JPH06273338A - Lb film for optical chemical sensor - Google Patents
Lb film for optical chemical sensorInfo
- Publication number
- JPH06273338A JPH06273338A JP6001793A JP6001793A JPH06273338A JP H06273338 A JPH06273338 A JP H06273338A JP 6001793 A JP6001793 A JP 6001793A JP 6001793 A JP6001793 A JP 6001793A JP H06273338 A JPH06273338 A JP H06273338A
- Authority
- JP
- Japan
- Prior art keywords
- dye
- film
- fluorescence intensity
- membrane potential
- monitor
- 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.)
- Withdrawn
Links
- 239000000126 substance Substances 0.000 title claims abstract description 123
- 230000003287 optical effect Effects 0.000 title claims abstract description 70
- 239000012528 membrane Substances 0.000 claims description 122
- 238000000034 method Methods 0.000 abstract description 29
- 230000006866 deterioration Effects 0.000 abstract description 23
- 230000007423 decrease Effects 0.000 abstract description 6
- 238000004040 coloring Methods 0.000 abstract 4
- 239000000975 dye Substances 0.000 description 194
- 239000010408 film Substances 0.000 description 82
- 239000000243 solution Substances 0.000 description 22
- 238000005259 measurement Methods 0.000 description 20
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- 239000007864 aqueous solution Substances 0.000 description 18
- 239000012488 sample solution Substances 0.000 description 14
- 230000035945 sensitivity Effects 0.000 description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- 150000002500 ions Chemical class 0.000 description 10
- 239000000758 substrate Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000011521 glass Substances 0.000 description 9
- 230000005284 excitation Effects 0.000 description 7
- 108010067973 Valinomycin Proteins 0.000 description 6
- FCFNRCROJUBPLU-UHFFFAOYSA-N compound M126 Natural products CC(C)C1NC(=O)C(C)OC(=O)C(C(C)C)NC(=O)C(C(C)C)OC(=O)C(C(C)C)NC(=O)C(C)OC(=O)C(C(C)C)NC(=O)C(C(C)C)OC(=O)C(C(C)C)NC(=O)C(C)OC(=O)C(C(C)C)NC(=O)C(C(C)C)OC1=O FCFNRCROJUBPLU-UHFFFAOYSA-N 0.000 description 6
- 238000012937 correction Methods 0.000 description 6
- JTXUVYOABGUBMX-UHFFFAOYSA-N didodecyl hydrogen phosphate Chemical compound CCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCC JTXUVYOABGUBMX-UHFFFAOYSA-N 0.000 description 6
- 238000012544 monitoring process Methods 0.000 description 6
- FCFNRCROJUBPLU-DNDCDFAISA-N valinomycin Chemical compound CC(C)[C@@H]1NC(=O)[C@H](C)OC(=O)[C@@H](C(C)C)NC(=O)[C@@H](C(C)C)OC(=O)[C@H](C(C)C)NC(=O)[C@H](C)OC(=O)[C@@H](C(C)C)NC(=O)[C@@H](C(C)C)OC(=O)[C@H](C(C)C)NC(=O)[C@H](C)OC(=O)[C@@H](C(C)C)NC(=O)[C@@H](C(C)C)OC1=O FCFNRCROJUBPLU-DNDCDFAISA-N 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000007850 fluorescent dye Substances 0.000 description 5
- -1 octadecyl rhodamine B Chemical compound 0.000 description 5
- 229940043267 rhodamine b Drugs 0.000 description 5
- 239000000470 constituent Substances 0.000 description 4
- VKOBVWXKNCXXDE-UHFFFAOYSA-N icosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCC(O)=O VKOBVWXKNCXXDE-UHFFFAOYSA-N 0.000 description 4
- 239000002356 single layer Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 229910000077 silane Inorganic materials 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- CITHEXJVPOWHKC-UUWRZZSWSA-N 1,2-di-O-myristoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCC CITHEXJVPOWHKC-UUWRZZSWSA-N 0.000 description 1
- OZSITQMWYBNPMW-GDLZYMKVSA-N 1,2-ditetradecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCC OZSITQMWYBNPMW-GDLZYMKVSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 150000003983 crown ethers Chemical class 0.000 description 1
- 239000002739 cryptand Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 125000005131 dialkylammonium group Chemical group 0.000 description 1
- 229960003724 dimyristoylphosphatidylcholine Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 238000007429 general method Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- DZVCFNFOPIZQKX-LTHRDKTGSA-M merocyanine Chemical compound [Na+].O=C1N(CCCC)C(=O)N(CCCC)C(=O)C1=C\C=C\C=C/1N(CCCS([O-])(=O)=O)C2=CC=CC=C2O\1 DZVCFNFOPIZQKX-LTHRDKTGSA-M 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 239000002120 nanofilm Substances 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
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- 239000001022 rhodamine dye Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 125000005504 styryl group Chemical group 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- GQIUQDDJKHLHTB-UHFFFAOYSA-N trichloro(ethenyl)silane Chemical compound Cl[Si](Cl)(Cl)C=C GQIUQDDJKHLHTB-UHFFFAOYSA-N 0.000 description 1
- 239000005050 vinyl trichlorosilane Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、試料中の特定成分を測
定するためのオプチカル化学センサーに使用されるラン
グミュアーブロージェット膜（ＬＢ膜）に関する。特
に、膜電位感受性色素と特定物質認識能を有する物質を
含有した超薄膜のＬＢ膜に関する。更に本発明は該ＬＢ
膜を使用したオプチカル化学センサーと、これを用いた
測定方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a Langmuir blow jet film (LB film) used in an optical chemical sensor for measuring a specific component in a sample. In particular, it relates to an ultrathin LB film containing a membrane potential sensitive dye and a substance having the ability to recognize a specific substance. Further, the present invention provides the LB
The present invention relates to an optical chemical sensor using a membrane and a measuring method using the same.
【０００２】[0002]
【従来の技術】近年、ＬＢ膜の製作技術が発展し、これ
によって、種々の薄膜設計が可能となってきている。Ｌ
Ｂ膜の作製技術については福田清成他編「ＬＢ膜とエレ
クトロニクス」（シーエムシー社刊）に詳細に説明され
ている。2. Description of the Related Art In recent years, techniques for manufacturing LB films have been developed and various thin film designs have become possible. L
The technology for producing the B film is described in detail in “LB film and electronics” (published by CMC) edited by Kiyonari Fukuda et al.
【０００３】本発明者は先にラングミュアーブロージェ
ット法（ＬＢ法）に従い、膜電位感受性色素および特定
物質認識能を有する物質を含有した両親媒性物質を水面
上に展開し、形成された単分子膜を圧縮した後に基板に
累積して形成されるオプチカル化学センサーにおいて、
累積時の表面圧を相転移前の表面圧として累積した膜か
らなるオプチカル化学センサーを提案した（特願平１−
１３５３１９）。このオプチカル化学センサーを用いる
ことにより、濃度変化に対する蛍光強度の変化をできる
だけ大きくすることができ、感度の向上を達成すること
ができた。The inventor of the present invention has previously developed an amphipathic substance containing a membrane potential sensitive dye and a substance having the ability to recognize a specific substance on the water surface according to the Langmuir blow jet method (LB method). In the optical chemical sensor formed by accumulating on the substrate after compressing the molecular film,
We proposed an optical chemical sensor consisting of a film in which the accumulated surface pressure was used as the surface pressure before the phase transition (Japanese Patent Application No. 1-
135319). By using this optical chemical sensor, the change in fluorescence intensity with respect to the change in concentration can be maximized, and the sensitivity can be improved.
【０００４】このオプチカル化学センサーは、試料溶液
を流しながら光源から所定波長の光を照射し、所定波長
の蛍光強度の変化をモニターして試料溶液中のイオン濃
度を測定するものである。即ち、試料溶液中に含まれる
特定のイオンが、ＬＢ膜に含まれるイオン選択性を有す
る特定物質を認識する物質に取り込まれると、ＬＢ膜の
膜電位が変化する。膜電位の変化に対応して膜電位感受
性色素の蛍光強度が変化するので、蛍光強度の変化をモ
ニターすることより、試料溶液中の特定のイオン濃度を
測定することが可能となる。This optical chemical sensor measures the ion concentration in the sample solution by irradiating light of a predetermined wavelength from a light source while flowing the sample solution and monitoring changes in fluorescence intensity of the predetermined wavelength. That is, when the specific ions contained in the sample solution are taken into the substance that recognizes the specific substance having ion selectivity contained in the LB film, the membrane potential of the LB film changes. Since the fluorescence intensity of the membrane potential sensitive dye changes according to the change of the membrane potential, it is possible to measure the specific ion concentration in the sample solution by monitoring the change of the fluorescence intensity.
【０００５】他方、ボルフバイス等は両親媒性物質とし
てアラキン酸等を用い、同様のオプチカル化学センサー
の研究を進めている（Ｏ．Ｓ．Ｗｏｌｆｂｅｉｓ，ｅ
ｔ．ａｌ．，Ａｎａｌｙｔｉｃａ Ｃｈｉｍｉｃａ Ａ
ｃｔａ，１９８（１９８７）１−１２）。しかし、上記
で得られたセンサーの研究をさらに進めてみると、未だ
次のような問題が残されていることが判明した。On the other hand, Wolffice et al. Have been studying a similar optical chemical sensor using arachidic acid and the like as an amphipathic substance (OS Wolfbeis, e.
t. al. , Analytica Chimica A
Cta, 198 (1987) 1-12). However, further research on the sensor obtained above revealed that the following problems still remain.
【０００６】上記オプチカル化学センサーを用いて長時
間の測定を行うと膜が劣化し、等濃度の溶液を測定して
いても感度が次第に低下するため光学的変化を単純に濃
度に変換するだけでは正確な測定ができなくなる。ま
た、オプチカル化学センサーで測定する蛍光強度の変化
量は非常に小さいためその変化量と同レベルまたはそれ
以上の感度で劣化の程度を検知しないと十分な補正が行
えないことである。通常、このような劣化に基づく蛍光
強度の経時変化をモニターし、補正するためには標準の
溶液を準備し、それを適当な間隔で繰返し測定する。し
かし、このような方法は、標準液を予め準備し、適当な
間隔でこの標準溶液の濃度を測定しなければならず、簡
便性の点においてオプチカル化学センサーの応用の幅を
狭めることになる。しかも、その標準液を測定するため
に試料溶液の測定を中断しなければならない等の不都合
も有する。When a long-time measurement is performed using the above-mentioned optical chemical sensor, the film deteriorates, and the sensitivity gradually decreases even when measuring a solution having an equal concentration. Therefore, the optical change is simply converted into the concentration. Accurate measurement is not possible. Further, since the amount of change in fluorescence intensity measured by the optical chemical sensor is very small, sufficient correction cannot be performed unless the degree of deterioration is detected at the same level of sensitivity as the amount of change or higher. Usually, in order to monitor and correct the change with time in fluorescence intensity due to such deterioration, a standard solution is prepared, and it is repeatedly measured at appropriate intervals. However, such a method requires preparing a standard solution in advance and measuring the concentration of the standard solution at appropriate intervals, which narrows the range of application of the optical chemical sensor in terms of simplicity. Moreover, there is a disadvantage that the measurement of the sample solution must be interrupted in order to measure the standard solution.
【０００７】[0007]
【発明が解決しようとする課題】従って、本発明の課題
はオプチカル化学センサーの劣化による蛍光強度の減少
を簡便に、且つできるだけ正確に補正し、試料の濃度を
正しく測定できるようなＬＢ膜を提供することである。
更に、このＬＢ膜を使用したオプチカル化学センサーを
用いた補正の方法を提供することである。望ましくは連
続的な測定を中断することなく補正が行える方法を提供
することである。SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide an LB film which can correct the decrease in fluorescence intensity due to the deterioration of an optical chemical sensor easily and as accurately as possible and can measure the sample concentration correctly. It is to be.
Furthermore, it is to provide a method of correction using an optical chemical sensor using this LB film. It is desirable to provide a method by which correction can be performed without interrupting continuous measurements.
【０００８】[0008]
【課題を解決するための手段】上記課題は以下の（１）
から（６）によって解決される。[Means for Solving the Problems] The above problems are as follows (1)
To (6).
【０００９】（１）両親媒性物質からなる単分子膜を支
持体上に移し取って調製されるラングミュアー・ブロジ
ェット膜において、該膜中に膜電位感受性色素及び特定
物質認識能を有する物質を含有させ、更に膜電位感受性
色素と異なる光学特性を有し、且つ膜電位不感受性のモ
ニター色素（以下モニター色素と称する）を含有させた
ことを特徴とするラングミュアー・ブロジェット膜。 （２）上記（１）のＬＢ膜であって、モニター色素が膜
電位感受性色素と同じ荷電を有し、かつ両親媒性の蛍光
色素からなることを特徴とするＬＢ膜。 （３）支持体上に形成された上記（１）又は（２）のＬ
Ｂ膜を用いたオプチカル化学センサー。(1) In a Langmuir-Blodgett membrane prepared by transferring a monomolecular film made of an amphipathic substance onto a support, a substance having a membrane potential-sensitive dye and a substance-recognizing ability in the membrane. A Langmuir-Blodgett film, which further comprises a monitor dye (hereinafter, referred to as a monitor dye) which contains a dye, which has optical characteristics different from those of the dye sensitive to a membrane potential and is insensitive to the membrane potential. (2) The LB film according to (1) above, wherein the monitor dye has the same charge as the membrane potential sensitive dye and is composed of an amphipathic fluorescent dye. (3) L of the above (1) or (2) formed on a support
Optical chemical sensor using B film.
【００１０】（４）上記（３）のオプチカル化学センサ
ーを用いた測定において、特定物質の濃度によって誘起
される膜電位感受性色素の光学的変化を、前記モニター
色素の蛍光強度を基にして補正することを特徴とする測
定方法。(4) In the measurement using the optical chemical sensor of the above (3), the optical change of the membrane potential sensitive dye induced by the concentration of the specific substance is corrected based on the fluorescence intensity of the monitor dye. A measuring method characterized by the above.
【００１１】（５）前記モニター色素と膜電位感受性色
素の両方が蛍光色素からなるオプチカル化学センサーに
おいて、該モニター色素の蛍光波長を膜電位感受性色素
の励起波長と重なるように選択し、該モニター色素を励
起して膜電位感受性色素の蛍光強度を測定することによ
ってセンサーの劣化に基づく蛍光強度の減少を補正する
ことを特徴とする測定方法。(5) In an optical chemical sensor in which both the monitor dye and the membrane potential sensitive dye are fluorescent dyes, the fluorescence wavelength of the monitor dye is selected so as to overlap with the excitation wavelength of the membrane potential sensitive dye, and the monitor dye A method for measuring a fluorescence intensity of a dye which is sensitive to a membrane potential, and the decrease in the fluorescence intensity due to deterioration of a sensor is corrected by exciting the dye.
【００１２】（６）ラングミュアー・ブロジェット法に
従い、膜電位感受性色素及び特定物質認識能を有する物
質を含有した両親媒性物質を基板に累積して形成される
オプチカル化学センサーを用いた測定において、前記膜
電位感受性色素を空気中に露出した状態で該色素の蛍光
強度を測定し、これを基にして蛍光強度の減少を補正す
ることを特長とする測定方法。 以下に本発明を更に詳細に説明する。まず本発明のＬＢ
膜について述べる。(6) In the measurement using an optical chemical sensor formed by accumulating an amphipathic substance containing a membrane potential sensitive dye and a substance capable of recognizing a specific substance on a substrate according to the Langmuir-Blodgett method A measuring method characterized by measuring the fluorescence intensity of the dye with the membrane potential-sensitive dye exposed to the air, and correcting the decrease in the fluorescence intensity based on this. The present invention will be described in more detail below. First, the LB of the present invention
The membrane will be described.
【００１３】オプチカル化学センサーに使用し得る本発
明のＬＢ膜は、その構成成分として膜電位感受性色素、
特定物質認識能を有する物質、両親媒性物質及び膜電位
感受性色素に対して不活性で、膜電位感受性色素と異な
る光学特性を有し、且つ膜電位不感受性のモニター色素
を含有する。ここで、不活性とは、膜電位感受性色素を
消光させる作用を持たないことを意味する。本発明のＬ
Ｂ膜は、以下のようにして調製される。即ち、両親媒性
物質中に、膜電位感受性色素、特定物質認識能を有する
物質、及びモニター色素を含有したクロロホルム等の有
機溶媒の溶液を調製し、これを水面上に展開することに
よって単分子膜を形成する。このとき上記膜電位感受性
色素、特定物質認識能を有する物質、及びモニター色素
は、溶液中で両親媒性物質中に均一に分散されているの
で単分子膜中においても一様に分散され均一な単分子膜
が形成される。このように展開された単分子膜は、適切
な支持体上に移し取られ、１層又はそれ以上の層に累積
される。このときの支持体は、ＬＢ膜を１層以上適切に
累積させることができるものであれば特に限定されな
い。このような支持体の例にはガラス基板等がある。こ
のようにして調製されたＬＢ膜を模式図で表わすと図１
のようになる。図１では１層のＬＢ膜を示した。The LB film of the present invention which can be used in an optical chemical sensor has a membrane potential sensitive dye as its constituent component,
It contains a substance having the ability to recognize a specific substance, an amphipathic substance, and a monitor dye which is inactive to a membrane potential sensitive dye, has optical characteristics different from those of the membrane potential sensitive dye, and is insensitive to the membrane potential. Here, inactive means having no action of quenching the membrane potential sensitive dye. L of the present invention
The B film is prepared as follows. That is, a solution of an organic solvent such as chloroform containing a membrane potential sensitive dye, a substance having the ability to recognize a specific substance, and a monitor dye in an amphipathic substance is prepared, and the solution is spread on the water surface to form a single molecule. Form a film. At this time, since the membrane potential sensitive dye, the substance having the ability to recognize a specific substance, and the monitor dye are uniformly dispersed in the amphipathic substance in the solution, they are evenly dispersed even in the monomolecular film. A monolayer is formed. The thus-developed monolayer is transferred onto an appropriate support and accumulated in one or more layers. The support at this time is not particularly limited as long as it can properly accumulate one or more LB films. Examples of such a support include a glass substrate and the like. The LB film thus prepared is shown in a schematic diagram in FIG.
become that way. In FIG. 1, a single-layer LB film is shown.
【００１４】本発明のＬＢ膜に使用される両親媒性物質
はＬＢ膜のマトリックスとなるものであればとくに限定
されない。例えばジドデシルリン酸、ジアルキルリン
酸、ジアルキルアンモニウム塩、ジミリストイルフォス
ファチジルコリン、ジミリストイルフォスファチジン
酸、アラキン酸、ステアリン酸等種々の両親媒性物質を
挙げることができる。The amphipathic substance used in the LB film of the present invention is not particularly limited as long as it serves as a matrix of the LB film. Examples thereof include various amphipathic substances such as didodecylphosphoric acid, dialkylphosphoric acid, dialkylammonium salt, dimyristoylphosphatidylcholine, dimyristoylphosphatidic acid, arachidic acid, and stearic acid.
【００１５】膜電位感受性色素としてはシアニン系色
素、メロシアニン系色素、ローダミン系色素、オキソノ
ール系色素、スチリル系色素等を用いることができ、ま
たジャーナル・オブ・メンブラン・バイオロジー（Ｊ．
Ｍｅｍｂｒａｎｅ Ｂｉｏｌ．）Ｖｏｌ．１９，１〜３
６（１９７４）に記載されている膜電位感受性色素を使
用することも出来る。その濃度も０．１〜５ｍｏｌ％の
範囲で適宜変更することが出来る。Cyanine dyes, merocyanine dyes, rhodamine dyes, oxonol dyes, styryl dyes and the like can be used as the membrane potential sensitive dyes, and the Journal of Membrane Biology (J.
Membrane Biol. ) Vol. 19, 1-3
6 (1974) can also be used. The concentration can also be appropriately changed within the range of 0.1 to 5 mol%.
【００１６】特定物質認識能を有する分子としてはバリ
ノマイシンを用いることができる。これ以外にも電気化
学第５３巻、Ｎｏ．１２，９４２〜９４６（１９８５）
に記載されているクラウンエーテル類やクリブタンド
類、さらにはジャーナル・オブ・アメリカン・ケミカル
・ソサエティー、第１１０巻、Ｎｏ．２，５７１〜５７
７（１９８８）に記載されているスフェランド類を使用
することが出来る。これらは分子はイオン選択性を有
し、カリウム、カルシウム、ナトリウム、リチウム、マ
グネシウム等のイオンを選択する機能を有する。これら
特定物質認識能を有する物質の濃度としては１〜５０ｍ
ｏｌ％が選択可能である。Valinomycin can be used as a molecule having the ability to recognize a specific substance. Other than this, Electrochemistry Vol. 53, No. 12,942-946 (1985)
Crown ethers and cryptands described in "Journal of American Chemical Society", Vol. 110, No. 2,571-57
7 (1988) can be used. These molecules have ion selectivity, and have a function of selecting ions such as potassium, calcium, sodium, lithium, and magnesium. The concentration of these substances with specific substance recognition ability is 1 to 50 m
ol% can be selected.
【００１７】本発明のＬＢ膜は、両親媒性であり、且つ
上記膜電位感受性色素に対して不活性で上記膜電位感受
性色素と異なった光学特性を有し、しかも膜電位不感受
性のモニター色素を含有することを特徴とする。ここで
膜電位不感受性とは、ＬＢ膜の膜電位の変化に対して色
素の蛍光強度が影響されないことを言う。The LB film of the present invention is a monitor dye that is amphipathic, inactive to the above-mentioned membrane potential-sensitive dye, has different optical characteristics from the above-mentioned membrane potential-sensitive dye, and is insensitive to the membrane potential. It is characterized by containing. Here, the membrane potential insensitivity means that the fluorescence intensity of the dye is not affected by the change in the membrane potential of the LB membrane.
【００１８】本発明のＬＢ膜に使用できるモニター色素
は、上記の条件を満足するものであれば特に限定されな
いが、例えばＮＫ３１６７、ＮＫ２５６０、ＮＫ２６６
５、ＮＫ３１７５（日本感光色素社製）等を挙げること
ができる。本発明において、前述したように調製された
ＬＢ膜は、オプチカル化学センサーとして使用し得る。The monitor dye that can be used in the LB film of the present invention is not particularly limited as long as it satisfies the above conditions. For example, NK3167, NK2560, NK266.
5, NK3175 (manufactured by Nippon Senshoku Co., Ltd.) and the like. In the present invention, the LB film prepared as described above can be used as an optical chemical sensor.
【００１９】本発明のＬＢ膜をオプチカル化学センサー
として使用する場合の支持体は、オプチカル化学センサ
ーに使用できるものであればとくに限定されないが、例
えば、シラン処理したガラス基板等を挙げることができ
る。The support used when the LB film of the present invention is used as an optical chemical sensor is not particularly limited as long as it can be used for an optical chemical sensor, and examples thereof include a silane-treated glass substrate.
【００２０】オプチカル化学センサー用のＬＢ膜を調製
するためには、水面上に展開された単分子膜を、本発明
者等が先に提案した方法、即ち、相転移を起こす前の圧
力で支持体上に累積する。In order to prepare an LB film for an optical chemical sensor, the monolayer film developed on the water surface is supported by the method previously proposed by the present inventors, that is, by the pressure before the phase transition. Accumulate on the body.
【００２１】本発明のＬＢ膜には、モニター色素が新た
に添加されている。これは、本発明のＬＢ膜をオプチカ
ル化学センサーとして使用した場合、オプチカル化学セ
ンサーの劣化を該色素でモニターし、補正するためであ
る。本発明のＬＢ膜をオプチカル化学センサーに用いる
場合、該モニター色素には以下のような性質が求められ
る。第１に、モニター色素は共存する膜電位感受性色素
と異なった光学特性を有することが望ましい。A monitor dye is newly added to the LB film of the present invention. This is because when the LB film of the present invention is used as an optical chemical sensor, deterioration of the optical chemical sensor is monitored and corrected by the dye. When the LB film of the present invention is used in an optical chemical sensor, the monitor dye is required to have the following properties. First, it is desirable that the monitor dye have different optical characteristics from the coexisting membrane potential sensitive dye.
【００２２】すなわち膜電位感受性色素の吸収波長や蛍
光波長と干渉しない光学特性を有することが必要であ
る。又、同時に膜電位に対しては光学的変化が小さいこ
とが望ましい。第２に、モニター色素は蛍光性のものが
望ましい。That is, it is necessary to have optical characteristics that do not interfere with the absorption wavelength or fluorescence wavelength of the membrane potential sensitive dye. At the same time, it is desirable that the optical change is small with respect to the membrane potential. Second, the monitor dye is preferably fluorescent.
【００２３】膜電位感受性色素としては吸光度の変化や
蛍光強度の変化を利用するものがあるが、変化量として
は蛍光強度の変化の方が大きいため蛍光性のものが用い
られている。このような観点から、オプチカル化学セン
サーには蛍光性の色素が用いられており、モニター色素
も蛍光性のものが好ましい。特に、ＬＢ膜のような超薄
膜において色素の吸光度を高精度に測定するには非常に
高精度の装置を必要とするため、吸光度の変化を利用す
るような色素を使用するとその変化量が小さいためセン
サーの利用範囲を狭めることになる。第３に、モニター
色素は両親媒性のものが好ましい。ＬＢ膜中に色素を分
散させるにはＬＢ膜のマトリックスとなる両親媒性物質
と同様の両親媒性の物質が望ましいからである。Some membrane potential-sensitive dyes utilize changes in absorbance and changes in fluorescence intensity. However, since the change in fluorescence intensity is greater than that in fluorescence, a fluorescent one is used. From such a viewpoint, a fluorescent dye is used in the optical chemical sensor, and the monitor dye is also preferably a fluorescent dye. In particular, in an ultra-thin film such as an LB film, a highly accurate device is required to measure the absorbance of a dye with high accuracy. Therefore, when a dye that utilizes the change in absorbance is used, the amount of change is small. Therefore, the usage range of the sensor is narrowed. Third, the monitor dye is preferably amphipathic. This is because, in order to disperse the dye in the LB film, an amphipathic substance similar to the amphipathic substance that serves as the matrix of the LB film is desirable.
【００２４】イオン性の水溶性色素をＬＢ膜材料とイオ
ン結合させて保持する方法もあるが、接する溶液のイオ
ン強度により解離し流出することがあるのであまり好ま
しくない。また、脂溶性の場合には凝集の恐れがある。
このような観点からオプチカル化学センサーにおいて、
膜電位感受性色素はＬＢ膜の構成材料と同じ両親媒性色
素を用いている。従って、モニター用の色素も両親媒性
であることが望ましい。There is also a method in which an ionic water-soluble dye is ion-bonded to the LB film material and held, but it is not preferable because it may be dissociated and flowed out depending on the ionic strength of the solution in contact therewith. If it is fat-soluble, it may aggregate.
From such a viewpoint, in the optical chemical sensor,
As the membrane potential sensitive dye, the same amphiphilic dye as the constituent material of the LB film is used. Therefore, it is desirable that the monitor dye is also amphipathic.
【００２５】また、モニター色素の親水基も膜電位感受
性色素と同種の荷電を有することが望ましい。反対の荷
電を有した場合に膜電位感受性色素とモニター色素が静
電的なイオン結合をして蛍光が消光されることがあるか
らである。膜電位感受性色素と類似の性質の色素を用い
ることにより本発明のＬＢ膜の劣化の程度を簡便な補正
で見積もれることが期待される。Further, it is desirable that the hydrophilic group of the monitor dye also has the same type of charge as the membrane potential sensitive dye. This is because, when they have opposite charges, the membrane potential-sensitive dye and the monitor dye may electrostatically bond with each other to quench the fluorescence. It is expected that the degree of deterioration of the LB film of the present invention can be estimated by a simple correction by using a dye having properties similar to those of the membrane potential sensitive dye.
【００２６】以上のように、本発明のＬＢ膜を使用した
オプチカル化学センサーのモニター色素は、両親媒性で
あり、且つ膜電位感受性色素に対して不活性で、しかも
膜電位感受性色素と異なった光学特性を有する蛍光性の
ものが好ましい。このような色素の例としては、先に挙
げたＮＫ３１６７、ＮＫ２５６０、ＮＫ２６６５、ＮＫ
３１７５（日本感光色素社製）等がある。次に本発明の
ＬＢ膜を使用したオプチカル化学センサーの測定方法に
ついて述べる。As described above, the monitor dye of the optical chemical sensor using the LB film of the present invention is amphipathic, inactive to the membrane potential sensitive dye, and different from the membrane potential sensitive dye. A fluorescent substance having optical characteristics is preferable. Examples of such dyes include NK3167, NK2560, NK2665 and NK mentioned above.
3175 (manufactured by Nippon Senshoku Co., Ltd.) and the like. Next, the measuring method of the optical chemical sensor using the LB film of the present invention will be described.
【００２７】本発明のＬＢ膜を用いたオプチカル化学セ
ンサーは、上述のようにＬＢ単分子膜を、例えばシラン
処理したガラス基板上に累積して形成される。このセン
サーを用いて、例えば図２に示したようなフローセルを
組立て、試料中のイオンを測定する。図２において、２
１はシラン処理を施したガラス基板、２２はＬＢ膜、２
３はスペーサ、２４はガラスである。本方法では、モニ
ター色素の蛍光強度の変化とセンサーの劣化に起因する
センサーの感度の変化を同時に測定する。The optical chemical sensor using the LB film of the present invention is formed by accumulating the LB monomolecular film on, for example, a silane-treated glass substrate as described above. Using this sensor, for example, a flow cell as shown in FIG. 2 is assembled and ions in the sample are measured. In FIG. 2, 2
1 is a silane-treated glass substrate, 22 is an LB film, 2
3 is a spacer and 24 is glass. In this method, a change in fluorescence intensity of a monitor dye and a change in sensor sensitivity due to sensor deterioration are simultaneously measured.
【００２８】実際の測定では、予め２種類の既知濃度Ｃ
1 、Ｃ2 の試料溶液を調製しておき、それぞれの試料溶
液に対して膜電位感受性色素の蛍光強度とモニター色素
の蛍光強度の時間変化を測定する。次いで、モニター色
素の蛍光強度と既知濃度に対する膜電位感受性色素の蛍
光強度とを対比させたグラフを作成する。このグラフ
は、例えば図３に示すようになる。本発明のＬＢ膜を用
いたオプチカル化学センサーでは、モニター色素の蛍光
強度に対して、膜電位感受性色素の蛍光強度はほぼ直線
関係になる。In actual measurement, two known concentrations C
Sample solutions of C 1 and C 2 are prepared in advance, and the time change of the fluorescence intensity of the membrane potential sensitive dye and the fluorescence intensity of the monitor dye is measured for each sample solution. Then, a graph is prepared by comparing the fluorescence intensity of the monitor dye with the fluorescence intensity of the membrane potential sensitive dye at a known concentration. This graph is as shown in FIG. 3, for example. In the optical chemical sensor using the LB film of the present invention, the fluorescence intensity of the membrane potential sensitive dye has a substantially linear relationship with the fluorescence intensity of the monitor dye.
【００２９】次に、未知試料の濃度を測定する場合は、
該モニター色素の蛍光強度（ＦMt）からグラフにより既
知濃度での膜電位感受性色素の蛍光強度（Ｆ1t及び
Ｆ2t）を求める。未知試料の蛍光強度（Ｆxt）と既知濃
度での蛍光強度（Ｆ1t及びＦ2t）から濃度（Ｃx ）を求
める。Next, when measuring the concentration of an unknown sample,
From the fluorescence intensity (F Mt ) of the monitor dye, the fluorescence intensity (F 1t and F 2t ) of the membrane potential sensitive dye at a known concentration is determined by a graph. The concentration (C x ) is obtained from the fluorescence intensity (F xt ) of the unknown sample and the fluorescence intensity (F 1t and F 2t ) at a known concentration.
【００３０】本方法のオプチカル化学センサーに用いら
れるＬＢ膜に使用できる好ましい両親媒性物質、膜電位
感受性色素、特定物質認識能を有する物質、及びモニタ
ー色素としては、前述の化合物を挙げることができる。
次にセンサーの感度の劣化による変化を更に高精度に検
出する方法について述べる。As the preferable amphipathic substance, membrane potential sensitive dye, substance having specific substance recognition ability, and monitor dye which can be used for the LB film used in the optical chemical sensor of the present method, the above compounds can be mentioned. .
Next, a method for detecting the change due to the deterioration of the sensitivity of the sensor with higher accuracy will be described.
【００３１】本方法において、センサーの感度の劣化の
経時変化を更に高精度に検出するためには、モニター色
素の蛍光波長と膜電位感受性色素の励起波長を重なるよ
うにすればよい。モニター色素は蛍光を発するが、その
蛍光波長と膜電位感受性色素の励起波長が重なっていれ
ばエネルギー移動が起こり、モニター色素の蛍光が膜電
位感受性色素によって吸収され膜電位感受性色素が蛍光
を発することになる。即ち、モニター色素及び膜電位感
受性色素が劣化によってその蛍光強度を減少すると、そ
の変化量が、膜電位感受性色素の蛍光において重畳され
て観測されることになるので、膜電位感受性色素の劣化
に起因する蛍光強度の変化のみを観測した場合に比べ蛍
光強度の変化は大きくなる。従って、オプチカル化学セ
ンサーの劣化の程度がより正確に検出できることにな
る。In this method, in order to detect the deterioration of the sensitivity of the sensor over time with higher accuracy, the fluorescence wavelength of the monitor dye and the excitation wavelength of the membrane potential sensitive dye may be overlapped. The monitor dye emits fluorescence, but if the fluorescence wavelength and the excitation wavelength of the membrane potential sensitive dye overlap, energy transfer occurs, the fluorescence of the monitor dye is absorbed by the membrane potential sensitive dye, and the membrane potential sensitive dye emits fluorescence. become. That is, when the fluorescence intensity of the monitor dye and the membrane potential sensitive dye is reduced due to deterioration, the amount of change is observed in the fluorescence of the membrane potential sensitive dye in a superposed manner. The change in fluorescence intensity is larger than that in the case where only the change in fluorescence intensity is observed. Therefore, the degree of deterioration of the optical chemical sensor can be detected more accurately.
【００３２】本方法を用いる場合、通常の測定において
は膜電位感受性色素を励起し、その蛍光強度を測定す
る。センサーの劣化の程度を測定する場合にはモニター
色素を励起し、膜電位感受性色素の発する蛍光の強度を
測定する。When the present method is used, in a usual measurement, a membrane potential sensitive dye is excited and its fluorescence intensity is measured. When measuring the degree of sensor deterioration, the monitor dye is excited and the intensity of fluorescence emitted by the membrane potential sensitive dye is measured.
【００３３】実際の測定では、予め２種類の既知濃度の
試料溶液を調製しておき、それぞれの試料溶液に対して
膜電位感受性色素を励起して観測される蛍光強度とモニ
ター色素を励起して観測される蛍光強度の時間変化を測
定する。このグラフは、例えば図４のようになる。この
場合、比は下式のように表されるので、膜電位発生によ
る膜電位感受性色素の発光効率の変化はキャンセルで
き、上記図４の２つの直線はほぼ平行になる。In the actual measurement, two kinds of sample solutions having known concentrations were prepared in advance, and the fluorescence intensity and the monitor dye observed by exciting the membrane potential sensitive dye were excited for each sample solution. The time change of the observed fluorescence intensity is measured. This graph is as shown in FIG. 4, for example. In this case, since the ratio is expressed by the following equation, the change in the luminous efficiency of the membrane potential sensitive dye due to the generation of the membrane potential can be canceled, and the two straight lines in FIG. 4 become almost parallel.
【００３４】[0034]
【数１】 [Equation 1]
【００３５】本発明のＬＢ膜を用いたセンサーにおいて
は、条件を設定することにより、上記の関係を図４に示
すような直線関係にすることができる。劣化によるセン
サーの補正をより簡便に行なうため、このような直線関
係が得られるようにＬＢ膜に含まれる各物質を選択する
ことが好ましい。直線関係を得るための好ましい物質に
は、両親媒性物質、膜電位感受性色素、及び特定物質認
識能を有する物質としては前述の化合物を挙げることが
できる。また、好ましいモニター色素としては、例え
ば、ＮＫ３０４５（日本感光色素）等がある。In the sensor using the LB film of the present invention, the above relationship can be made linear as shown in FIG. 4 by setting the conditions. In order to more easily correct the sensor due to deterioration, it is preferable to select each substance contained in the LB film so as to obtain such a linear relationship. Preferred substances for obtaining a linear relationship include amphipathic substances, membrane potential-sensitive dyes, and compounds having the ability to recognize a specific substance described above. Further, as a preferable monitor dye, for example, NK3045 (Japan Photosensitive Dye) or the like can be used.
【００３６】次に、未知試料の濃度を測定する場合は、
蛍光強度の比（ＦMt’）から既知濃度での膜電位感受性
色素の蛍光強度（Ｆ1t’及びＦ2t’）をグラフより求め
る。膜電位感受性色素を励起して観測される未知試料の
蛍光強度（Ｆxt’）と前記既知濃度での膜電位感受性色
素の蛍光強度（Ｆ1t’及びＦ2t’）から濃度（Ｃx ’）
を求める。Next, when measuring the concentration of an unknown sample,
The fluorescence intensity (F 1t 'and F 2t ') of the membrane potential sensitive dye at a known concentration is determined from the ratio of the fluorescence intensity (F Mt ') from a graph. The fluorescence intensity (F xt ') of an unknown sample observed by exciting the membrane potential-sensitive dye and the fluorescence intensity (F 1t ' and F 2t ') of the membrane potential-sensitive dye at the known concentration to the concentration (C x ')
Ask for.
【００３７】次に、両親媒性物質、膜電位感受性色素、
及び機能性物質を含有したＬＢ膜を使用したオプチカル
化学センサーの劣化を非常に簡便に検出する方法につい
て述べる。本方法は、オプチカル化学センサーの空気中
の蛍光強度が水中での蛍光強度に比べ倍以上の値が得ら
れることに基づいている。Next, an amphipathic substance, a membrane potential sensitive dye,
And, a method for very easily detecting deterioration of an optical chemical sensor using an LB film containing a functional substance will be described. This method is based on the fact that the fluorescence intensity in the air of the optical chemical sensor is more than double the fluorescence intensity in water.
【００３８】例えば、両親媒性物質としてジドデシルリ
ン酸を用い、膜電位感受性色素としてオクタデシルロー
ダミンＢ、特定物質認識能を有する物質とてバリノマイ
シンを用いたＬＢ膜からなるオプチカル化学センサーの
蛍光を水中と空気中で測定した場合、その蛍光スペクト
ルは図５に示すようになる。図からわかるように空気中
で測定した場合の蛍光強度は、水中のそれに比べ５８０
ｎｍの波長で約２倍である。このことは、センサーの劣
化による蛍光強度の変化が、空気中でより正確に測定で
きることを意味している。即ち、センサーの劣化の程度
をモニターする場合には空気中にセンサーを晒して蛍光
を測定すればよい。これにより高精度に劣化の程度を検
出することができる。従って、実際の測定では、空気中
での蛍光強度を適宜測定しながら試料溶液を測定し、空
気中の蛍光強度を基に、上述のモニター色素を用いた場
合と同様に補正をすれば、高精度に試料溶液の濃度を求
めることが可能となる。For example, the fluorescence of an optical chemical sensor consisting of LB film using didodecyl phosphate as an amphipathic substance, octadecyl rhodamine B as a membrane potential sensitive dye, and valinomycin as a substance capable of recognizing a specific substance is used as fluorescence in water and air. When measured in, the fluorescence spectrum is as shown in FIG. As can be seen from the figure, the fluorescence intensity when measured in air is 580 compared to that in water.
It is about double at the wavelength of nm. This means that changes in fluorescence intensity due to sensor deterioration can be measured more accurately in air. That is, in order to monitor the degree of deterioration of the sensor, the fluorescence may be measured by exposing the sensor to the air. This makes it possible to detect the degree of deterioration with high accuracy. Therefore, in the actual measurement, the sample solution is measured while appropriately measuring the fluorescence intensity in the air, and if the correction is performed in the same manner as in the case where the above monitor dye is used based on the fluorescence intensity in the air, the high It is possible to accurately determine the concentration of the sample solution.
【００３９】一般的な方法として、既知濃度の溶液にセ
ンサーを浸漬し、蛍光強度の変化をモニターする方法も
あるが、既知濃度の溶液が必要であること及び蛍光強度
も本方法に比べ弱いので、センサーの操作上の簡便性及
び測定精度の観点から本方法の方が有効である。As a general method, there is a method of immersing the sensor in a solution having a known concentration and monitoring the change in fluorescence intensity. However, since a solution having a known concentration is required and the fluorescence intensity is weaker than this method. This method is more effective from the viewpoints of operational simplicity of the sensor and measurement accuracy.
【００４０】[0040]
【作用】本発明のＬＢ膜は、その構成成分として膜電位
感受性色素、特定物質認識能を有する物質、両親媒性物
質およびモニター色素を含有する。また、本発明のＬＢ
膜は、前記構成成分をクロロホルム等の有機溶媒に溶解
した溶液を水面上に展開して単分子膜とし、これを適切
な支持体上に１層乃至それ以上累積して形成される。こ
のようにして形成された本発明のＬＢ膜はオプチカル化
学センサーとして使用し得る。The LB film of the present invention contains, as its constituent components, a membrane potential sensitive dye, a substance capable of recognizing a specific substance, an amphipathic substance and a monitor dye. In addition, the LB of the present invention
The membrane is formed by spreading a solution of the above-mentioned constituents in an organic solvent such as chloroform on the water surface to form a monomolecular film, and accumulating one or more layers on a suitable support. The LB film of the present invention thus formed can be used as an optical chemical sensor.
【００４１】また、本発明のＬＢ膜を使用したオプチカ
ル化学センサーは、蛍光性のモニター色素を含有してお
り、この蛍光強度を測定することにより該センサーの補
正を容易に、しかも測定操作を中断することなく行なう
ことができる。Further, the optical chemical sensor using the LB film of the present invention contains a fluorescent monitor dye, and by measuring the fluorescence intensity, the sensor can be easily corrected and the measurement operation can be interrupted. You can do it without doing.
【００４２】更に、上記オプチカル化学センサーに含有
される蛍光性のモニター色素の蛍光波長を膜電位感受性
色素の励起波長と重なるように該モニター色素を選択
し、且つ膜電位感受性色素やモニター色素を適切に選択
する等の設定条件を適切に行なうことによって、より高
精度にセンサーの補正を行なうことができる。Further, the monitor dye is selected so that the fluorescence wavelength of the fluorescent monitor dye contained in the optical chemical sensor overlaps with the excitation wavelength of the membrane potential sensitive dye, and the membrane potential sensitive dye or the monitor dye is appropriately selected. The sensor can be corrected with higher accuracy by appropriately performing the setting conditions such as selection to.
【００４３】ＬＢ膜を使用したオプチカル化学センサー
は、空気中と水中で蛍光強度が大きく異なるため、空気
中の蛍光強度を用いてセンサーの感度の補正を行なうこ
とが可能である。Since the optical chemical sensor using the LB film has a great difference in fluorescence intensity between air and water, it is possible to correct the sensitivity of the sensor by using the fluorescence intensity in air.
【００４４】[0044]
実施例１ ＬＢ膜の調製 Example 1 Preparation of LB membrane
【００４５】両親媒性物質であるジドデシルリン酸（相
互薬工）中に、膜電位感受性色素としてオクタデシルロ
ーダミンＢ（モレキュラープローブ）を２ｍｏｌ％、機
能性物質とてバリノマイシン（シグマ）を１０ｍｏｌ
％、さらにモニター色素として両親媒性色素のＮＫ３１
６７（日本感光色素）を２ｍｏｌ％含むクロロホルム溶
液を調製した。この溶液を純水上に展開し、単分子膜を
形成した。この単分子膜を相転移が起きる前の圧力（１
７ｍＮ）でフローセル用ガラス基板に移し取る作業を繰
り返しＬＢ膜を４層累積し本発明のＬＢ膜とした。ガラ
ス基板はビニルトリクロロシランで疎水処理を施したも
のを用いた。この方法で基板の両側に累積されたＬＢ膜
は、溶液と接触し測定に用いる面側のみからの蛍光を測
定するため、溶液と接触しない側のＬＢ膜は有機溶剤で
除去した。 実施例２2 mol% of octadecyl rhodamine B (molecular probe) as a membrane potential sensitive dye and 10 mol of valinomycin (sigma) as a functional substance in didodecyl phosphate (mutual drug) which is an amphipathic substance.
%, And NK31 which is an amphipathic dye as a monitor dye
A chloroform solution containing 2 mol% of 67 (Japan Photosensitive Dye) was prepared. This solution was spread on pure water to form a monomolecular film. The pressure (1
The work of transferring to a glass substrate for a flow cell at 7 mN) was repeated and four LB films were accumulated to obtain an LB film of the present invention. The glass substrate used was one that was subjected to a hydrophobic treatment with vinyltrichlorosilane. The LB films accumulated on both sides of the substrate by this method measure the fluorescence only from the surface side which is in contact with the solution and used for the measurement, so the LB film on the side not in contact with the solution was removed with an organic solvent. Example 2
【００４６】実施例１で調製したＬＢ膜は、オプチカル
化学センサーとして使用できる。従って、これを図２に
示すようにフローセルに装着し、このフローセルを蛍光
光度計に組み込んで蛍光強度の測定を行った。なお、試
料溶液は外部からローラーポンプを用いて連続的にフロ
ーセルに導入した。膜電位感受性色素の蛍光強度の測定
には波長５４０ｎｍの光で該色素を励起し、５８５ｎｍ
の波長で蛍光強度を測定した。またオプチカル化学セン
サーの劣化をモニターするため、モニター色素ＮＫ３１
６７を波長７４１ｎｍの光で励起し、９００ｎｍの波長
で該モニター色素の蛍光強度を測定した。The LB film prepared in Example 1 can be used as an optical chemical sensor. Therefore, this was attached to a flow cell as shown in FIG. 2, and this flow cell was incorporated into a fluorometer to measure the fluorescence intensity. The sample solution was continuously introduced into the flow cell from the outside using a roller pump. To measure the fluorescence intensity of a membrane potential sensitive dye, the dye is excited with light having a wavelength of 540 nm and 585 nm.
The fluorescence intensity was measured at the wavelength of. In order to monitor the deterioration of the optical chemical sensor, the monitor dye NK31
67 was excited with light having a wavelength of 741 nm, and the fluorescence intensity of the monitor dye was measured at a wavelength of 900 nm.
【００４７】センサーを１時間エージングしてある程度
安定させた後、２４時間連続的に測定した．結果を図６
に示す。図６は時間に対してモニター色素の蛍光強度、
１０-3ＭＫＣｌ水溶液及び１０-2ＭＫＣｌ水溶液を測定
した場合の膜電位感受性色素の蛍光強度をプロットした
ものである。このグラフからモニター色素の蛍光強度及
び膜電位感受性色素の蛍光強度が同じように減衰してい
ることがわかる。この実験結果から、モニター色素の蛍
光強度に対して、１０-3ＭＫＣｌ水溶液及び１０-2ＭＫ
Ｃｌ水溶液を測定した場合の膜電位感受性色素の蛍光強
度をプロットしたものが図７である。両水溶液とも良い
直線関係が得られた。この図７を使用して補正をするこ
とができる。以下に図７を基にして説明する。The sensor was aged for 1 hour to be stabilized to some extent, and then continuously measured for 24 hours. The result is shown in Fig. 6.
Shown in. FIG. 6 shows the fluorescence intensity of the monitor dye with respect to time,
2 is a plot of fluorescence intensities of dyes sensitive to membrane potential when measuring 10 −3 MKCl aqueous solution and 10 −2 MKCl aqueous solution. From this graph, it can be seen that the fluorescence intensity of the monitor dye and the fluorescence intensity of the membrane potential sensitive dye are similarly attenuated. From the results of this experiment, it was found that the fluorescence intensity of the monitor dye was 10 -3 MKCl aqueous solution and 10 -2 MK
FIG. 7 is a plot of the fluorescence intensities of the membrane potential sensitive dyes when Cl aqueous solution was measured. A good linear relationship was obtained with both aqueous solutions. Correction can be made using this FIG. The following is a description based on FIG. 7.
【００４８】まず、２種類の既知濃度の試料（１０-3Ｍ
ＫＣｌ水溶液及び１０-2ＭＫＣｌ水溶液）を測定し、図
７に示したようなモニター色素の蛍光強度と、膜電位感
受性色素の蛍光強度の関係をプロットしておく。次に、
未知濃度の試料をセルに導入し、モニター色素の蛍光強
度（ＦMt）及び膜電位感受性色素の蛍光強度（Ｆx ）を
測定する。図７の関係から、モニター色素の蛍光強度が
ＦMtのときの既知濃度での膜電位感受性色素の蛍光強度
（図７ではＦ1t及びＦ2t）を求めることができるので測
定したＦxtとグラフから求めたＦ1t及びＦ2t並びに既知
濃度（本実施例の場合１０-2ＭＫＣｌと１０-3ＭＫＣ
ｌ）から測定試料の濃度Ｃx を求めることができる。First, two kinds of samples with known concentrations (10 −3 M
A KCl aqueous solution and a 10 −2 MKCl aqueous solution) are measured, and the relationship between the fluorescence intensity of the monitor dye and the fluorescence intensity of the membrane potential sensitive dye as shown in FIG. 7 is plotted. next,
A sample of unknown concentration is introduced into the cell, and the fluorescence intensity (F Mt ) of the monitor dye and the fluorescence intensity (F x ) of the membrane potential sensitive dye are measured. From the relationship of FIG. 7, F xt and graphs the fluorescence intensity measured it is possible to obtain the (in FIG. 7 F 1t and F 2t) of the membrane potential-sensitive dye in a known concentration at the fluorescence intensity of the monitor dye F Mt F 1t and F 2t obtained from the above and known concentrations (10 −2 MKCl and 10 −3 MKC in this example)
The concentration C x of the measurement sample can be obtained from l).
【００４９】また、本実施例のＬＢ膜を使用したオプチ
カル化学センサーはＫ+ イオンに応答するので１０-2Ｍ
ＫＣｌと１０-3ＭＫＣｌ水溶液を測定したときの蛍光強
度の差を感度とすることができる。更に、数式で取扱う
場合について説明する。The optical chemical sensor using the LB film of this embodiment is K +. 10 -2 M because it responds to ions
The difference in fluorescence intensity when measuring KCl and an aqueous solution of 10 −3 MKCl can be used as the sensitivity. Further, a case of handling by a mathematical formula will be described.
【００５０】ＬＢ膜を使用したオプチカル化学センサー
の時間ｔでの膜電位感受性色素の蛍光強度（Ｆ）は、測
定する試料の濃度（Ｃ）、時間ｔでのセンサーの感度
（Ｓt）、及び時間ｔでの蛍光強度の定数項（Ｆt ）と
すると、以下の式（１）で表わされることがわかってい
る（M.Shimomura, A.Honnma, N.Tajima, S.Kondo, E.Sh
inohara, K.Koshiishi, 4th Int. Meeting of Chem. Se
ns. ）。 Ｆ＝Ｆt −Ｓt ・ｌｏｇＣ （１） 従って、既知濃度Ｃ1 及びＣ2 を時間ｔで測定した場合
は、 溶液１ Ｆ1t＝Ｆt −Ｓt ・ｌｏｇＣ1 （２） 溶液２ Ｆ2t＝Ｆt −Ｓt ・ｌｏｇＣ2 （３） となる。The fluorescence intensity (F) of the membrane potential sensitive dye at the time t of the optical chemical sensor using the LB film is the concentration of the sample to be measured (C), the sensitivity of the sensor at the time t (S t ), and It is known that if the constant term (F t ) of the fluorescence intensity at time t is expressed by the following equation (1) (M.Shimomura, A.Honnma, N.Tajima, S.Kondo, E.Sh)
inohara, K. Koshiishi, 4th Int. Meeting of Chem. Se
ns.). F = F t −S t · log C (1) Therefore, when known concentrations C 1 and C 2 are measured at time t, solution 1 F 1t = F t −S t · log C 1 (2) solution 2 F 2t = F t -S t · logC 2 (3) to become.
【００５１】ここでＦ1tおよびＦ2tは、時間ｔにおける
既知濃度の溶液１および２の膜電位感受性色素の蛍光強
度、Ｃ1 およびＣ2 は、既知濃度の溶液１および２の濃
度（本実施例の場合は、それぞれ１０-3ＭＫＣｌおよび
１０-2ＭＫＣｌ）である。時間ｔで未知試料Ｃx を測定
したときの膜電位感受性色素の蛍光強度をＦxtとすると Ｆxt＝Ｆt −Ｓt ・ｌｏｇＣx （４） ここでＦt は時間ｔt での蛍光強度の定数項、Ｓt は時
間ｔt での感度である。Here, F 1t and F 2t are the fluorescence intensities of the membrane potential sensitive dyes of solutions 1 and 2 of known concentration at time t, and C 1 and C 2 are the concentrations of solutions 1 and 2 of known concentration (in this embodiment). In the case of the examples, they are 10 −3 MKCl and 10 −2 MKCl, respectively. Let F xt be the fluorescence intensity of the membrane potential sensitive dye when the unknown sample C x is measured at time t, F xt = F t −St t log C x (4) where F t is the fluorescence intensity at time t t The constant term, S t, is the sensitivity at time t t .
【００５２】Ｆ1tを時間ｔでの溶液１を測定したときの
膜電位感受性色素の蛍光強度とすると図７よりＦ1tはモ
ニター色素の蛍光強度（ＦM ）の関数として表され、本
実験ではほぼ直線的な関係が得られたので Ｆ1t＝ＡＦMt＋Ｂ （５） となる。よって式（２）及び（５）から、 Ｆ1t＝Ｆt −Ｓt ・ｌｏｇＣ1 ＝ＡＦMt＋Ｂ （６） ここで、Ａ及びＢは図７の直線（ａ）の傾き及び切片で
ある。従って、 Ｆt ＝ＡＦMt＋Ｂ＋Ｓt ・ｌｏｇＣ1 （７） ここで、Ｃ及びＤは図７の直線（ｂ）の傾き及び切片で
ある。となる。同様に、 Ｆ2t＝ＣＦMt＋Ｄ （８） と表され、従って、 Ｆ2t＝Ｆt −Ｓt ・ｌｏｇＣ2 ＝ＣＦMt＋Ｄ （９） Ｆt ＝ＣＦMt＋Ｄ＋Ｓt ・ｌｏｇＣ2 （１０） となる。式（７）及び（１０）よりＦt とＳt を求める
と、Ｓt は（７）−（１０）より、[0052] F 1t than 7 When the fluorescence intensity of the membrane potential-sensitive dye when measuring the solution 1 in F 1t time t is expressed as a function of the fluorescence intensity of the monitor dye (F M), in this experiment Since a substantially linear relationship was obtained, F 1t = AF Mt + B (5) Therefore, from Equations (2) and (5), F 1t = F t −St t log C 1 = A F Mt + B (6) Here, A and B are the slopes and intercepts of the straight line (a) in FIG. 7. Therefore, F t = A F Mt + B + S t · log C 1 (7) Here, C and D are the slope and intercept of the straight line (b) in FIG. 7. Becomes Similarly, it is expressed as F 2t = CF Mt + D (8), and thus F 2t = F t −St t logC 2 = CF Mt + D (9) F t = CF Mt + D + S t log C 2 (10) Become. When F t and S t are obtained from the equations (7) and (10), S t is obtained from (7)-(10).
【００５３】[0053]
【数２】 となり、Ｆt は式（１２）を式（９）に代入することに
より、[Equation 2] And F t is obtained by substituting the equation (12) into the equation (9),
【００５４】[0054]
【数３】 となる。[Equation 3] Becomes
【００５５】従って、式（４）に式（１２）及び（１
３）を代入し、時間ｔでの膜電位感受性色素を直接励起
して観測される蛍光強度（Ｆxt）とモニター色素を励起
して観測される蛍光強度ＦMtを測定することにより、サ
ンプルの濃度を演算で求めることができる。Therefore, equations (12) and (1) are added to equation (4).
3) is substituted, and the fluorescence intensity (F xt ) observed by directly exciting the membrane potential-sensitive dye at time t and the fluorescence intensity F Mt observed by exciting the monitor dye are measured, and The concentration can be calculated.
【００５６】なお本実験では既知濃度のサンプルとして
１０-3ＭＫＣｌおよび１０-2ＭＫＣｌを用いたがこれに
限定されるわけでなく、濃度は測定しようとするサンプ
ルにあわせて種々選択が可能である。また、センサーの
膜構成によってはモニター色素の蛍光強度と膜電位感受
性色素の蛍光強度およびセンサーの感度が直線的な関係
を示さない場合もあり得る。その場合には他の近似式、 例えば Ｆ1t＝Ａ′ＦMt 2 ＋Ｂ′ＦMt＋Ｃ′ Ｆ1t＝Ｄ′ｅｘｐ｛Ｅ′ＦMt｝＋Ｇ′ のような種々の関数を用いて最小２剰法などで曲線のフ
ィッティングを行い、これらの式から上記Ｆt 及びＳt
を導き出して用いればよい。In this experiment, 10 -3 MKCl and 10 -2 MKCl were used as samples of known concentration, but the sample is not limited to this, and the concentration can be variously selected according to the sample to be measured. . Further, depending on the membrane structure of the sensor, there is a case where the fluorescence intensity of the monitor dye, the fluorescence intensity of the membrane potential sensitive dye and the sensitivity of the sensor do not show a linear relationship. In that case, another approximation formula, for example, F 1t = A′F Mt 2 + B'F Mt + C 'F 1t = D'exp {E'F Mt } + G' is used to perform curve fitting by the least square method or the like, and from these equations, F t and S t
Should be derived and used.
【００５７】以上のように、本発明のＬＢ膜を用いたオ
プチカル化学センサーでは、モニター色素の蛍光強度及
び膜電位感受性色素の蛍光強度を測定するだけで容易に
センサーの補正が行なえ、未知試料の濃度を求めること
ができる。As described above, in the optical chemical sensor using the LB film of the present invention, the sensor can be easily corrected by simply measuring the fluorescence intensity of the monitor dye and the fluorescence intensity of the membrane potential sensitive dye, and the unknown sample The concentration can be determined.
【００５８】なお本実験ではモニター色素としてＮＫ３
１６７を用いたが、これに限定されるものではない。即
ち、両親媒性で膜電位感受性色素と異なった光学特性を
持ち且つ蛍光性のものであるならば使用可能である。こ
のような色素としては、例えばＮＫ２５６０、ＮＫ２６
６５、ＮＫ３１７５などがある。上記色素をＬＢ膜に含
有する場合の該色素の濃度は、０．１〜２０ｍｏｌ％の
間で適宜変更することが出来る。 実施例３In this experiment, NK3 was used as a monitor dye.
Although 167 was used, it is not limited to this. That is, if it is amphipathic, has different optical characteristics from the membrane potential sensitive dye, and is fluorescent, it can be used. Examples of such dyes include NK2560 and NK26.
65, NK3175, etc. When the above dye is contained in the LB film, the concentration of the dye can be appropriately changed within the range of 0.1 to 20 mol%. Example 3
【００５９】両親媒性物質であるジドデシルリン酸中
に、膜電位感受性色素としてオクタデシルローダミンＢ
を２ｍｏｌ％、機能性物質とてバリノマイシンを１０ｍ
ｏｌ％、さらに経時変化モニター用色素として両親媒性
色素のＮＫ３０４５（日本感光色素）を１０ｍｏｌ％含
むクロロホルム溶液を調製し、実施例１と同様にＬＢ膜
を調製した。これをフローセルに装着し、蛍光光度計に
組み込んで蛍光強度の測定を行った。なお、試料溶液は
外部からローラーポンプを用いて連続的にフローセルに
導入した。膜電位感受性色素の蛍光強度の測定には波長
５４０ｎｍの光で膜電位感受性色素を励起し、５８５ｎ
ｍの波長で該色素の蛍光強度を測定した。またオプチカ
ル化学センサーの劣化をモニターするため色素ＮＫ３０
４５を波長４８５ｎｍの光で励起し、膜電位感受性色素
の発する蛍光強度を測定した。Octadecyl rhodamine B was added as a membrane potential sensitive dye in didodecyl phosphate which is an amphipathic substance.
Is 2 mol% and valinomycin is 10 m as a functional substance.
Then, a chloroform solution containing ol% and 10 mol% of amphiphilic dye NK3045 (Japan Photosensitive Dye) as a dye for aging monitoring was prepared, and an LB film was prepared in the same manner as in Example 1. This was attached to a flow cell and incorporated into a fluorometer to measure fluorescence intensity. The sample solution was continuously introduced into the flow cell from the outside using a roller pump. To measure the fluorescence intensity of the membrane potential sensitive dye, the membrane potential sensitive dye was excited with light having a wavelength of 540 nm to obtain 585n.
The fluorescence intensity of the dye was measured at the wavelength of m. In addition, to monitor the deterioration of the optical chemical sensor, the dye NK30
45 was excited with light having a wavelength of 485 nm, and the fluorescence intensity emitted by the membrane potential sensitive dye was measured.
【００６０】測定は、センサーを１時間エージングして
ある程度安定させた後、２４時間連続的に行った。本実
施例においても、センサーはＫ+ イオンに応答するので
１０-2ＭＫＣｌと１０-3ＭＫＣｌの水溶液を測定した場
合の、膜電位感受性色素を直接励起して発せられる蛍光
強度の差を感度として、実施例２と同様に図８を作成し
た。但し、本実施例においては、モニター色素の蛍光強
度を直接測定していないため、実施例２の図７のモニタ
ー色素の蛍光強度の代わりに、モニター色素を励起して
観測された膜電位感受性色素の蛍光強度と膜電位感受性
色素を直接励起して観測された蛍光強度の比を取りそれ
を横軸とした。本実施例においては、図８に示すように
蛍光強度の比に対し、１０-3ＭＫＣｌ、１０-2ＭＫＣｌ
溶液を測定した場合の、膜電位感受性色素を直接励起し
て観測される蛍光強度との関係を示した。本実施例にお
いては、両者とも平行の関係を示した。The measurement was carried out continuously for 24 hours after aging the sensor for 1 hour to stabilize it for a while. Also in this embodiment, the sensor is K +. Since it responds to ions, the difference between the fluorescence intensities emitted by directly exciting the membrane potential-sensitive dye in the case of measuring an aqueous solution of 10 −2 MKCl and 10 −3 MKCl is taken as sensitivity, and FIG. Created. However, in this Example, since the fluorescence intensity of the monitor dye was not directly measured, instead of the fluorescence intensity of the monitor dye of FIG. 7 of Example 2, the membrane potential sensitive dye observed by exciting the monitor dye was observed. The abscissa is the ratio of the fluorescence intensity observed by directly exciting the dye sensitive to membrane potential and the fluorescence intensity observed. In this example, as shown in FIG. 8, the ratio of fluorescence intensity was 10 −3 MKCl, 10 −2 MKCl.
The relationship with the fluorescence intensity observed by directly exciting the membrane potential-sensitive dye when the solution was measured is shown. In this embodiment, both have a parallel relationship.
【００６１】未知の濃度の試料を測定する場合にはこの
図８を基にして、実施例２と同様にモニター用色素の蛍
光強度の比（ＦMt’）から１０-3ＭＫＣｌ水溶液の膜電
位感受性色素の蛍光強度（Ｆ1t’）と１０-2ＫＣｌ水溶
液の膜電位感受性色素の蛍光強度（Ｆ2t’）を求め測定
試料の濃度（Ｃx ’）を求めることができる。In the case of measuring a sample of unknown concentration, based on this FIG. 8, the membrane potential of the 10 −3 MKCl aqueous solution was calculated from the ratio of the fluorescence intensities (F Mt ′) of the monitoring dyes as in Example 2. The fluorescence intensity (F 1t ') of the sensitive dye and the fluorescence intensity (F 2t ') of the membrane potential sensitive dye of the 10 -2 KCl aqueous solution can be obtained to obtain the concentration (C x ') of the measurement sample.
【００６２】演算で濃度を求める場合は、本実施例にお
いても図８に示されるような平行関係が得られたことか
ら実施例２の式（７）及び（１０）のＦMtを上記蛍光色
素の比に置き換えることにより実施例２と同様に演算に
よって導き出すことができる。When the concentration is calculated by calculation, the parallel relationship as shown in FIG. 8 is obtained also in the present embodiment. Therefore, F Mt of the formulas (7) and (10) of the second embodiment is set to the above fluorescent dye. It can be derived by calculation as in the second embodiment by substituting the ratio
【００６３】以上のように、本発明のＬＢ膜を用いたオ
プチカル化学センサーでは、モニター色素を励起して観
測される膜電位感受性色素の蛍光強度と膜電位感受性色
素を直接励起して観測された蛍光強度の比及び膜電位感
受性色素の蛍光強度を測定するだけで容易にセンサーの
補正が行なえ、未知試料の濃度を求めることができる。As described above, in the optical chemical sensor using the LB film of the present invention, the fluorescence intensity of the membrane potential sensitive dye observed by exciting the monitor dye and the direct excitation of the membrane potential sensitive dye were observed. By simply measuring the ratio of the fluorescence intensity and the fluorescence intensity of the membrane potential sensitive dye, the sensor can be easily corrected and the concentration of the unknown sample can be obtained.
【００６４】なお本実験ではモニター色素としてＮＫ３
０４５を用いたが、これに限定されるものではない。即
ち、両親媒性で膜電位感受性色素の励起波長とモニター
用色素の蛍光波長が重なる光学特性を有するものである
ならば使用することができる。モニター色素をＬＢ膜に
含有する場合の該色素の濃度は、０．５〜５０ｍｏｌ％
の間で適宜変更することが出来る。 実施例４ モニター色素を用いずに本発明のＬＢ膜を使用したオプ
チカル化学センサーの劣化の程度を補正する方法につい
て述べる。In this experiment, NK3 was used as a monitor dye.
Although 045 was used, it is not limited to this. That is, it can be used as long as it is an amphipathic substance having an optical characteristic in which the excitation wavelength of the membrane potential sensitive dye and the fluorescence wavelength of the monitoring dye overlap. When the monitor dye is contained in the LB film, the concentration of the dye is 0.5 to 50 mol%.
Can be changed as appropriate. Example 4 A method for correcting the degree of deterioration of an optical chemical sensor using the LB film of the present invention without using a monitor dye will be described.
【００６５】両親媒性物質であるジドデシルリン酸中
に、膜電位感受性色素としてオクタデシルローダミンＢ
を２ｍｏｌ％、機能性物質としてバリノマイシンを１０
ｍｏｌ％含むクロロホルム溶液を調製し、実施例１と同
様にＬＢ膜を調製した。これをフローセルに装着し、蛍
光光度計に組み込んで蛍光強度の測定を行った。試料液
は外部からローラーポンプを用いて連続的にフローセル
に導入した。膜電位感受性色素の励起光は、波長５４０
ｎｍの光を使用し、５８５ｎｍの波長の蛍光強度を測定
した。またセンサーの劣化に起因する蛍光強度の減少を
補正するため、ローラーポンプで空気をセルに送り込
み、その時の蛍光強度を測定した。Octadecyl rhodamine B as a membrane potential sensitive dye in didodecyl phosphate which is an amphipathic substance.
2 mol% and valinomycin 10 as a functional substance
A chloroform solution containing mol% was prepared, and an LB film was prepared in the same manner as in Example 1. This was attached to a flow cell and incorporated into a fluorometer to measure fluorescence intensity. The sample solution was continuously introduced into the flow cell from the outside using a roller pump. The excitation light of the membrane potential sensitive dye has a wavelength of 540
nm light was used to measure the fluorescence intensity at a wavelength of 585 nm. In order to correct the decrease in fluorescence intensity due to sensor deterioration, air was blown into the cell with a roller pump and the fluorescence intensity at that time was measured.
【００６６】センサーを１時間エージングしてある程度
安定させた後、２４時間測定した。本実施例においても
本センサーは、Ｋ+ イオンに応答するので１０-2ＭＫＣ
ｌと１０-3ＭＫＣｌ水溶液を測定したときの蛍光強度を
実施例２と同様にグラフを作成した（図９）。但し、空
気中で測定した蛍光強度を横軸に取った。本実施例にお
いてもよい直線関係が得られた。The sensor was aged for 1 hour to be stabilized to some extent, and then measured for 24 hours. Also in this embodiment, this sensor is K + 10 -2 MKC as it responds to ions
A graph was prepared in the same manner as in Example 2 with respect to the fluorescence intensity when 1 and 10 −3 MKCl aqueous solution were measured (FIG. 9). However, the fluorescence intensity measured in air was plotted on the horizontal axis. A good linear relationship was obtained also in this example.
【００６７】未知の濃度の試料を測定した場合にはこの
図９を基にして、実施例２と同様に空気中で測定したと
きの蛍光強度（ＦMt”）から１０-3ＭＫＣｌ水溶液の膜
電位感受性色素の蛍光強度（Ｆ1t”）と１０-2ＭＫＣｌ
水溶液の膜電位感受性色素の蛍光強度（Ｆ2t”）を求
め、測定試料の濃度（Ｃx ”）を演算により求めること
ができる。When a sample having an unknown concentration is measured, based on FIG. 9, the fluorescence intensity (F Mt ″) measured in air as in Example 2 indicates the film of 10 −3 MKCl aqueous solution. Fluorescence intensity of voltage sensitive dye (F 1t ") and 10 -2 MKCl
The fluorescence intensity (F 2t ″) of the membrane potential sensitive dye in the aqueous solution can be obtained, and the concentration (C x ″) of the measurement sample can be calculated.
【００６８】演算で濃度を求める場合は、本実施例にお
いても図９に示されるような直線関係が得られたことか
ら実施例２の式（７）及び（１０）のＦMtを空気中で測
定した時の蛍光強度（ＦMt”）に置き換えることにより
実施例２と同様に行える。When the concentration is calculated by calculation, the linear relationship as shown in FIG. 9 is obtained also in the present embodiment, and therefore F Mt of the equations (7) and (10) of the second embodiment is set in the air. The same procedure as in Example 2 can be performed by substituting the measured fluorescence intensity (F Mt ″).
【００６９】[0069]
【発明の効果】以上詳述したようにＬＢ膜を使用したオ
プチカル化学センサーに本発明のＬＢ膜を使用すること
によって、測定を中断することなく該センサーの劣化を
高感度に検出することができ、濃度の補正が可能にな
る。As described in detail above, by using the LB film of the present invention in the optical chemical sensor using the LB film, deterioration of the sensor can be detected with high sensitivity without interrupting the measurement. , The density can be corrected.
【図１】本発明のＬＢ膜の模式図。FIG. 1 is a schematic diagram of an LB film of the present invention.
【図２】本発明のＬＢ膜を組み込んだフローセルの断面
図。FIG. 2 is a sectional view of a flow cell incorporating the LB film of the present invention.
【図３】本発明のＬＢ膜を用いたオプチカル化学センサ
ーにおいて、モニター色素の蛍光強度に対して、既知濃
度の溶液を測定した場合の膜電位感受性色素の蛍光強度
をプロットした図。FIG. 3 is a diagram in which, in the optical chemical sensor using the LB film of the present invention, the fluorescence intensity of a membrane potential sensitive dye when a solution having a known concentration is measured is plotted against the fluorescence intensity of a monitor dye.
【図４】本発明のＬＢ膜を用いたオプチカル化学センサ
ーにおいて、蛍光強度比に対して、既知濃度の溶液を測
定した場合の膜電位感受性色素の蛍光強度をプロットし
た図。FIG. 4 is a graph plotting the fluorescence intensity of a membrane potential sensitive dye when a solution of known concentration is measured against the fluorescence intensity ratio in the optical chemical sensor using the LB film of the present invention.
【図５】両親媒性物質としてジドデシルリン酸、膜電位
感受性色素としてオクタデシルローダミンＢ、特定物質
認識能を有する物質とてバリノマイシンを用いたＬＢ膜
からなるオプチカル化学センサーの蛍光を水中と空気中
で測定した場合の蛍光スペクトル。FIG. 5: Measurement of fluorescence of an optical chemical sensor consisting of LB membrane using didodecyl phosphate as an amphipathic substance, octadecyl rhodamine B as a membrane potential sensitive dye, and valinomycin as a substance capable of recognizing a specific substance in water and air. Fluorescence spectrum when
【図６】本発明のＬＢ膜を使用したオプチカル化学セン
サーを用いて１０-2ＭＫＣｌと１０-3ＭＫＣｌ水溶液を
測定したときのモニター色素及び膜電位感受性色素の蛍
光強度の時間変化を表わした図。FIG. 6 is a diagram showing changes in fluorescence intensity of a monitor dye and a membrane potential-sensitive dye with time when 10 −2 MKCl and 10 −3 MKCl aqueous solutions were measured using an optical chemical sensor using the LB film of the present invention. .
【図７】本発明のＬＢ膜を使用したオプチカル化学セン
サーにおいて、モニター色素の蛍光強度と膜電位感受性
色素の蛍光強度関係を、１０-2ＭＫＣｌと１０-3ＭＫＣ
ｌ水溶液について表わした図。FIG. 7 shows the relationship between the fluorescence intensity of a monitor dye and the fluorescence intensity of a membrane potential sensitive dye in an optical chemical sensor using the LB film of the present invention, which is 10 −2 MKCl and 10 −3 MKC.
1 is a diagram showing an aqueous solution.
【図８】本発明のＬＢ膜を使用したオプチカル化学セン
サーにおいて、蛍光強度の比に対して１０-2ＭＫＣｌと
１０-3ＭＫＣｌ水溶液の既知濃度試料を測定したときの
膜電位感受性色素の蛍光強度をプロットした図。FIG. 8: Fluorescence intensity of a membrane potential sensitive dye when a known concentration sample of 10 −2 MKCl and 10 −3 MKCl aqueous solution was measured with respect to the ratio of fluorescence intensity in the optical chemical sensor using the LB film of the present invention. The figure which plotted.
【図９】実施例４で調製したＬＢ膜を使用したオプチカ
ル化学センサーにおいて、空気中での膜電位感受性色素
の蛍光強度に対して１０-2ＭＫＣｌと１０-3ＭＫＣｌ水
溶液の既知濃度試料を測定したときの膜電位感受性色素
の蛍光強度をプロットした図。FIG. 9: Measurement of known concentration samples of 10 −2 MKCl and 10 −3 MKCl aqueous solutions with respect to the fluorescence intensity of a membrane potential sensitive dye in air in an optical chemical sensor using the LB membrane prepared in Example 4. The figure which plotted the fluorescence intensity of the membrane potential sensitive dye at the time of doing.
１１…両親媒性物質、１２…特定物質認識能を有する物
質、１３…膜電位感受性色素、１４…モニター色素、１
５…支持体、２１…ガラス基板、２２…ＬＢ膜、２３…
スペーサ、２４…ガラス11 ... amphipathic substance, 12 ... substance having specific substance recognition ability, 13 ... membrane potential sensitive dye, 14 ... monitor dye, 1
5 ... Support, 21 ... Glass substrate, 22 ... LB film, 23 ...
Spacer, 24 ... Glass
Claims (1)
上に移し取って調製されるラングミュアー・ブロジェッ
ト膜において、該膜中に膜電位感受性色素及び特定物質
認識能を有する物質を含有させ、更に膜電位感受性色素
と異なる光学特性を有し、且つ膜電位不感受性のモニタ
ー色素を含有させたことを特徴とするラングミュアー・
ブロジェット膜。1. A Langmuir-Blodgett membrane prepared by transferring a monomolecular film made of an amphipathic substance onto a support, wherein a membrane potential-sensitive dye and a substance capable of recognizing a specific substance are contained in the membrane. Langmuir characterized in that it contains a monitor dye having optical properties different from those of a dye sensitive to a membrane potential and being insensitive to a membrane potential.
Blodgett film.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6001793A JPH06273338A (en) | 1993-03-19 | 1993-03-19 | Lb film for optical chemical sensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6001793A JPH06273338A (en) | 1993-03-19 | 1993-03-19 | Lb film for optical chemical sensor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH06273338A true JPH06273338A (en) | 1994-09-30 |
Family
ID=13129876
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6001793A Withdrawn JPH06273338A (en) | 1993-03-19 | 1993-03-19 | Lb film for optical chemical sensor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH06273338A (en) |
-
1993
- 1993-03-19 JP JP6001793A patent/JPH06273338A/en not_active Withdrawn
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