JP4273228B2 - Hydrogen peroxide determination using self-supporting clay thin film - Google Patents
Hydrogen peroxide determination using self-supporting clay thin film Download PDFInfo
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は、自立粘土薄膜を用いた過酸化水素の定量方法に関するものであり、更に詳しくは、酵素触媒及びルミノールが均一に分散している自立粘土薄膜を用いることにより、簡便な方法で、水溶液中の過酸化水素を定量することを可能とする新しい過酸化水素の定量方法に関するものである。
本発明は、ルミノール発光法による過酸化水素の検出及び定量方法の技術分野において、従来法におけるような、触媒及びルミノールを溶液状態で保存すると、これらが不活性化し、過酸化水素の適正な検出及び定量ができないという問題点があることをふまえ、触媒及びルミノール分子を粘土薄膜中に固定化することにより、耐久性を高めた、自立粘土薄膜を用いた新しい過酸化水素の定量方法を提供するものとして有用である。
The present invention relates to a method for quantifying hydrogen peroxide using a self-supporting clay thin film, and more specifically, by using a self-supporting clay thin film in which an enzyme catalyst and luminol are uniformly dispersed, an aqueous solution can be obtained by a simple method. The present invention relates to a new method for quantifying hydrogen peroxide that makes it possible to quantitate hydrogen peroxide in it.
In the technical field of detection and quantification of hydrogen peroxide by the luminol luminescence method, the present invention deactivates the catalyst and luminol when stored in a solution state as in the conventional method, and the appropriate detection of hydrogen peroxide. In view of the problem that it cannot be quantified, a new method for the determination of hydrogen peroxide using a self-supporting clay thin film is provided by immobilizing the catalyst and luminol molecules in the clay thin film. Useful as a thing.
一般に、ペルオキシダーゼを始めとする酵素は、生体触媒として有用であり、きわめて高選択的、特異的に反応を司るが、耐熱性に劣るという欠点がある。しかしながら、有機物の熱安定性は、それが無機物で包接されたときには、一般には、かなり高くなることが知られている。酵素には、室温で長時間保存すると、活性が低下するものがある。したがって、酵素反応を利用して分析を行う場合、測定直前に、その都度、溶液の調製をしなければならない。この場合、測定手順が面倒になる他、無駄になる溶液あるいは試薬が発生し、経済的に好ましくないだけではなく、廃液あるいは廃薬品が発生し、それらの処分に手間がかかるという問題点がある。 In general, enzymes such as peroxidase are useful as biocatalysts, and they are highly selective and specific in their reactions, but have the disadvantage of poor heat resistance. However, it is known that the thermal stability of an organic substance is generally much higher when it is clad with an inorganic substance. Some enzymes have reduced activity when stored at room temperature for long periods of time. Therefore, when an analysis is performed using an enzyme reaction, a solution must be prepared immediately before the measurement. In this case, there are problems that the measurement procedure becomes troublesome, and wasteful solutions or reagents are generated, which is not only economically undesirable, but also waste liquids or chemicals are generated, and it takes time to dispose of them. .
一方、微量の過酸化水素を検出あるいは定量することは、特に、臨床検査分野において、重要な位置を占めるようになった。それは、臨床検査において測定すべき生体成分に特異的に作用する酸化酵素を作用させた場合に発生する過酸化水素を定量することにより、過酸化水素の前駆体である目的成分の量を求めることができるからである。しかも、このような酵素を利用した分析方法は、その高い特異性のため、盛んに用いられるようになった。 On the other hand, detection or quantification of trace amounts of hydrogen peroxide has become an important position, especially in the field of clinical examination. It determines the amount of the target component that is a precursor of hydrogen peroxide by quantifying the amount of hydrogen peroxide that is generated when an oxidase that specifically acts on the biological component to be measured in a clinical test. Because you can. Moreover, analytical methods using such enzymes have been actively used due to their high specificity.
また、分析対象物質を高感度に検出・定量するために、抗原、抗体、受容体等の特異的補足剤を用いた特異的な結合反応を利用することが知られており、代表的な測定方法として、サンドイッチ型免疫測定法が知られている。前記結合反応の検出用物質としては、ラジオアイソトープ、酵素、蛍光性物質、及び発光性物質が知られている（特許文献１）。酵素を検出用物質として使用する方法は、ラジオアイソトープを用いる免疫測定法のような危険性はなく、特異的な結合反応を利用できることから、広く利用されている。 In addition, it is known to use a specific binding reaction using specific supplements such as antigens, antibodies, and receptors in order to detect and quantify analytes with high sensitivity. As a method, a sandwich type immunoassay is known. As a substance for detecting the binding reaction, a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance are known (Patent Document 1). A method using an enzyme as a detection substance is widely used because there is no danger like an immunoassay using a radioisotope and a specific binding reaction can be used.
過酸化水素の検出及び定量方法において、化学発光反応の触媒としてペルオキシダーゼ、フェリシアン化カリウムなどを使用し、化学発光基質としてルミノールを用いる方法が検討されている（特許文献２、特許文献３）。これらは、分析対象に、触媒、及び化学発光基質を溶解させた試薬を混合させて分析するものであり、水溶液の調製と混合等の取扱いの手間が避けられないという問題点、及び化学種、特に、ルミノールの不活性化が著しいため、水溶液を長期間保存しておけないという問題点、があった。そして、例えば、化粧品及び医薬品分野においては、例えば、好適な球状の有機複合粘土鉱物（特許文献４、特許文献５）、粘土鉱物と酸と酵素とを混合した湿潤性水虫の治療薬の製造（特許文献６、特許文献７）等、が提案されており、粘土と有機化合物を複合化させることが行われている。
In a method for detecting and quantifying hydrogen peroxide, a method using peroxidase, potassium ferricyanide, or the like as a chemiluminescent reaction catalyst and luminol as a chemiluminescent substrate has been studied (
一方、粘土は、水やアルコールに分散し、その分散液をガラス板の上に広げ、静置乾燥することにより粒子の配向の揃った膜を形成することが知られており、この方法でＸ線回折用の定方位試料が調製されてきた（非特許文献１）。しかしながら、ガラス板上に膜を形成した場合、ガラス板から粘土膜を剥がすことが困難であり、また、粘土膜を剥がす際に膜に亀裂が生じるなど、自立膜として得ることが難しかった。また、ガラス板から粘土膜を剥がせたとしても、膜が脆く、強度が不足であり、しかも、ピンホールのない均一の厚さの膜を調製することは困難であった。 On the other hand, it is known that clay is dispersed in water or alcohol, and the dispersion is spread on a glass plate and left to dry to form a film with uniform particle orientation. A fixed orientation sample for line diffraction has been prepared (Non-Patent Document 1). However, when a film is formed on a glass plate, it is difficult to peel the clay film from the glass plate, and when the clay film is peeled off, it is difficult to obtain a self-supporting film because a crack occurs in the film. Further, even if the clay film is peeled off from the glass plate, the film is brittle, the strength is insufficient, and it is difficult to prepare a film having a uniform thickness without a pinhole.
最近、ラングミュアーブロジェット法（Langmuir-Blodgette Method ）を応用した粘土薄膜の作製が行われている（非特許文献２）。しかし、この方法でも、粘土薄膜は、ガラス等の材料でできた基板表面上に形成されるものであり、自立膜としての強度を得ることができなかった。更に、従来、例えば、機能性粘土薄膜等を調製する方法が種々報告されている。例えば、ハイドロタルサイト系層間化合物の水分散液を膜状化して乾燥することからなる粘土薄膜の製造方法（特許文献８）、層状粘土鉱物と燐酸又は燐酸基との反応を促進させる熱処理を施すことによる層状粘土鉱物が持つ結合構造を配向固定した層状粘土鉱物薄膜の製造方法（特許文献９）、スメクタイト系粘土鉱物と２価以上の金属の錯化合物を含有する皮膜処理用水性組成物（特許文献１０）、等をはじめ、多数の事例が存在する。 Recently, a clay thin film using the Langmuir-Blodgette Method has been produced (Non-Patent Document 2). However, even in this method, the clay thin film is formed on the surface of the substrate made of a material such as glass, and the strength as a self-supporting film cannot be obtained. Furthermore, conventionally, for example, various methods for preparing functional clay thin films have been reported. For example, a method for producing a clay thin film comprising forming an aqueous dispersion of a hydrotalcite-based intercalation compound into a film and drying it (Patent Document 8), and a heat treatment for promoting the reaction between the layered clay mineral and phosphoric acid or phosphate groups A method for producing a layered clay mineral thin film in which the bonded structure of the layered clay mineral is oriented and fixed (Patent Document 9), an aqueous composition for film treatment containing a complex compound of a smectite clay mineral and a divalent or higher metal (Patent Document 9) There are many cases including literature 10).
しかしながら、これまで、自立膜として利用可能な機械的強度を有し、粘土粒子の積層を高度に配向させた膜の開発例はなかった。以上のように、これまで、自立膜として利用可能な機械的強度を有し、粘土粒子の積層を高度に配向させた膜はなく、有機複合粘土鉱物を自立膜として用いる技術は開発されてこなかったのが実情である。更に、ルミノール発光法による過酸化水素の検出及び定量方法において、触媒及びルミノールを溶液状態で保存すると、これらが不活性化し、過酸化水素の適正な検出及び定量ができないという問題点があった。そのため、測定する度に、溶液を調製する手間がかかるという問題があり、当技術分野においては、その解決が強く求められていた。 However, until now, there has been no development example of a film having mechanical strength that can be used as a free-standing film and having a highly oriented laminate of clay particles. As described above, there has been no film that has mechanical strength that can be used as a free-standing film and that has a highly oriented laminate of clay particles, and no technology that uses organic composite clay minerals as a free-standing film has been developed. The fact is. Furthermore, in the method for detecting and quantifying hydrogen peroxide by the luminol luminescence method, if the catalyst and luminol are stored in a solution state, they are inactivated, and hydrogen peroxide cannot be properly detected and quantified. Therefore, there is a problem that it takes time and effort to prepare a solution for each measurement, and there has been a strong demand for a solution in this technical field.
このような状況の中で、本発明者らは、上記従来技術に鑑みて、ルミノール発光法による過酸化水素の検出及び定量方法における上記諸問題を抜本的に解決し得る新しい定量技術を開発することを目標として鋭意研究を積み重ねた結果、（１）触媒及びルミノールの不活性化を防ぐために、触媒及びルミノール分子を粘土薄膜中に固定化することにより、耐久性を高めることができること、（２）また、粘土薄膜の調製方法について、触媒及びルミノールとともに粘土分散水溶液を調製し、これを種々の固液分離方法、すなわち、遠心分離、ろ過、真空乾燥、凍結真空乾燥、加熱蒸発法で乾燥固化させることにより、粘土粒子が配向し、粘土粒子間に触媒及びルミノールが固定化された粘土薄膜が得られ、触媒及びルミノール分子の不活性化が抑制されるとともに、分析手順を簡便にできること、（３）一方、単に、粘土、触媒、及びルミノールのみからでは粘土薄膜の機械的強度は高いものではなく、ある種の有機分子をバインダーとして加えることにより、粘土薄膜の機械的強度が向上すること、（４）また、粘土薄膜は、触媒あるいはルミノールが均一に膜に分散して存在しているため、一定の面積の粘土薄膜を切り取ることによって、用いる触媒あるいはルミノールを一定量用いることが可能であること、等の新規知見を見出し、本発明を完成するに至った。
本発明は、過酸化水素の簡便な検出あるいは定量を可能とする粘土薄膜を用いた新規過酸化水素の定量方法を提供することを目的とするものである。
Under such circumstances, the present inventors have developed a new quantification technique capable of drastically solving the above problems in the method for detecting and quantifying hydrogen peroxide by the luminol luminescence method in view of the above-described conventional technology. As a result of intensive research with the goal of (1) in order to prevent inactivation of the catalyst and luminol, the durability can be enhanced by immobilizing the catalyst and luminol molecules in the clay thin film, (2 ) Also, regarding the method of preparing the clay thin film, an aqueous dispersion of clay is prepared together with a catalyst and luminol, and this is dried and solidified by various solid-liquid separation methods, that is, centrifugation, filtration, vacuum drying, freeze vacuum drying, and heat evaporation. To obtain a clay thin film in which clay particles are oriented and the catalyst and luminol are immobilized between the clay particles, and the catalyst and luminol molecules are deactivated. (3) On the other hand, the mechanical strength of the clay thin film is not high only from clay, catalyst, and luminol, and certain organic molecules are added as a binder. The mechanical strength of the clay thin film is improved. (4) Since the clay thin film is present in which the catalyst or luminol is uniformly dispersed in the film, by cutting out the clay thin film of a certain area, The inventors have found new findings such as the fact that a certain amount of catalyst or luminol can be used, and have completed the present invention.
An object of the present invention is to provide a novel method for quantifying hydrogen peroxide using a clay thin film that enables simple detection or quantification of hydrogen peroxide.
上記課題を解決するための本発明は、以下の技術手段から構成される。
（１）酵素触媒、及び化学発光に係わる物質を添加した自立粘土薄膜を用いて、水溶液中の過酸化水素を簡便に検出あるいは定量することを可能とする過酸化水素の定量方法であって、
１）これらの複数の異なる活性を有する物質を取り込んだ一枚の自立粘土薄膜を用いることにより、複数の化学反応を同時に、あるいは逐次的に行わせるようにして分析手順を簡便化すること、
２）上記複数の異なる活性を有する物質として、酵素としてのペルオキシダーゼと、化学発光に係わる物質としてのルミノールを用いること、を特徴とする過酸化水素の定量方法。
（２）該薄膜の粘土成分が、天然又は合成粘土の、雲母、バーミキュライト、モンモリロナイト、鉄モンモリロナイト、バイデライト、サポナイト、ヘクトライト、スチーブンサイト、又はノントロナイトであることを特徴とする、前記（１）に記載の過酸化水素の定量方法。
（３）自立粘土薄膜として、（１）ペルオキシダーゼ及び粘土を水あるいは水を主成分とする分散媒である液体に分散し、均一な粘土分散液を調製する、（２）この分散液を静置し、粘土粒子を沈積させるとともに、分散媒である液体を固液分離手段で分離して、膜状に形成し、複合粘土膜を作製する、（３）更に、任意に、１１０から２００℃の温度条件下で乾燥し、自立膜を得る、ことにより作製された複合粘土膜を用いることを特徴とする、前記（１）に記載の過酸化水素の定量方法。
（４）薄膜中のペルオキシダーゼの、全固体に対する重量割合が１パーセントから３０パーセントである、前記（１）又は（３）に記載の過酸化水素の定量方法。
（５）薄膜調製時にルミノールを添加し、ルミノールの全固体に対する重量割合が０．１パーセントから５パーセントである、前記（１）又は（３）に記載の過酸化水素の定量方法。
（６）薄膜調製時にグルコースオキシダーゼを添加し、グルコースオキシダーゼの、全固体に対する重量割合が１パーセントから１５パーセントである、前記（１）又は（３）に記載の過酸化水素の定量方法。
The present invention for solving the above problems comprises the following technical means.
(1) A method for quantifying hydrogen peroxide, which enables simple detection or quantification of hydrogen peroxide in an aqueous solution using an enzyme catalyst and a self-supporting clay thin film to which a substance related to chemiluminescence is added,
1) By using a single self-supporting clay thin film incorporating a plurality of substances having a plurality of different activities, a plurality of chemical reactions can be performed simultaneously or sequentially to simplify the analysis procedure .
2) as a substance having a plurality of different activity, peroxidase as an enzyme, that have use luminol as substances involved in chemiluminescence determination method of hydrogen peroxide, characterized in.
(2) The clay component of the thin film is mica, vermiculite, montmorillonite, iron montmorillonite, beidellite, saponite, hectorite, stevensite, or nontronite, which is natural or synthetic clay (1) ) For determining hydrogen peroxide.
(3) As a self-supporting clay thin film, (1) Disperse peroxidase and clay in water or a liquid that is a dispersion medium containing water as a main component to prepare a uniform clay dispersion. (2) Let this dispersion stand still Then, the clay particles are deposited, and the liquid as the dispersion medium is separated by solid-liquid separation means to form a film, thereby producing a composite clay film. (3) Further, optionally, 110 to 200 ° C. The method for quantifying hydrogen peroxide according to (1) above, wherein the composite clay film produced by drying under a temperature condition of (1) to obtain a self-supporting film is used.
(4) The method for quantifying hydrogen peroxide according to (1) or (3) above, wherein the weight ratio of peroxidase in the thin film to the total solid is 1 to 30 percent.
(5) The method for quantifying hydrogen peroxide according to (1) or (3) above, wherein luminol is added during the preparation of the thin film, and the weight ratio of luminol to the total solid is from 0.1 percent to 5 percent.
(6) The method for quantifying hydrogen peroxide according to (1) or (3) above, wherein glucose oxidase is added during the preparation of the thin film, and the weight ratio of glucose oxidase to the total solid is 1 to 15 percent.
次に、本発明について更に詳細に説明する。
本発明者らは、粘土分散水溶液を調製し、これにペルオキシダーゼ等の有機試薬を適量混合し、均一な分散液を得た後、この分散液を静置し、粘土粒子を沈積させるとともに、種々の固液分離方法、すなわち、遠心分離、ろ過、真空乾燥、凍結真空乾燥、又は加熱蒸発法で分離し、膜状に形成した後、支持体から剥離することにより、粘土粒子が配向し、粘土粒子間隙にペルオキシダーゼ等の有機試薬が均一に分布した粘土薄膜を自立膜として得られることを見出し、更に、この自立粘土薄膜を用いた水溶液中の過酸化水素の定量方法を見出した。すなわち、本発明は、希薄で均一な粘土及びペルオキシダーゼを含む分散水溶液を調製し、該分散液を水平に静置し、粘土粒子をゆっくりと沈積させるとともに、分散媒である液体を種々の固液分離方法、例えば、遠心分離、ろ過、真空乾燥、凍結真空乾燥、又は加熱蒸発法で分離し、膜状に形成した後、これを支持体から剥離すること、その際に、均一な厚さで自立膜として用いるに十分な強度を得るための製造条件を採用すること、これにより、粘土粒子の積層を高度に配向させた粘土薄膜の粒子間隙にペルオキシダーゼを均一に分布させた複合粘土膜を自立膜として得ること、及びこの複合粘土膜を用いて水溶液中の過酸化水素を定量すること、を特徴とするものである。
Next, the present invention will be described in more detail.
The present inventors prepared an aqueous clay dispersion, mixed an appropriate amount of an organic reagent such as peroxidase to obtain a uniform dispersion, and then left this dispersion to deposit clay particles. Solid-liquid separation method, that is, separation by centrifugation, filtration, vacuum drying, freeze vacuum drying, or heat evaporation method, and after forming into a film shape, the clay particles are oriented by peeling off from the support. The present inventors have found that a clay thin film in which an organic reagent such as peroxidase is uniformly distributed in the interstices can be obtained as a self-supporting film, and further has found a method for quantifying hydrogen peroxide in an aqueous solution using the self-supporting clay thin film. That is, the present invention prepares a dispersion aqueous solution containing dilute and uniform clay and peroxidase, and horizontally disperses the dispersion to slowly deposit clay particles, and to disperse the liquid as a dispersion medium into various solid-liquids. After separation by a separation method, for example, centrifugation, filtration, vacuum drying, freeze vacuum drying, or heat evaporation, and forming into a film, this is peeled off from the support, with a uniform thickness. Adopting manufacturing conditions to obtain sufficient strength to use as a self-supporting membrane, thereby making it possible to self-support a composite clay membrane in which peroxidase is evenly distributed in the interstices of the clay thin film with highly oriented clay particles. It is characterized in that it is obtained as a film, and hydrogen peroxide in an aqueous solution is quantified using this composite clay film.
本発明では、粘土として、天然あるいは合成物、好ましくは天然あるいは合成スメクタイトの何れか、あるいはそれらの混合物を用い、水あるいは水を主成分とする液体に加え、希薄で均一な分散液を調製する。粘土分散液の濃度は、好適には０．５から１０重量パーセント、より好ましくは１から３重量パーセントである。このとき、粘土分散液の濃度が薄すぎる場合、乾燥に時間がかかり過ぎるという問題点がある。また、粘土分散液の濃度が濃過ぎる場合、よく粘土が分散しないため、粘土粒子の配向が悪く、均一な膜ができないという問題がある。次に、秤量したペルオキシダーゼ粉末を粘土分散液に加え、均一なペルオキシダーゼ粘土分散液を調製する。ペルオキシダーゼの全固体に対する割合は、１パーセントから３０パーセントであり、好ましくは５パーセントから２０パーセントである。このとき、ペルオキシダーゼの割合が低過ぎる場合、ペルオキシダーゼの添加の効果が現れず、また、ペルオキシダーゼの割合が高過ぎる場合、調製した膜中でペルオキシダーゼと粘土の分布が不均一になり、やはり添加効果が薄れる。 In the present invention, as clay, either natural or synthetic, preferably natural or synthetic smectite, or a mixture thereof is used, and in addition to water or a liquid mainly composed of water, a dilute and uniform dispersion is prepared. . The concentration of the clay dispersion is suitably from 0.5 to 10 weight percent, more preferably from 1 to 3 weight percent. At this time, if the concentration of the clay dispersion is too thin, there is a problem that it takes too much time to dry. Further, when the concentration of the clay dispersion is too high, the clay does not disperse well, so that there is a problem that the orientation of the clay particles is poor and a uniform film cannot be formed. Next, the weighed peroxidase powder is added to the clay dispersion to prepare a uniform peroxidase clay dispersion. The ratio of peroxidase to total solids is from 1 percent to 30 percent, preferably from 5 percent to 20 percent. At this time, if the ratio of peroxidase is too low, the effect of addition of peroxidase does not appear, and if the ratio of peroxidase is too high, the distribution of peroxidase and clay becomes uneven in the prepared membrane, and the effect of addition is still Fade.
本発明において、薄膜調製時に、分散液に適量のルミノールを添加すると、ペルオキシダーゼ及びルミノールが均一に分散した粘土膜を得ることができる。このルミノールとペルオキシダーゼが共存する粘土薄膜は、過酸化水素水溶液と接すると、ルミノール反応により化学発光し、過酸化水素水溶液中の過酸化水素の定量に用いることが可能である。このとき、ペルオキシダーゼは、ルミノール反応の酵素触媒として働く。添加するルミノールの全固体に対する重量割合は、０．１パーセントから５パーセントであり、このとき、ルミノールの割合が低過ぎる場合、ルミノールの添加の効果が現れず、高い発光強度が得られない。また、ルミノールの割合が高過ぎる場合、調製した膜中でルミノールと粘土の分布が不均一になり、発光挙動が不安定になるという問題点がある。 In the present invention, a clay film in which peroxidase and luminol are uniformly dispersed can be obtained by adding an appropriate amount of luminol to the dispersion during the preparation of the thin film. When this clay thin film in which luminol and peroxidase coexist is in contact with an aqueous hydrogen peroxide solution, chemiluminescence is generated by the luminol reaction and can be used for the determination of hydrogen peroxide in the aqueous hydrogen peroxide solution. At this time, peroxidase acts as an enzyme catalyst for the luminol reaction. The weight ratio of the added luminol to the total solid is 0.1% to 5%. At this time, if the ratio of luminol is too low, the effect of addition of luminol does not appear, and high emission intensity cannot be obtained. Moreover, when the ratio of luminol is too high, there is a problem that the distribution of luminol and clay becomes non-uniform in the prepared film and the luminescence behavior becomes unstable.
また、本発明では、薄膜調製時に、分散液に適量のグルコースオキシダーゼを添加すると、粘土薄膜の乾燥時及び乾燥後の粘土膜のひび割れを防ぐ効果がある。グルコースオキシダーゼの、全固体に対する重量割合は、１パーセントから１５パーセントであり、このとき、グルコースオキシダーゼの割合が低過ぎる場合、グルコースオキシダーゼの添加の効果が現れず、また、グルコースオキシダーゼの割合が高過ぎる場合、調製した膜中でグルコースオキシダーゼと粘土の分布が不均一になり、やはり添加効果が薄れる。 In the present invention, when an appropriate amount of glucose oxidase is added to the dispersion during thin film preparation, there is an effect of preventing cracking of the clay film during and after drying of the clay thin film. The weight ratio of glucose oxidase to the total solid is 1% to 15%. At this time, if the ratio of glucose oxidase is too low, the effect of adding glucose oxidase does not appear, and the ratio of glucose oxidase is too high. In this case, the distribution of glucose oxidase and clay becomes uneven in the prepared membrane, and the effect of addition is also reduced.
次に、このペルオキシダーゼ粘土分散液を水平に静置し、粘土粒子をゆっくりと沈積させるとともに、例えば、分散液である液体をゆっくりと蒸発させ、膜状に形成する。この場合、好適には、例えば、種々の固液分離方法、好適には、例えば、遠心分離、ろ過、真空乾燥、凍結真空乾燥、加熱蒸発法の何れか、あるいはこれらの方法を組み合わせて乾燥粘土薄膜を得る。これらの方法のうち、例えば、加熱蒸発法を用いる場合、真空引きにより事前に脱気した分散液を平坦なトレイ、好ましくはプラスチック製あるいは金属製のトレイ等の支持体に注ぎ、水平を保った状態で、強制送風式オーブン中で３０から７０℃の温度条件下、好ましくは３０から５０℃の温度条件下で、３時間から半日間程度、好ましくは３時間から５時間乾燥して粘土薄膜を得る。分散液を事前に脱気しない場合、粘土薄膜に気泡に由来する孔ができ易くなるという問題点がある。 Next, this peroxidase clay dispersion is left to stand horizontally to slowly deposit clay particles, and for example, the liquid as the dispersion is slowly evaporated to form a film. In this case, preferably, for example, various solid-liquid separation methods, preferably, for example, centrifugation, filtration, vacuum drying, freeze vacuum drying, heat evaporation method, or a combination of these methods is used to dry clay. Get a thin film. Among these methods, for example, when using the heating evaporation method, the dispersion liquid previously deaerated by evacuation is poured onto a support such as a flat tray, preferably a plastic or metal tray, and kept horizontal. The clay thin film is dried in a forced air oven for 30 to 70 ° C., preferably 30 to 50 ° C. for about 3 hours to half a day, preferably 3 to 5 hours. obtain. When the dispersion is not degassed in advance, there is a problem that pores derived from bubbles are easily formed in the clay thin film.
また、乾燥条件は、液体分を蒸発によって取り除くのに十分であるように設定される。このとき、温度が低過ぎると乾燥に時間がかかるという問題点がある。また、温度が高過ぎると分散液の対流が起こり、粘土粒子の配向度が低下するという問題点がある。粘土薄膜がトレイ等の支持体から自然に剥離しない場合は、１１０から２００℃の温度条件下で乾燥し、剥離を容易にして自立膜を得る。このとき、温度が低過ぎる場合には、粘土薄膜の支持体からの剥離が起こりにくいという問題点がある。温度が高過ぎる場合には、粘土薄膜の乾燥による亀裂が発生しやすくなるという問題点がある。本発明において、粘土粒子の積層を高度に配向させるとは、粘土粒子の単位構造層（厚さ約１ナノメートル）を、層面の向きを一にして積み重ね、層面に垂直な方向に高い周期性を持たせることを意味する。このような粘土粒子の配向を得るためには、希薄で均一な粘土分散液を水平に静置し、粘土粒子をゆっくりと沈積させるとともに、例えば、分散媒である液体をゆっくりと蒸発させ、膜状に形成する必要がある。このプロセスにおける好適な製造条件を示すと、粘土分散液中の粘土の濃度は、好ましくは０．５から１０重量パーセント、より好ましくは１から３重量パーセントであり、また、加熱乾燥法による乾燥条件は、好ましくは強制送風式オーブン中で３０℃から７０℃の温度条件下、より好ましくは３０℃から５０℃の温度条件下で、３時間から半日間程度の乾燥、より好ましくは３時間から５時間程度の乾燥である。 The drying conditions are set to be sufficient to remove the liquid component by evaporation. At this time, if the temperature is too low, there is a problem that drying takes time. Further, when the temperature is too high, convection of the dispersion occurs, and there is a problem that the degree of orientation of the clay particles is lowered. If the clay thin film is not naturally separated from the support of the tray or the like, and dried at a temperature of from 1 10 200 ° C., to obtain a self-supporting film to facilitate peeling. At this time, when the temperature is too low, there is a problem that peeling of the clay thin film from the support hardly occurs. When the temperature is too high, there is a problem that cracks due to drying of the clay thin film are likely to occur. In the present invention, highly oriented lamination of clay particles means that unit structure layers (thickness of about 1 nanometer) of clay particles are stacked with the direction of the layer surface being the same, and high periodicity in a direction perpendicular to the layer surface. Means to have. In order to obtain such clay particle orientation, a thin and uniform clay dispersion is allowed to stand horizontally, and the clay particles are slowly deposited. For example, the liquid as a dispersion medium is slowly evaporated to form a film. It is necessary to form in a shape. In terms of suitable production conditions in this process, the clay concentration in the clay dispersion is preferably 0.5 to 10 weight percent, more preferably 1 to 3 weight percent, and the drying conditions by the heat drying method Is preferably dried in a forced air oven at a temperature of 30 ° C. to 70 ° C., more preferably at a temperature of 30 ° C. to 50 ° C. for about 3 hours to half a day, more preferably 3 hours to 5 hours. Dry for about an hour.
この粘土薄膜は、例えば、はさみ、カッター等で容易に円、正方形、長方形などの任意の大きさ、形状に切り取ることができる。本発明の粘土薄膜は、好適には、厚さは１ｍｍよりも薄く、面積は１ｃｍ2 よりも大きい。また、粘土薄膜の主要構成成分は、好適には、例えば、雲母、バーミキュライト、モンモリロナイト、鉄モンモリロナイト、バイデライト、サポナイト、ヘクトライト、スチーブンサイト、又はノントロナイトである。また、本発明の粘土薄膜は、粘土粒子の積層が高度に配向し、ピンホールの存在しないことを特徴とし、フレキシビリティーに優れ、２５０℃以上６００℃までの高温においても構造変化しないことを特徴とする。この粘土薄膜は、自立膜として用いることが可能であり、２５０℃を超える高温条件下で使用が可能であり、フレキシビリティーに優れており、ピンホールの存在しない緻密な材料であり、かつ気体・液体のバリアー性に優れているといった特徴を有する。 This clay thin film can be easily cut into an arbitrary size and shape such as a circle, a square, and a rectangle with, for example, scissors and a cutter. The clay thin film of the present invention preferably has a thickness of less than 1 mm and an area of more than 1 cm 2 . The main constituent of the clay thin film is preferably mica, vermiculite, montmorillonite, iron montmorillonite, beidellite, saponite, hectorite, stevensite or nontronite. In addition, the clay thin film of the present invention is characterized in that the lamination of clay particles is highly oriented and pinholes are not present, and it has excellent flexibility and does not change its structure even at high temperatures from 250 ° C. to 600 ° C. Features. This clay thin film can be used as a free-standing film, can be used under high-temperature conditions exceeding 250 ° C., has excellent flexibility, is a dense material free of pinholes, and is a gas -It has the feature that it has excellent liquid barrier properties.
更に、この複合粘土膜は、耐熱性を有するペルオキシダーゼを酵素触媒として用いることが可能であり、フレキシビリティーに優れており、ピンホールの存在しない緻密な材料であり、かつバリアー性に優れているといった特徴を有する。したがって、この複合粘土膜は、高温条件下でフレキシビリティーに優れた自立膜として広範に使用することができる。この複合粘土膜中のペルオキシダーゼの熱安定性は、上記複合化で著しく改善されており、耐熱型の酵素触媒としての利用も見込める。ペルオキシダーゼが粘土粒子間隙に均一に分散することの意義は、膜強度の均一性を実現するとともに、複合粘土膜をルミノール反応に基づく過酸化水素の定量に利用する際に、高い定量再現性を実現できることである。また、薄膜調製時に、グルコースオキシダーゼを添加すると、グルコースオキシダーゼが粘土粒子外表面と親和性があるため、グルコースオキシダーゼが粘土粒子外表面に付着した状態で粘土粒子が積層し、結果として、グルコースオキシダーゼが粘土粒子間隙を満たし、粘土粒子を結合する役割を果たす。そのため、グルコースオキシダーゼの配合により複合粘土膜の亀裂の発生が抑えられ、それにより、自立膜として利用可能な優れた特性を有する粘土薄膜が得られる。 Furthermore, this composite clay film can use heat-resistant peroxidase as an enzyme catalyst, has excellent flexibility, is a dense material free of pinholes, and has excellent barrier properties. It has the following characteristics. Therefore, this composite clay film can be widely used as a self-supporting film excellent in flexibility under high temperature conditions. The thermal stability of peroxidase in this composite clay film has been remarkably improved by the above-mentioned complexation, and can be expected to be used as a heat-resistant enzyme catalyst. The significance of the uniform dispersion of peroxidase in the clay particle gap is to realize the uniformity of membrane strength and the high quantitative reproducibility when using composite clay membrane for the determination of hydrogen peroxide based on luminol reaction. It can be done. In addition, when glucose oxidase is added at the time of thin film preparation, since glucose oxidase has affinity with the outer surface of the clay particles, the clay particles are laminated with glucose oxidase adhering to the outer surface of the clay particles. Fills the clay particle gap and plays a role in bonding the clay particles. Therefore, the formation of cracks in the composite clay film is suppressed by the addition of glucose oxidase, thereby obtaining a clay thin film having excellent characteristics that can be used as a self-supporting film.
本発明では、粘土として、天然あるいは合成スメクタイトの均一な分散液Ａを調製する。このとき、粘土分散液の濃度は０．５から１０重量パーセント、好ましくは１から３重量パーセントである。この分散液Ａに、粘土に対して５から５０重量パーセントの触媒、必要に応じて粘土に対して３から２０重量パーセントのバインダーを加え、再び激しく振とうし、均一な分散液Ｂを得る。この分散液Ｂに、ルミノール水溶液を添加し、均一な分散液Ｃを得る。この分散液Ｃを平坦なトレイに注ぎ、水平を保った状態で、強制送風式オーブン中で３０から７０℃の温度条件下、好ましくは４０から５０℃の温度条件下で、３時間から半日間程度、好ましくは３時間から５時間乾燥して均一な粘土薄膜を得る。この薄膜を裁断し、蛍光発光分析用セルに入れ、ｐＨ７．８に調整した０．０１Ｍ−ｔｒｉｓ−ＨＣｌ緩衝液を０．５ｃｍ3 加え、濃度未知の過酸化水素水溶液３ｃｍ3 を加え、直ちにフォトカウンター内に入れ、膜の発光強度を測定する。発光強度は、過酸化水素水溶液を加えた後、１０秒後から５分後、好ましくは２分から４分後に測定する。 In the present invention, a uniform dispersion A of natural or synthetic smectite is prepared as clay. At this time, the concentration of the clay dispersion is 0.5 to 10 weight percent, preferably 1 to 3 weight percent. To this dispersion A, 5 to 50 percent by weight of catalyst with respect to the clay and optionally 3 to 20 percent by weight of binder with respect to the clay are added and shaken vigorously again to obtain a uniform dispersion B. A luminol aqueous solution is added to the dispersion B to obtain a uniform dispersion C. The dispersion C is poured into a flat tray and kept in a horizontal condition in a forced air oven at a temperature of 30 to 70 ° C., preferably 40 to 50 ° C. for 3 hours to half a day. Dry to a degree, preferably 3 to 5 hours to obtain a uniform clay film. This thin film is cut, placed in a fluorescence emission analysis cell, 0.5 M 3 of 0.01 M tris-HCl buffer adjusted to pH 7.8 is added, 3 cm 3 of an aqueous hydrogen peroxide solution of unknown concentration is added, and the photo is immediately taken. Place in the counter and measure the emission intensity of the film. The luminescence intensity is measured after 10 seconds and 5 minutes, preferably 2 to 4 minutes after adding the hydrogen peroxide solution.
本発明により、（１）酵素触媒、及び化学発光に係わる物質を粘土薄膜に取り込むことにより、長期間に渡って、熱、光、あるいは空気中酸素等による活性の低下を防止するとともに、コンパクト化することによって取扱いの便を向上させることができる、（２）また、本発明は、複数の異なる活性を有する物質を一枚の粘土薄膜に取り込むことにより、複数の化学プロセスを同時に、あるいは逐次的に行わせ、分析手順を簡便化することができる、（３）長期間にわたって劣化なく保管できる自立粘土薄膜を用いた、手順が簡便で、省資源、低環境負荷な溶液中の化学種の定量法を提供することができる、という格別の効果が奏される。 According to the present invention, (1) by incorporating a substance related to an enzyme catalyst and chemiluminescence into a clay thin film, it is possible to prevent a decrease in activity due to heat, light, oxygen in the air, etc. over a long period of time and to make it compact. (2) In addition, the present invention can incorporate a plurality of chemical processes simultaneously or sequentially by incorporating a plurality of substances having different activities into a single clay thin film. (3) Quantitative determination of chemical species in a solution that is simple, resource-saving, and environmentally friendly, using a self-supporting clay thin film that can be stored without deterioration over a long period of time. The special effect is that the law can be provided.
次に、実施例に基づいて本発明を具体的に説明するが、本発明は、以下の実施例によって何ら限定されるものではない。 EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the following Examples.
粘土として、合成スメクタイト（スメクトンＳＡ、クニミネ工業株式会社製）を０．７ｇ、６０ｃｍ3 の蒸留水に加え、密封容器に入れ、激しく振とうし、均一な分散液Ａを得た。この分散液Ａに触媒としてペルオキシダーゼ（和光純薬工業株式会社製）を０．１５ｇ、バインダーとしてグルコースオキシダーゼ（和光純薬工業株式会社製）を０．１５ｇ加え、再び激しく振とうし、均一な分散液Ｂを得た。次に、ルミノール（東京化成株式会社製）０．０１ｇを１０ｃｍ3 の蒸留水に加え、均一な溶液Ｃを得た。このとき、０．１Ｎ水酸化ナトリウム水溶液を滴下し、アルカリ性とすることでルミノールを溶解した。次に、分散液Ｂに、水溶液Ｃを加え、密封容器に入れて、激しく振とうし、均一な分散液Ｄを得た。この分散液Ｄを平坦なトレイに注ぎ、分散液を水平に静置し、粘土粒子をゆっくりと沈積させるとともに、トレイの水平を保った状態で、強制送風式オーブン中で５０℃の温度条件下で５時間乾燥して厚さ約４０マイクロメートルの粘土薄膜を得た。この薄膜を１カ月常温、あるいは冷蔵して保存後、０．９ｃｍ×２．５ｃｍの長方形に裁断し、蛍光発光分析用セルに入れ、ｐＨ７．８に調整した０．０１Ｍ−ｔｒｉｓ−ＨＣｌ緩衝液を０．５ｃｍ3 加え、任意濃度の過酸化水素水溶液３ｃｍ3 を加え、直ちにフォトカウンター内に入れ、膜の発光強度を測定した。発光強度は、過酸化水素水溶液を加えた後、３分２０秒後に測定した。得られた発光強度を過酸化水素の濃度に対してプロットした結果を図１に示す。得られた発光強度と過酸化水素濃度との関係は二次曲線でよく表わすことが可能であり、その研量線に基づいて発光強度から過酸化水素濃度を計算することが可能であることが分かった。 As clay, synthetic smectite (Smecton SA, manufactured by Kunimine Industries Co., Ltd.) was added to 0.7 g of 60 cm 3 of distilled water, placed in a sealed container, and shaken vigorously to obtain uniform dispersion A. To this dispersion A, 0.15 g of peroxidase (manufactured by Wako Pure Chemical Industries, Ltd.) as a catalyst and 0.15 g of glucose oxidase (manufactured by Wako Pure Chemical Industries, Ltd.) as a binder were added, and the mixture was shaken vigorously and uniformly dispersed. Liquid B was obtained. Next, 0.01 g of luminol (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to 10 cm 3 of distilled water to obtain a uniform solution C. At this time, 0.1N sodium hydroxide aqueous solution was dropped to make the solution alkaline, thereby dissolving luminol. Next, the aqueous solution C was added to the dispersion B, put into a sealed container, and vigorously shaken to obtain a uniform dispersion D. The dispersion D is poured into a flat tray, the dispersion is left to stand horizontally, clay particles are slowly deposited, and the tray is kept in a horizontal condition in a forced air oven at 50 ° C. And dried for 5 hours to obtain a clay thin film having a thickness of about 40 micrometers. This thin film was stored at room temperature or refrigerated for 1 month, then cut into a 0.9 cm × 2.5 cm rectangle, placed in a fluorescence emission analysis cell, and adjusted to pH 7.8 0.01 M-tris-HCl buffer. the 0.5 cm 3 was added, the addition of hydrogen peroxide aqueous solution 3 cm 3 of an arbitrary concentration, immediately placed in a photo counter to determine the emission intensity of the film. The emission intensity was measured 3 minutes and 20 seconds after adding the aqueous hydrogen peroxide solution. The results of plotting the obtained emission intensity against the concentration of hydrogen peroxide are shown in FIG. The relationship between the obtained emission intensity and the hydrogen peroxide concentration can be well expressed by a quadratic curve, and it is possible to calculate the hydrogen peroxide concentration from the emission intensity based on the calibration curve. I understood.
粘土として、天然モンモリロナイト（クニピアＰ、クニミネ工業株式会社製）を０．７ｇ、６０ｃｍ3 の蒸留水に加え、密封容器に入れ、激しく振とうし、均一な分散液Ａを得た。この分散液Ａに触媒としてペルオキシダーゼ（和光純薬工業株式会社製）を０．１５ｇ、バインダーとしてグルコースオキシダーゼ（和光純薬工業株式会社製）を０．１５ｇ加え、再び激しく振とうし、均一な分散液Ｂを得た。次に、ルミノール（東京化成株式会社製）０．０１ｇを１０ｃｍ3 の蒸留水に加え、均一な溶液Ｃを得た。このとき、０．１Ｎ水酸化ナトリウム水溶液を滴下し、アルカリ性とすることでルミノールを溶解した。次に、分散液Ｂに、水溶液Ｃを加え、密封容器に入れて、激しく振とうし、均一な分散液Ｄを得た。この分散液Ｄを平坦なトレイに注ぎ、分散液を水平に静置し、粘土粒子をゆっくりと沈積させるとともに、トレイを水平を保った状態で、強制送風式オーブン中で５０℃の温度条件下で５時間乾燥して厚さ約４０マイクロメートルの粘土薄膜を得た。この薄膜を１カ月常温、あるいは冷蔵して保存後、０．９ｃｍ×２．５ｃｍの長方形に裁断し、蛍光発光分析用セルに入れ、ｐＨ７．８に調整した０．０１Ｍ−ｔｒｉｓ−ＨＣｌ緩衝液を０．５ｃｍ3 加え、任意濃度の過酸化水素水溶液３ｃｍ3 を加え、直ちにフォトカウンター内に入れ、膜の発光強度を測定した。発光強度は、過酸化水素水溶液を加えた後、３分２０秒後に測定した。得られた発光強度と過酸化水素の濃度には線形の関係が得られ、この検量線に基づいて発光強度から過酸化水素濃度を計算することが可能であることが分かった。 As a clay, natural montmorillonite (Kunipia P, manufactured by Kunimine Kogyo Co., Ltd.) was added to 0.7 g of 60 cm 3 of distilled water, placed in a sealed container, and shaken vigorously to obtain a uniform dispersion A. To this dispersion A, 0.15 g of peroxidase (manufactured by Wako Pure Chemical Industries, Ltd.) as a catalyst and 0.15 g of glucose oxidase (manufactured by Wako Pure Chemical Industries, Ltd.) as a binder were added, and the mixture was shaken vigorously and uniformly dispersed. Liquid B was obtained. Next, 0.01 g of luminol (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to 10 cm 3 of distilled water to obtain a uniform solution C. At this time, 0.1N sodium hydroxide aqueous solution was dropped to make the solution alkaline, thereby dissolving luminol. Next, the aqueous solution C was added to the dispersion B, put into a sealed container, and vigorously shaken to obtain a uniform dispersion D. The dispersion D is poured into a flat tray, the dispersion is left to stand horizontally, clay particles are slowly deposited, and the tray is kept in a horizontal condition in a forced air oven at 50 ° C. And dried for 5 hours to obtain a clay thin film having a thickness of about 40 micrometers. This thin film was stored at room temperature or refrigerated for 1 month, then cut into a 0.9 cm × 2.5 cm rectangle, placed in a fluorescence emission analysis cell, and adjusted to pH 7.8 0.01 M-tris-HCl buffer. the 0.5 cm 3 was added, the addition of hydrogen peroxide aqueous solution 3 cm 3 of an arbitrary concentration, immediately placed in a photo counter to determine the emission intensity of the film. The emission intensity was measured 3 minutes and 20 seconds after adding the aqueous hydrogen peroxide solution. It was found that a linear relationship was obtained between the obtained emission intensity and the hydrogen peroxide concentration, and it was possible to calculate the hydrogen peroxide concentration from the emission intensity based on this calibration curve.
比較例１
粘土、ルミノール、ペルオキシダーゼ、及びグルコースオキシダーゼを含む水溶液系を冷蔵庫に保存し、これを１ｃｍ3 蛍光発光分析用セルに入れ、ｐＨ７．８に調整した０．０１Ｍ−ｔｒｉｓ−ＨＣｌ緩衝液を０．５ｃｍ3 加え、任意濃度の過酸化水素水溶液３ｃｍ3 を加え、直ちにフォトカウンター内に入れ、膜の発光強度を測定した。発光強度は、過酸化水素水溶液を加えた後、３分２０秒後に測定したが発光強度は０であった。
Comparative Example 1
An aqueous solution system containing clay, luminol, peroxidase, and glucose oxidase is stored in a refrigerator, and this is placed in a 1 cm 3 fluorescence emission analysis cell, and 0.01 M tris-HCl buffer adjusted to pH 7.8 is added to 0.5 cm. 3 and 3 cm 3 of an aqueous hydrogen peroxide solution having an arbitrary concentration were added and immediately placed in a photocounter, and the emission intensity of the film was measured. The emission intensity was measured 3 minutes and 20 seconds after the addition of the aqueous hydrogen peroxide solution, but the emission intensity was 0.
参考例１
実施例１において、グルコースオキシダーゼを用いないで粘土薄膜を調製した。この場合、実施例１と比べて均一な膜はできなかった。
Reference example 1
In Example 1, a clay thin film was prepared without using glucose oxidase. In this case, a uniform film could not be formed as compared with Example 1 .
（１）粘土薄膜の製造
粘土として、０．７０グラムの天然モンモリロナイト（クニピアＰ、クニミネ工業株式会社製）を６０ｃｍ3 の蒸留水に加え、プラスチック製密封容器にテフロン（登録商標）回転子とともに入れ、激しく振とうし、均一な分散液を得た。この分散液に、０．３０グラムのペルオキシダーゼ粉末（和光純薬工業株式会社製）を加え、この分散液を、底面が平坦であり、底面の形状が正方形であり、その一辺の長さが約１０ｃｍのポリプロピレン製トレイに注ぎ、分散液を水平に静置し、粘土粒子をゆっくりと沈積させるとともに、トレイの水平を保った状態で、強制送風式オーブン中で５０℃の温度条件下で５時間乾燥して、厚さ約４０マイクロメートルの均一な褐色粘土薄膜を得た。
(1) Manufacture of clay thin film As clay, 0.70 grams of natural montmorillonite (Kunipia P, manufactured by Kunimine Kogyo Co., Ltd.) is added to 60 cm 3 of distilled water and placed in a plastic sealed container with a Teflon (registered trademark) rotor. The mixture was shaken vigorously to obtain a uniform dispersion. To this dispersion, 0.30 grams of peroxidase powder (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and this dispersion was made to have a flat bottom surface, a square bottom surface, and a side length of about Pour into a 10cm polypropylene tray, leave the dispersion horizontally, slowly deposit clay particles, and keep the tray horizontal, in a forced air oven at 50 ° C for 5 hours. Dried to obtain a uniform brown clay thin film having a thickness of about 40 micrometers.
（２）粘土薄膜の特性
この粘土薄膜のＸ線回折チャートを図２に示す。底面反射ピーク００１がｄ＝４．１６及び１．５３ｎｍの位置に観察された。前者はモンモリロナイト（クニピアＰ）の層間にペルオキシダーゼが入り込んだ構造、後者はモンモリロナイト（クニピアＰ）の構造に対応する。この結果から上記方法で作製した粘土薄膜では、モンモリロナイト（クニピアＰ）層間にペルオキシダーゼが存在し、粘土層に抱接されていることが分かる。このペルオキシダーゼ複合粘土膜（ペルオキシダーゼ−クニピアＰ薄膜）のＴＧ−ＤＴＡチャートを図３に示す。ここで、ペルオキシダーゼの全固体に対する重量割合は３０％である。ＴＧ曲線より、室温から２００℃までに吸着水の脱水による重量減少が観察される。また、２００から４５０℃にかけて大きな重量減少が観察される。モンモリロナイト（クニピアＰ、図４）とペルオキシダーゼ（図５）のＴＧ−ＤＴＡチャートを比較すると、モンモリロナイト（クニピアＰ）では２００から４５０℃に大きな重量減少は観察されないのに対し、ペルオキシダーゼではこの温度範囲に大きな重量減少が観察されることが分かる。このことから、この重量減少は、ペルオキシダーゼの分解に伴う変化と考えられる。また、ＤＴＡ曲線から、上記粘土薄膜では、ペルオキシダーゼのみで測定した場合よりも、分解が高温側に移っていることが分かる。
(2) Properties of clay thin film FIG. 2 shows an X-ray diffraction chart of this clay thin film. Bottom reflection peaks 001 were observed at d = 4.16 and 1.53 nm. The former corresponds to a structure in which peroxidase enters between layers of montmorillonite (Kunipia P), and the latter corresponds to the structure of montmorillonite (Kunipia P). From this result, it can be seen that in the clay thin film prepared by the above method, peroxidase is present between the montmorillonite (Kunipia P) layers and is embraced by the clay layer. A TG-DTA chart of this peroxidase composite clay film (peroxidase-Kunipia P thin film) is shown in FIG. Here, the weight ratio of peroxidase to the total solid is 30%. From the TG curve, weight reduction due to dehydration of adsorbed water is observed from room temperature to 200 ° C. A large weight loss is observed from 200 to 450 ° C. When comparing the TG-DTA charts of montmorillonite (Kunipia P, Fig. 4) and peroxidase (Fig. 5), no significant weight loss is observed from 200 to 450 ° C for montmorillonite (Kunipia P), whereas peroxidase is in this temperature range. It can be seen that a large weight loss is observed. From this, this weight reduction is considered to be a change accompanying degradation of peroxidase. Moreover, from the DTA curve, it can be seen that the above-mentioned clay thin film has been decomposed to a higher temperature side than when measured with peroxidase alone.
以上詳述したように、本発明は、自立粘土薄膜を用いた過酸化水素の定量方法に係るものであり、本発明で用いる、複合粘土薄膜は、その中のペルオキシダーゼの熱安定性が著しく改善されており、耐熱型の酵素触媒としての利用も見込める。酵素を膜化するとともに、任意の大きさに揃えて切り取り保存することにより、必要なときに必要な量の試薬を利用することが可能となり、手順が簡便になるほか、廃液及び廃薬品量を減らすことができる。したがって、本発明は、簡便で、環境にやさしい分析プロセスとして利用できる。本発明は、長期間に渡って劣化なく保管できる自立粘土薄膜を用いた、手順が簡便で、省資源、低環境負荷な溶液中の化学種の定量法を提供するものであり、特に、臨床検査及び臨床化学の分野等で利用し得るものとして有用である。 As described above in detail, the present invention relates to a method for quantifying hydrogen peroxide using a self-supporting clay thin film, and the composite clay thin film used in the present invention has a markedly improved thermal stability of peroxidase therein. It can be used as a heat-resistant enzyme catalyst. By forming the enzyme into a membrane and cutting and storing it in an arbitrary size, it becomes possible to use the necessary amount of reagent when necessary, simplifying the procedure, and reducing the amount of waste liquid and chemicals. Can be reduced. Therefore, the present invention can be used as a simple and environmentally friendly analysis process. The present invention provides a method for quantifying chemical species in a solution that uses a self-supporting clay thin film that can be stored without deterioration over a long period of time, has a simple procedure, is resource-saving, and has a low environmental impact. It is useful as one that can be used in the field of examination and clinical chemistry.
Claims (6)
１）これらの複数の異なる活性を有する物質を取り込んだ一枚の自立粘土薄膜を用いることにより、複数の化学反応を同時に、あるいは逐次的に行わせるようにして分析手順を簡便化すること、
２）上記複数の異なる活性を有する物質として、酵素としてのペルオキシダーゼと、化学発光に係わる物質としてのルミノールを用いること、を特徴とする過酸化水素の定量方法。 A hydrogen peroxide quantification method capable of easily detecting or quantifying hydrogen peroxide in an aqueous solution using an enzyme catalyst and a self-supporting clay thin film to which a substance related to chemiluminescence is added,
1) By using a single self-supporting clay thin film incorporating a plurality of substances having a plurality of different activities, a plurality of chemical reactions can be performed simultaneously or sequentially to simplify the analysis procedure .
2) as a substance having a plurality of different activity, peroxidase as an enzyme, that have use luminol as substances involved in chemiluminescence determination method of hydrogen peroxide, characterized in.
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