位を高い値に
するものの場合とでは、ベース電位及び前記所定電位間
の電位差に違いが生じるので、リポソームの膜電位変化
が起こる周期も違ってくる。すなわち、ベース電位及び
前記所定電位間の電位差が大きい程膜電位変化が繰り返
される周期が長くなる。In the liposome provided in the bioelement, the membrane potential rises or falls due to stimulation of a chemical substance. When the membrane potential of the liposome reaches a predetermined potential, the membrane potential-oriented ion channel incorporated in the liposome changes from a closed state to an open state (depending on the type of ion channel, from an open state to a closed state). When such a state change occurs in the ion channel, the membrane potential of the liposome returns to the non-stimulated potential (hereinafter, referred to as “base potential”). This return closes the channel again. In this liposome, the change in membrane potential is repeated between the base potential and the predetermined potential. This base potential varies depending on the type and concentration of the chemical substance that stimulates the liposome. Therefore, there is a difference in the potential difference between the base potential and the predetermined potential between the case where the type and the concentration of the chemical substance make the base potential of the liposome low, and the case where the base potential of the liposome makes the base potential high. Therefore, the cycle at which the change in the membrane potential of the liposome occurs also differs. That is, the larger the potential difference between the base potential and the predetermined potential, the longer the cycle in which the membrane potential change is repeated.
【００１６】したがって、この膜電位変化の周期を測定
することが出来れば、また化学物質の種類やその濃度に
対応するこの周期を予め測定しておけば、これらを照合
することにより、いま測定している化学物質の判別や定
量が可能になる。Therefore, if the period of the change in the membrane potential can be measured, and if the period corresponding to the type of the chemical substance and its concentration is measured in advance, these can be measured by comparing them. It is possible to determine and quantify the chemical substances used.
【００１７】しかし、リポソームはその直径が大きくと
も数μｍであるので膜電位変化を直接測定することは現
在のところ出来ない。この発明のバイオ素子に具わる膜
電位感受性蛍光色素はこれを補う。However, since liposomes have a diameter of at most several μm, it is not possible at present to directly measure changes in membrane potential. The membrane potential-sensitive fluorescent dye provided in the bioelement of the present invention compensates for this.
【００１８】この膜電位感受性蛍光色素は、リポソーム
の膜電位変化に応じてリポソーム周囲での配向具合が変
化する。この配向具合の変化は外部からは蛍光強度の変
化として認識できる。この結果、化学物質に応答する蛍
光強度変化ひいては蛍光強度変化の周期が得られる。The orientation of the membrane potential-sensitive fluorescent dye around the liposome changes according to the change in the membrane potential of the liposome. This change in orientation can be recognized from the outside as a change in fluorescence intensity. As a result, a change in the fluorescence intensity in response to the chemical substance and a cycle of the change in the fluorescence intensity are obtained.
【００１９】また、この出願の第二発明の化学物質の判
別定量方法では、化学物質の刺激による蛍光強度変化を
測定した後、例えばこの蛍光強度変化の周期、蛍光強度
変化の振幅を種々の化学物質について予め測定しておい
た周期や振幅に照合させることにより、化学物質の判別
及び又は定量ができる。In the method for discriminating and quantifying a chemical substance according to the second invention of this application, after measuring the change in the fluorescence intensity due to the stimulation of the chemical substance, for example, the cycle of the change in the fluorescence intensity and the amplitude of the change in the fluorescence intensity are measured by various chemical methods. The chemical substance can be discriminated and / or quantified by collating the substance with a previously measured cycle or amplitude.
【００２０】[0020]
【実施例】以下、第一発明のバイオ素子の実施例及び第
二発明の化学物質の判別定量方法の実施例について併せ
て説明する。なお、この実施例では、リポソームを構成
するため使用する材料としてコレステロール（関東化学
社製）及びホスファチジルコリン（Ｓｉｇｍａ社製）を
用い、膜電位感受性蛍光色素としてｄｉｓＣ（３）５
（日本感光色素社製）を用い、膜電位配向性イオンチャ
ンネル構成用蛋白質としてアラメチシン（Ｓｉｇｍａ社
製）を用いている。しかし、これら使用材料はこの発明
の範囲内の好適例にすぎない。また、以下の実施例中で
用いる装置名及び、使用材料の濃度、使用量、処理時
間、処理温度等の数値的条件、処理方法等はこの発明の
範囲内の好適例にすぎない。また、以下の説明をいくつ
かの図を参照して行うがこれら図はこの発明を理解でき
る程度に概略的に示してあるにすぎない。 １．リポソーム懸濁液の調整 ナス型フラスコ中に５ｍｌのクロロホルムと５ｍｇのコ
レステロールと２０ｍｇのホスファチジルコリンとを入
れこれらを混合する。その後、ロータリーエバポレータ
ーを用いこの混合溶液からクロロホルムを減圧除去す
る。EXAMPLES Hereinafter, examples of the biodevice of the first invention and examples of the method for discriminating and quantifying chemical substances of the second invention will be described together. In this example, cholesterol (manufactured by Kanto Chemical Co., Ltd.) and phosphatidylcholine (manufactured by Sigma) were used as materials used to construct liposomes, and disC (3) 5 was used as a membrane potential-sensitive fluorescent dye.
(Manufactured by Nippon Kogaku Dyestuffs Co., Ltd.), and alamethicin (manufactured by Sigma) is used as a protein for constituting a membrane potential-oriented ion channel. However, these materials are only preferred examples within the scope of the present invention. Further, the names of the apparatuses used in the following examples, the numerical conditions such as the concentration of the materials used, the amount used, the processing time, the processing temperature, etc., and the processing methods are merely preferred examples within the scope of the present invention. The following description is made with reference to several figures, which are merely schematic enough to provide an understanding of the invention. 1. Preparation of liposome suspension 5 ml of chloroform, 5 mg of cholesterol and 20 mg of phosphatidylcholine are placed in an eggplant-shaped flask and mixed. Thereafter, chloroform is removed from this mixed solution under reduced pressure using a rotary evaporator.
【００２１】次に、このフラスコ中に、濃度が１００ｍ
ＭのＫＣｌ水溶液を５ｍｌ加え、その後これを３７℃の
温度下でｖｏｒｔｅｘミキサーで撹拌しリポソーム懸濁
液を得る。この懸濁液中にリポソームが構成されている
ことは、光学顕微鏡を用いて確認した。Next, the concentration of 100 m
5 ml of M KCl aqueous solution is added, and then the mixture is stirred at 37 ° C. with a vortex mixer to obtain a liposome suspension. The formation of liposomes in this suspension was confirmed using an optical microscope.
【００２２】次に、このリポソーム懸濁液を遠心分離器
により１０，０００Ｇの条件で遠心を行った後上澄みを
採取する。Next, the liposome suspension is centrifuged at 10,000 G by a centrifuge, and the supernatant is collected.
【００２３】次に、ｄｉｓＣ（３）５膜電位感受性蛍光
色素を０．９１ｍＭの濃度で含むｄｉｓＣ（３）水溶液
２０μｌと、アラメチシンを１０-6ｇ／ｍｌの濃度で含
むアラメチシン水溶液１ｍｌとの混合液に、上記上澄み
０．９ｍｌを添加する。この水溶液を説明の都合上以下
「実験用水溶液」と称する。この実験用水溶液中におい
て、蛋白質であるアラメチシンがリポソームのリン脂質
二分子膜に組み込まれる。図１はこの実験水溶液中での
アラメチシンと、リポソームと、膜電位感受性蛍光色素
ｄｉｓＣ（３）５との関係を模式的に示した図である。
図１において１１はリポソームのリン脂質二分子膜、１
３はアラメチシン、１５は膜電位感受性蛍光色素ｄｉｓ
Ｃ（３）５である。アラメチシン１３を組み込んだリポ
ソームと膜電位感受性蛍光色素であるｄｉｓＣ（３）５
とは、ｄｉｓＣ（３）５が当該リポソーム周囲に分布す
る状態で、実験水溶液中に含まれている。 ２．蛍光強度変化の測定 次に、ホールスライドグラスを用意し、そのホール中に
濃度が５ｍＭのＫＣｌ水溶液をいれる。そして、上述の
実験水溶液からマイクロピペットを用いて１個のリポソ
ーム（アラメチシン組込みリポソーム）を実験水溶液と
共に採取し濃度が５ｍＭのＫＣｌ水溶液を満たしてある
上述のホール中に移す。Next, a mixture of 20 μl of a disC (3) aqueous solution containing a disC (3) 5 membrane potential-sensitive fluorescent dye at a concentration of 0.91 mM and 1 ml of an alamethicin aqueous solution containing alamethicin at a concentration of 10 −6 g / ml. 0.9 ml of the above supernatant is added to the solution. This aqueous solution is hereinafter referred to as “experimental aqueous solution” for convenience of explanation. In this experimental aqueous solution, the protein alamethicin is incorporated into the phospholipid bilayer membrane of the liposome. FIG. 1 is a diagram schematically showing the relationship between alamethicin, liposomes, and a membrane potential-sensitive fluorescent dye disC (3) 5 in this experimental aqueous solution.
In FIG. 1, reference numeral 11 denotes a liposome phospholipid bilayer membrane,
3 is alamethicin, 15 is a membrane potential sensitive fluorescent dye dis
C (3) 5. Liposomes incorporating alamethicin 13 and disC (3) 5, a membrane potential sensitive fluorescent dye
Means that disC (3) 5 is distributed around the liposome and is contained in the experimental aqueous solution. 2. Measurement of Fluorescence Intensity Change Next, a hole slide glass is prepared, and a 5 mM KCl aqueous solution is placed in the hole. Then, one liposome (aramethicin-incorporated liposome) is collected from the experimental aqueous solution using a micropipette together with the experimental aqueous solution and transferred to the above-mentioned hole filled with a 5 mM KCl aqueous solution.
【００２４】次に、このリポソームを公知のＧ励起法で
蛍光顕微鏡を用いて観察する。この蛍光顕微鏡にはビデ
オカメラが接続してある。さらにこのビデオカメラの出
力はピアス（株）製の画像情報処理システムＬＡ−５２
５Ｒに入力される構成としてある。Next, the liposome is observed by a known G excitation method using a fluorescence microscope. A video camera is connected to the fluorescence microscope. Further, the output of this video camera is an image information processing system LA-52 manufactured by Pierce Co., Ltd.
It is configured to be input to 5R.
【００２５】この画像情報処理システムは、時間経過に
伴う蛍光強度を、横軸に時間及び縦軸に蛍光強度を取っ
た特性図として出力する。この測定では蛍光強度がパル
ス状にかつ周期的に変化する特性が得られた。図２にそ
の特性図の一例を示した。各パルスでの最小値（図２中
ｆAで示す値。）はアラメチシン組込みリポソームでの
例えばベース電位と考えることが出来、最大値（図２中
ｆBで示す値。）はこのリポソームでイオンチャンネル
が状態を変化させるときの膜電位と考えることが出来
る。そして、図２に示した特性図では、周期が分り、ま
た単位時間当たりのパルス数が分るので（もちろん周期
の逆数からでも良いが）蛍光強度変化の周波数すなわち
リポソームの膜電位の変化の周波数が分ると考えること
が出来る。This image information processing system outputs the fluorescence intensity over time as a characteristic diagram in which the horizontal axis represents time and the vertical axis represents fluorescence intensity. In this measurement, a characteristic was obtained in which the fluorescence intensity changed in a pulsed manner and periodically. FIG. 2 shows an example of the characteristic diagram. Minimum at each pulse (value shown in FIG. 2 f A.) Can be considered, for example, the base potential at alamethicin embedded liposomes, the maximum value (the value shown in FIG. 2 f B.) Ion in the liposome It can be considered as the membrane potential when the channel changes state. In the characteristic diagram shown in FIG. 2, the period is known, and the number of pulses per unit time is known (of course, the reciprocal of the period may be used). Can be considered.
【００２６】上述の実施例では濃度が１００ｍＭのＫＣ
ｌ水溶液での蛍光強度変化の周波数を求めたが、これと
は別にＫＣｌ水溶液の代わりに、種々の濃度のキニーネ
水溶液、種々の濃度のＨＣｌ水溶液、種々の濃度のＮａ
Ｃｌ水溶液、種々の濃度のサッカロース水溶液で実施例
と同様に蛍光強度変化を測定しそれぞれでの蛍光強度変
化の周波数を算出した。この結果を、横軸にキニーネ、
ＨＣｌなどの濃度（単位はモル濃度の対数表示）をとり
縦軸に蛍光強度変化の周波数（Ｈｚ）をとって図３に示
した。なお、図３においてＩで示す特性がキニーネのも
の、IIで示す特性がＨＣｌのもの、III で示す特性がＮ
ａＣｌのもの、ＩＶで示す特性がサッカロースのもので
ある。In the above-described embodiment, KC having a concentration of 100 mM was used.
The frequency of the change in the fluorescence intensity in the aqueous solution was determined. Apart from this, instead of the KCl aqueous solution, various concentrations of quinine aqueous solution, various aqueous HCl solutions, and various concentrations of Na aqueous solution were used.
Fluorescence intensity changes were measured in the same manner as in the examples using Cl aqueous solution and sucrose aqueous solutions of various concentrations, and the frequency of the fluorescence intensity change was calculated for each. This result is plotted on the horizontal axis with quinine,
FIG. 3 shows the concentration of HCl or the like (the unit is expressed in logarithm of the molar concentration), and the vertical axis shows the frequency (Hz) of the change in the fluorescence intensity. In FIG. 3, the characteristic indicated by I is quinine, the characteristic indicated by II is HCl, and the characteristic indicated by III is N.
The characteristics indicated by aCl and IV are those of saccharose.
【００２７】図３から明らかなように、化学物質の濃度
がＬｏｇ濃度でいって−４より高濃度であると例えばキ
ニーネに対する実施例のバイオ素子での蛍光強度変化の
周波数と、サッカロースに対する同周波数とは明らかに
違ってくる。また、各化学物質それぞれの濃度を増加さ
せるに従い実施例のバイオ素子での蛍光強度変化の周波
数が高くなる。これらのことから、この実施例のバイオ
素子はある種の化学物質間での物質判別、各化学物質の
定量を行うことが出来ると考えられる。As is apparent from FIG. 3, when the concentration of the chemical substance is higher than -4 in terms of Log concentration, for example, the frequency of the fluorescence intensity change in the bioelement of the embodiment for quinine and the same frequency for saccharose It is obviously different. Further, as the concentration of each chemical substance is increased, the frequency of the change in the fluorescence intensity in the bioelement of the embodiment increases. From these facts, it is considered that the bioelement of this embodiment can perform substance discrimination between certain kinds of chemical substances and quantitative determination of each chemical substance.
【００２８】上述においてはこの出願の各発明の実施例
について説明したがこれら発明は上述の実施例に限られ
ない。Although the embodiments of each invention of this application have been described above, these inventions are not limited to the above embodiments.
【００２９】上述の実施例では膜電位配向性イオンチャ
ンネルをアラメチシンで構成していた。しかし、アラメ
チシンの代わりにモナゾマイシンなどの他のイオンチャ
ンネルを用いてバイオ素子を構築した場合も実施例と同
様の効果が得られることは明らかである。In the above-described embodiment, the membrane potential-oriented ion channel is constituted by alamethicin. However, it is clear that the same effect as in the example can be obtained when a bioelement is constructed using another ion channel such as monazomycin instead of alamethicin.
【００３０】また、リポソームを構成する材料、膜電位
感受性蛍光色素は実施例のものに限られず他の好適なも
のでも良い。リポソームを構成する材料の他の例として
は例えばスフィンゴミエリン、酸化コレステロール等を
挙げることが出来る。また、膜電位感受性色素の他の例
としては例えば日本感光色素社製の商品名がＲＨ４６
１、同ＲＨ１５５等を挙げることが出来る。The material constituting the liposome and the membrane potential-sensitive fluorescent dye are not limited to those of the embodiment, but may be other suitable ones. Other examples of the material constituting the liposome include sphingomyelin, cholesterol oxide and the like. Further, as another example of the membrane potential-sensitive dye, for example, RH46 is a product name of Nippon Kogaku Dyestuffs Co., Ltd.
1 and RH155.
【００３１】[0031]
【発明の効果】上述した説明からも明らかなように、こ
の出願の第一発明のバイオ素子によれば化学物質に応答
して蛍光強度が変化する特性を示す。このため、化学物
質の判別定量のセンサとして用いることが出来る。さら
に、生物を模倣した情報処理形態を持つバイオコンピュ
ータが実現された場合、その入力装置或はその出力装置
の構築に寄与することが期待出来る。As is clear from the above description, the biodevice according to the first invention of the present application exhibits the characteristic that the fluorescence intensity changes in response to a chemical substance. Therefore, it can be used as a sensor for discriminating and quantifying chemical substances. Furthermore, when a biocomputer having an information processing mode imitating a living thing is realized, it can be expected to contribute to the construction of the input device or the output device.
【図１】実施例のバイオ素子を模式的に示した図であ
る。FIG. 1 is a diagram schematically showing a bioelement of an example.
【図２】実施例のバイオ素子で観測される蛍光強度変化
の特性の一例を示した図である。FIG. 2 is a diagram illustrating an example of a characteristic of a change in fluorescence intensity observed in the bioelement of the example.
【図３】実施例のバイオ素子の蛍光強度変化の周波数
が、化学物質の種類及び濃度にどのように依存するかを
示した特性図である。FIG. 3 is a characteristic diagram showing how the frequency of a change in the fluorescence intensity of the bioelement of an example depends on the type and concentration of a chemical substance.
１１：リポソームの二分子膜 １３：膜電位配向性イオンチャンネル １５：膜電位感受性蛍光色素 11: Bilayer membrane of liposome 13: Membrane potential oriented ion channel 15: Membrane potential sensitive fluorescent dye
───────────────────────────────────────────────────── フロントページの続き (72)発明者 海部 勝晶 東京都港区虎ノ門１丁目７番12号 沖電 気工業株式会社内 (72)発明者 加藤 雅一 東京都港区虎ノ門１丁目７番12号 沖電 気工業株式会社内 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Katsuaki Kaibe 1-7-12 Toranomon, Minato-ku, Tokyo Inside Oki Electric Industry Co., Ltd. (72) Masakazu Kato 1-7-12 Toranomon, Minato-ku, Tokyo No.Oki Electric Industry Co., Ltd.
Claims (2)
んだリポソームと、該リポソームの膜電位に応答する膜
電位感受性蛍光色素とを水溶液中に含んで成ることを特
徴とするバイオ素子。1. A biodevice comprising, in an aqueous solution, a liposome incorporating a membrane potential-oriented ion channel and a membrane potential-sensitive fluorescent dye responsive to the membrane potential of the liposome.
んだリポソームと、該リポソームの膜電位に応答する膜
電位感受性蛍光色素と、化学物質とを水溶液中に混在さ
せ、該化学物質の種類及び濃度並びに前記イオンチャン
ネルの開放及び閉鎖に起因する前記リポソームの電位変
化に応じて前記膜電位感受性色素で生じる蛍光強度変化
を測定し、該蛍光強度変化に基いて前記化学物質の判別
及び定量の双方又は一方を行うことを特徴とする化学物
質の判別定量方法。2. A liposome incorporating a membrane potential-oriented ion channel, a membrane potential-sensitive fluorescent dye responding to the membrane potential of the liposome, and a chemical substance mixed in an aqueous solution, and the type and concentration of the chemical substance and A change in the fluorescence intensity generated by the membrane potential-sensitive dye is measured in accordance with a change in the potential of the liposome caused by opening and closing of the ion channel, and the chemical substance is discriminated and / or quantified based on the change in the fluorescence intensity. And a method for discriminating and quantifying a chemical substance.
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|---|---|---|---|
| JP9699791A JP2573429B2 (en) | 1991-04-26 | 1991-04-26 | Bio element and method for discriminating and quantifying chemical substance using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9699791A JP2573429B2 (en) | 1991-04-26 | 1991-04-26 | Bio element and method for discriminating and quantifying chemical substance using the same |
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| Publication Number | Publication Date |
|---|---|
| JPH04326048A JPH04326048A (en) | 1992-11-16 |
| JP2573429B2 true JP2573429B2 (en) | 1997-01-22 |
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