JP2009058232A - Sensor equipped with polymer having molecule mold - Google Patents
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Abstract
Description
本発明は、微量物質を分離、濃縮そして検出するためのセンサー、およびそれを備える微量物質を分離、濃縮そして検出するためシステムに関する。 The present invention relates to a sensor for separating, concentrating and detecting trace substances, and a system for separating, concentrating and detecting trace substances comprising the same.
環境中に存在する化学物質、農薬、または、食品、医薬品などに存在する汚染微生物、汚染微生物由来の物質は、微量であるにもかかわらず、ヒトの健康に大きく影響し得る。一般に、これらの物質は、微量であるためにその測定が困難であるか、その測定に時間と費用とを要するか、またはその測定に専門的な技術が必要である。 Chemical substances present in the environment, agricultural chemicals, contaminated microorganisms present in foods, pharmaceuticals, etc., and substances derived from contaminated microorganisms can greatly affect human health, even in trace amounts. In general, these substances are difficult to measure because they are in minute amounts, require time and cost for the measurement, or require specialized techniques for the measurement.
環境中に存在する微量物質を測定する方法として、特許文献１は、ガス漏れ警報器、排気ガスモニタなどとして用いられるガス検知素子を記載している。このガス検知素子は、導電性高分子、例えば、ポリチオフェン、ポリピロール、またはポリアセチレンなどからなる膜を用い、この導電性の高分子が、ガス、例えば、ＮＯに接触した場合に変化する導電率または吸収スペクトルを測定する。 As a method for measuring trace substances present in the environment, Patent Document 1 describes a gas detection element used as a gas leak alarm, an exhaust gas monitor, or the like. This gas detection element uses a film made of a conductive polymer such as polythiophene, polypyrrole, or polyacetylene, and the conductivity or absorption that changes when the conductive polymer comes into contact with a gas such as NO. Measure the spectrum.
特許文献２は、食品や香料の品質検査、悪臭公害の検知、焦げ臭検知による火災警報機などに用いられる、導電性高分子を用いるからなる感応膜を備えるにおいセンサーを記載する。特許文献２は、英国アロマスキャン社およびネオトロニクス社が、感応膜にポリピロールを主体とした導電性ポリマーを用い、においと接触するときに変化する抵抗値変化を測定するおいセンサーを商品化していることを記載している。 Patent Document 2 describes an odor sensor provided with a sensitive film made of a conductive polymer used for quality inspection of foods and fragrances, detection of bad odor pollution, fire alarms by detection of burnt odor, and the like. In Patent Document 2, UK Aromascan and Neotronics commercialize a odor sensor that uses a conductive polymer mainly composed of polypyrrole as a sensitive film and measures the change in resistance value that changes when it comes into contact with odor. It is described.
食品、医薬品の微生物汚染は、企業イメージに致命的な打撃を与える。また、病院、老人介護施設における施設内微生物感染が社会問題化している。さらに、多様な抗菌商品の流通、需要の高まりに見られるように、一般家庭における衛生管理にも関心高まり、微生物汚染を簡単に測定できる技術の必要性が近年急速に高まっている。 Microbial contamination of food and pharmaceuticals has a fatal impact on corporate image. In addition, microbial infection in hospitals and elderly care facilities has become a social problem. Furthermore, as seen in the increase in the distribution and demand for various antibacterial products, interest in hygiene management in general households has increased, and the need for a technology that can easily measure microbial contamination has increased rapidly in recent years.
食中毒を引き起こす細菌は、一般に、公定法として、対象細菌の生理学的特性を検出する培地法を用いて検出されている。最近では、塩基配列、抗体などを利用した細菌の高感度迅速検出法も開発されている。 Bacteria causing food poisoning are generally detected using a medium method for detecting physiological characteristics of target bacteria as an official method. Recently, a highly sensitive rapid detection method for bacteria using nucleotide sequences, antibodies, and the like has been developed.
上記汚染微生物または汚染微生物由来の物質は、試料中に存在するＡＴＰを測定することによって間接的に測定され得る。ＡＴＰは、動物、植物、微生物などのすべての生物に存在するエネルギー物質であり、生体活性などを評価するマーカーとして用いられ得る。 The contaminating microorganism or the substance derived from the contaminating microorganism can be indirectly measured by measuring ATP present in the sample. ATP is an energy substance present in all living organisms such as animals, plants, and microorganisms, and can be used as a marker for evaluating biological activity and the like.
ＡＴＰを簡易に検出する方法として、ルシフェラーゼと蛍光検出とを組合せる方法がある。この方法では、例えば、測定対象、例えば、まな板を綿棒でふきとってＡＴＰを収集し、これにルシフェラーゼを作用させることにより発する蛍光を計測する。しかし、この方法は、用いるルシフェラーゼ、蛍光試薬が高価であって、その取り扱いに熟練を要し、さらに、蛍光検出に用いる装置が必要である。また、この方法は、ＡＴＰの検出に蛍光検出を用いているため、蛍光強度の経時的変化に起因して迅速な測定が要求される。特に問題となるのは、ルシフェラーゼの保存である。ルシフェラーゼの保存には、冷凍または冷蔵が必要で、保存可能な期間も短いという本質的な欠陥を備えている。 As a method for easily detecting ATP, there is a method of combining luciferase and fluorescence detection. In this method, for example, ATP is collected by wiping a measurement object, for example, a cutting board with a cotton swab, and fluorescence emitted by allowing luciferase to act on this is measured. However, in this method, the luciferase and fluorescent reagent to be used are expensive, requiring skill in handling them, and further, an apparatus used for fluorescence detection is required. In addition, since this method uses fluorescence detection to detect ATP, rapid measurement is required due to the change in fluorescence intensity over time. Of particular concern is the preservation of luciferase. Storage of luciferase requires refrigeration or refrigeration and has the inherent disadvantage of a short shelf life.
非特許文献１、非特許文献２、および非特許文献３は、微量物質を特異的に取り込む技術として、ポリピロールが過酸化されるとき、ゲスト分子が高分子マトリックス内に取り込まれ、分子鋳型を形成する「モレキュラーインプリンティング法」を記載する。非特許文献３は、この「モレキュラーインプリンティング法」を用い、ゲスト分子としてＬ−グルタミン酸を用い、ポリピロールを過酸化する過程でＬ−グルタミン酸を高分子マトリックス内に取り込み、得られたポリピロール膜が、Ｌ−グルタミン酸を取り込むが、Ｄ−グルタミン酸は取り込まれないことを示した。非特許文献４は、Ｌ−乳酸をプリントした過酸化ポリピロールコロイドを記載している。非特許文献５は、分子鋳型を利用した皮膚コレステロール計測法を記載している。非特許文献６は、自己組織化単分子膜修飾電極を用いるドーパミンの選択的定量法を記載している。また、特許文献３は、ステアリルメルカプタンを用いて作製した自己組織化単分子膜を利用するコレステロールセンサーを記載している。
本発明は、上記従来技術の課題を解決し、環境中に存在する化学物質、農薬、または、食品、医薬品などに存在する汚染微生物、汚染微生物由来の物質と、分子鋳型を有するポリマーとの相互作用によって引き起こされるこのポリマーの電気化学的性質を利用し、これら標的物質を簡便かつ効率良く、分離、濃縮、そして検知し得る手段を提供することを目的とする。 The present invention solves the above-mentioned problems of the prior art, and provides a mutual interaction between chemical substances present in the environment, agricultural chemicals, contaminating microorganisms present in foods, pharmaceuticals, etc., substances derived from contaminating microorganisms, and polymers having molecular templates. It is an object of the present invention to provide a means by which the target substance can be separated, concentrated and detected by utilizing the electrochemical properties of the polymer caused by the action.
上記汚染微生物を検出するための従来法は、培地法を用いるために時間を要する。また、上記酵素、抗体などを含む生体物質を利用する方法は、これら生体物質は、一般に、安定性に乏しいため、取り扱いに熟練を要し、高額な設備や専門的な作業をともない、コストパーフォーマンスにも乏しい。 The conventional method for detecting the contaminating microorganism requires time to use the culture medium method. In addition, the methods using biological materials including the above-mentioned enzymes, antibodies, etc. are generally low in stability, so that they require skill in handling, costly equipment and specialized work, It is poor in performance.
本発明は、標的分子に対して高い特異性を有する分子鋳型を備えたポリマーの電気化学的性質を利用し、上記従来技術の課題を解決する。本発明は、上記のような酵素などの生体物質を用いることなく、上記分子鋳型を備えたポリマーを利用することによって、コストパーフォーマンスに優れ、かつ、食品産業、病院、学校、福祉施設、一般家庭においても簡便に使用され得る微量標的物質測定様センサーおよびそれを備えたシステムを提供することを目的とする。本発明はまた、上記分子鋳型を備えたポリマーの電気化学的性質を利用した吸着剤を提供することを目的とする。 The present invention solves the above-mentioned problems of the prior art by utilizing the electrochemical properties of a polymer provided with a molecular template having high specificity for a target molecule. The present invention is excellent in cost performance by using a polymer provided with the molecular template without using a biological substance such as the enzyme as described above, and is suitable for food industry, hospital, school, welfare facility, It is an object of the present invention to provide a trace target substance measurement-like sensor that can be easily used at home and a system including the same. Another object of the present invention is to provide an adsorbent utilizing the electrochemical properties of the polymer provided with the molecular template.
本発明は、分子鋳型を有するポリマーとこの分子鋳型と相補的な微量標的物質との相互作用を鋭意検討し、分子鋳型技術の特徴である微量標的物質との特異性に加え、分子鋳型の電気的性質を利用することによって、この分子鋳型が微量標的物質を特異的に結合することに加え、分子鋳型の周りにこの微量標的物質を濃縮することを見出し、本発明を完成するに至った。 The present invention has intensively studied the interaction between a polymer having a molecular template and a trace target substance complementary to the molecular template. In addition to the specificity of the molecular template technology, By utilizing the physical properties, the present inventors have found that this molecular template specifically binds a trace amount of a target substance, and also concentrates the trace target substance around the molecular template, thereby completing the present invention.
本発明は、分子鋳型を有する過酸化ポリマー膜の電気化学的性質を利用する点を特徴の１つとし、センサーまたは吸着剤の一部として構成した点に特徴の１つを備えている。 The present invention is characterized in that it uses the electrochemical properties of a peroxide polymer film having a molecular template, and is characterized in that it is configured as a part of a sensor or an adsorbent.
より特定すれば、本発明は以下の項目に関する。 More specifically, the present invention relates to the following items.
（項目１）アニオン分子を検出するセンサーであって、電極、上記電極上に配置されたポリマー層であって、上記アニオン分子の立体構造に相補的な三次元構造を有する分子鋳型を備えたポリマー層、上記ポリマー層の導電性の変化を導出する導出部を備え、上記三次元構造が、上記ポリマー層を形成する際に上記ポリマーのモノマーを上記標的分子の存在下で重合する工程、および得られた重合体を過酸化する工程によって形成される、センサー。 (Item 1) A sensor for detecting an anionic molecule, comprising: an electrode, a polymer layer disposed on the electrode, and a molecular template having a three-dimensional structure complementary to the three-dimensional structure of the anionic molecule And a step of polymerizing the polymer monomer in the presence of the target molecule when the three-dimensional structure forms the polymer layer. A sensor formed by a process of peroxidizing a formed polymer.
（項目２）上記電極が第１の電位にあるとき、上記アニオン分子を上記分子鋳型に捕捉し、上記電極が上記第１の電位とは異なる第２の電位にあるとき、捕捉されたアニオン分子から酸化あるいは還元電流を生じる、項目１に記載のセンサー。 (Item 2) When the electrode is at a first potential, the anion molecule is captured by the molecular template, and when the electrode is at a second potential different from the first potential, the captured anion molecule is captured. Item 2. The sensor according to Item 1, wherein an oxidation or reduction current is generated from the catalyst.
（項目３）上記電極に接続される電源をさらに備え、上記電源が上記電極に上記第１の電位を印加することによって上記アニオン分子を上記分子鋳型に濃縮する、項目１に記載のセンサー。 (Item 3) The sensor according to item 1, further comprising a power source connected to the electrode, wherein the power source concentrates the anion molecule in the molecular template by applying the first potential to the electrode.
（項目４）上記導出部に接続され、上記導電性の変化を計測する検知部をさらに備える、項目１に記載のセンサー。 (Item 4) The sensor according to item 1, further comprising a detection unit connected to the deriving unit and measuring the change in conductivity.
（項目５）上記電極、上記ポリマー層、および上記導出部が感応部を構成し、上記感応部と、上記電源および上記検知部とが脱着可能に連結されている、項目４に記載のセンサー。 (Item 5) The sensor according to item 4, wherein the electrode, the polymer layer, and the lead-out portion constitute a sensitive portion, and the sensitive portion, the power source, and the detection portion are detachably connected.
（項目６）上記モノマーが、ピロール、アニリン、チオフェンおよびそれらの誘導体からなる群から選択される、項目１に記載のセンサー。 (Item 6) The sensor according to item 1, wherein the monomer is selected from the group consisting of pyrrole, aniline, thiophene and derivatives thereof.
（項目７）上記アニオン分子が、ＡＴＰ、Ｄ−アミノ酸、Ｌ−アミノ酸、無機アニオン、有機アニオン、およびこれら化合物を含む高分子化合物からなる群から選択される分子である、項目１に記載のセンサー。 (Item 7) The sensor according to item 1, wherein the anion molecule is a molecule selected from the group consisting of ATP, D-amino acid, L-amino acid, inorganic anion, organic anion, and a polymer compound containing these compounds. .
（項目８）上記アニオン分子が、ペプチド、ＤＮＡおよび糖からな群から選択される、項目１に記載のセンサー。 (Item 8) The sensor according to item 1, wherein the anion molecule is selected from the group consisting of peptides, DNA and sugars.
（項目９）上記ペプチドが、ベロ毒素、エンテロトキシンを含む細菌毒素である、項目８に記載のセンサー。 (Item 9) The sensor according to item 8, wherein the peptide is a bacterial toxin including verotoxin and enterotoxin.
上記電極としては、導電体である任意の材料が用いられ得、これには、金、炭素、白金、ＩＴＯなどが含まれるが、これらに制限されるわけではない。上記ポリマーからの電流を導出部に伝導し得る限り任意の導電体を用い得る。 The electrode may be any material that is a conductor, including but not limited to gold, carbon, platinum, ITO, and the like. Any conductor can be used as long as the current from the polymer can be conducted to the lead-out part.
本発明は、また、アニオン分子を吸着する吸着剤に関し、この吸着剤は、支持体、この支持体上に配置されたポリマー層であって、該アニオン分子の立体構造に相補的な三次元構造を有する分子鋳型を備えたポリマー層、このポリマー層の導電性の変化を導出する導出部を備え、上記三次元構造が、上記ポリマーの層を形成する際に上記ポリマーのモノマーを該標的分子の存在下で重合する工程、および得られた重合体を過酸化する工程によって形成される。 The present invention also relates to an adsorbent that adsorbs anion molecules, the adsorbent being a support, a polymer layer disposed on the support, and a three-dimensional structure complementary to the three-dimensional structure of the anion molecule A polymer layer having a molecular template having a derivation portion for deriving a change in conductivity of the polymer layer, and the three-dimensional structure converts the monomer of the polymer into the target molecule when forming the polymer layer. It is formed by a step of polymerizing in the presence and a step of peroxidizing the obtained polymer.
上記支持体は、任意の形状を有し得、微量物質の吸着剤として、例えば、円筒形のカラムに充填して使用する場合は、通常、球形または卵形などの形状であり、この上に分子鋳型を有するポリマー層が配置された吸着剤は、このカラムに効率的に充填される。 The support may have any shape, and when used as a trace substance adsorbent, for example, in a cylindrical column, it is usually in the shape of a sphere or egg, on which The adsorbent in which the polymer layer having the molecular template is arranged is efficiently packed in this column.
上記支持体が第１の電位にあるとき、上記アニオン分子を上記分子鋳型に捕捉し得、上記支持体が上記第１の電位より大きい第２の電位にあるとき、捕捉されたアニオン分子を解放し得る。 When the support is at a first potential, the anion molecules can be captured on the molecular template, and when the support is at a second potential greater than the first potential, the captured anion molecules are released. Can do.
本発明によって、物質の分離のみに限られていた、従来の分子鋳型を有するポリマーを、標的物質と、分子鋳型を有するポリマーとの相互作用によって生じる電気化学的事象を利用し、物質の分離のみならず、物質の濃縮、および感知することを可能にする吸着剤、センサー、およびこれらを含むシステムが提供される。 According to the present invention, a polymer having a conventional molecular template, which is limited only to the separation of a substance, is obtained by utilizing an electrochemical event generated by the interaction between a target substance and a polymer having a molecular template, and only the separation of the substance. Rather, an adsorbent, a sensor, and a system including them are provided that allow for the concentration and sensing of substances.
例として、本発明のセンサーは、従来のルシフェラーゼを用いたＡＴＰ測定法に対し、以下の利点を提供する。
ｉ）酵素を使用しないので、酵素反応のための試薬、およびその混合操作を必要とせず、酵素の失活を防ぐための冷蔵保存などの操作を必要とせず、蛍光強度の経時的変化、洗剤や消毒薬のなどの混入による酵素の失活を考慮する必要がない。
ｉｉ）酵素を含む試薬を保存するために設備が不要であるため、小規模事業所、一般家庭においても使用可能。
As an example, the sensor of the present invention provides the following advantages over the conventional ATP measurement method using luciferase.
i) Since no enzyme is used, a reagent for enzyme reaction and a mixing operation thereof are not required, and an operation such as refrigerated storage for preventing the inactivation of the enzyme is not required. There is no need to consider enzyme deactivation due to contamination with antiseptics.
ii) Since no equipment is required to store the reagent containing the enzyme, it can be used in small-scale offices and general households.
（実施形態１）ＡＴＰの分子鋳型を有するポリピロール膜を備えたセンサーの調製
電極上に、ＡＴＰの分子鋳型を有するポリピロール膜を調製する方法の概略を図１に示す。図１の（ａ）は、ＡＴＰ陰イオン存在下のピロールの酸化的重合反応の模式図であり、ポリピロール膜は、電極上を図１の（ａ）に示される右向きの矢印の方向に成長する。図１の（ｂ）は、重合後のポリピロール膜の模式図である（図中ＰＰｙは、ポリピロールの略である）。ポリピロールは、重合の過程で電極に電子を放出するためにそれ自体は陽電荷を有し、この陽電荷を補償するためにＡＴＰ陰イオンがポリマー内に取り込まれる（ドープされる）。
(Embodiment 1) Preparation of a sensor provided with a polypyrrole film having an ATP molecular template The outline of a method for preparing a polypyrrole film having an ATP molecular template on an electrode is shown in FIG. FIG. 1A is a schematic diagram of the oxidative polymerization reaction of pyrrole in the presence of an ATP anion, and the polypyrrole film grows on the electrode in the direction of the right-pointing arrow shown in FIG. . FIG. 1B is a schematic view of a polypyrrole film after polymerization (in the figure, PPy is an abbreviation for polypyrrole). Polypyrrole itself has a positive charge to release electrons to the electrode during the polymerization process, and an ATP anion is incorporated (doped) into the polymer to compensate for this positive charge.
そしてその後、ポリマーをさらに酸化（過酸化）すると、ポリマーが電気的に中性となるため、ＡＴＰは膜外に排出されてＡＴＰの分子鋳型が形成される（図１の（ｃ）：図中ＯＰＰｙは、過酸化ポリピロールの略である）。この過酸化は、ポリマーの硬化をも引き起こし、このＡＴＰの分子鋳型を安定化する。 Then, when the polymer is further oxidized (peroxidized), the polymer becomes electrically neutral, so that ATP is discharged out of the film and an ATP molecular template is formed ((c) in FIG. 1). OPPy is an abbreviation for peroxide polypyrrole). This peroxidation also causes the polymer to cure, stabilizing the ATP molecular template.
形成される鋳型の三次元構造は、過酸化反応の溶液組成、過酸化反応を引き起こすための電圧に依存して変動し得る。一般に、過酸化反応が徐々に進行するような条件下では、標的陰シオンの立体構造により緊密な三次元構造を有する分子鋳型が形成される。 The three-dimensional structure of the template formed may vary depending on the solution composition of the peroxidation reaction and the voltage to cause the peroxidation reaction. In general, under conditions where the peroxidation reaction proceeds gradually, a molecular template having a tight three-dimensional structure is formed by the three-dimensional structure of the target anion.
（実施形態２）センサーへの標的陰イオンの吸着とその検出
図２は、センサーへの標的陰イオンの吸着とその検出の概略を示す模式図である。
(Embodiment 2) Adsorption of target anion to sensor and its detection FIG. 2 is a schematic diagram showing the outline of adsorption of target anion to the sensor and its detection.
ＡＴＰは４価の陰イオンとして存在し、陽分極した電極とＡＴＰの間の静電的親和力を相互作用の一部とし、ＡＴＰと特異的な三次元構造を有するように形成された分子鋳型と特異的に結合する。ＡＴＰは、電極上の分子鋳型と結合することによって、試料溶液からセンサー表面上に効率良く濃縮される（図２の（ａ））。電極の周りに分子鋳型を配置することは、試料溶液からの標的物質の分子鋳型への結合、それによるセンサー表面への標的物質の濃縮に加え、分子鋳型を有するポリマーおよび電極に、高感度測定に好適な導電特性を与える。ＡＴＰの電気化学的検出は、ＡＴＰの蛍光検出法に比べて必ずしも高感度ではないが、このような試料溶液からセンサー表面上への濃縮によってＡＴＰの高感度検出が達成され得る。 ATP exists as a tetravalent anion, and the electrostatic affinity between the positive electrode and ATP is part of the interaction, and ATP is a molecular template formed to have a specific three-dimensional structure. Bind specifically. ATP is efficiently concentrated from the sample solution onto the sensor surface by binding to the molecular template on the electrode ((a) of FIG. 2). Placing the molecular template around the electrode is a sensitive measurement of the polymer and electrode with the molecular template in addition to the binding of the target substance from the sample solution to the molecular template, thereby concentrating the target substance on the sensor surface. To provide suitable conductive properties. The electrochemical detection of ATP is not necessarily highly sensitive compared to the fluorescence detection method of ATP, but highly sensitive detection of ATP can be achieved by such concentration from the sample solution onto the sensor surface.
ＡＴＰは、以下に示されるように、電極に電圧を印加することによってさらに試料溶液から濃縮され得る。 ATP can be further concentrated from the sample solution by applying a voltage to the electrode, as shown below.
分子鋳型に捕捉されたＡＴＰは、電極に付加的な電圧を印加することによって酸化され、そのときに生じる酸化電流の値（図２の（ｂ）において左向きの矢印で示される）から、ポリピロール膜に捕捉されたＡＴＰの量を測定でき、その結果、試料中のＡＴＰの量を算出することができる。 The ATP trapped in the molecular template is oxidized by applying an additional voltage to the electrode, and from the value of the oxidation current generated at that time (indicated by a left-pointing arrow in FIG. 2B), the polypyrrole film As a result, the amount of ATP in the sample can be calculated.
上記分子鋳型は、電極表面上に、複数の分子鋳型がマトリックス状に配置されたアレイとして形成され得る。アレイ内の分子鋳型の配置もまた、ＡＴＰの濃縮効率を変更するために改変され得る。 The molecular template may be formed as an array in which a plurality of molecular templates are arranged in a matrix on the electrode surface. The arrangement of molecular templates within the array can also be modified to alter the concentration efficiency of ATP.
（実施形態３）分子鋳型を有するポリマー膜を有するセンサーを備えたシステム
上記実施の形態１に記載のＡＴＰの分子鋳型を有するポリピロール膜を備えたセンサーは、ＡＴＰを含むアニオン系分子が、過酸化ポリピロール膜の分子鋳型に捕集されることによる過酸化ポリピロール膜の導電性、すなわち酸化還元特性の変化を電気化学的に計測することでアニオン系分子の高感度の測定を可能にするシステムの構築を可能にする。図１０は、このようなシステムの一例を示す。上記実施の形態１に従って、電極の周りにポリピロールなどの導電性高分子の分子鋳型を形成することにより、アニオン系分子の選択、濃縮に加えて、電気化学的測定時に導電性高分子に付与される特有の導電特性によって、アニオン系分子のより高感度の測定を可能にする。図１０に示されるシステムは、電極、電極上に形成された分子鋳型を有するポリマー層、およびケーブル４に接続され、このケーブル４を介して検知部６に導出される導出部（図示せず）を備える感応部５と、電圧／電流計を備える検知部６と、被検試料Ｓを拭き取ってサンプリングを行う綿棒Ｓ’を浸漬する試料溶解液を含む容器９とを備えている。感応部は、採取した試料を溶解した容器９に浸漬して、後の実施例で詳述される電気化学的測定を行う。
(Embodiment 3) System including a sensor having a polymer film having a molecular template The sensor including a polypyrrole film having an ATP molecular template according to Embodiment 1 described above has an anionic molecule containing ATP peroxidized. Construction of a system that enables highly sensitive measurement of anionic molecules by electrochemically measuring the change of conductivity, that is, redox characteristics of polypyrrole peroxide peroxide by being collected by molecular template of polypyrrole film Enable. FIG. 10 shows an example of such a system. According to the first embodiment, by forming a molecular template of a conductive polymer such as polypyrrole around the electrode, in addition to selection and concentration of anionic molecules, it is given to the conductive polymer during electrochemical measurement. The unique conductive properties allow for more sensitive measurement of anionic molecules. The system shown in FIG. 10 is connected to an electrode, a polymer layer having a molecular template formed on the electrode, and a cable 4, and a lead-out part (not shown) led to the detection part 6 via the cable 4. A sensing unit 6 having a voltage / ammeter, and a container 9 containing a sample solution for immersing a swab S ′ for wiping the sample S to be sampled. The sensitive part immerses the collected sample in a container 9 in which the sample is dissolved, and performs electrochemical measurement detailed in a later example.
（実施形態４）分子鋳型を有するポリマー膜を有するセンサーの選択吸着能評価
ポリマー内に取り込まれるドーパントとしてＨ３ＰＯ４またはＡＴＰを用い、実施形態１に従って、Ａｕ平面電極上にＯＰＰｙ／Ｈ３ＰＯ４膜またはＯＰＰｙ／ＡＴＰ膜を形成し、Ｈ３ＰＯ４の分子鋳型またはＡＴＰの分子鋳型を有するポリマーを備えた電極をそれぞれ得た。
(Embodiment 4) Evaluation of selective adsorption ability of a sensor having a polymer film having a molecular template Using H 3 PO 4 or ATP as a dopant incorporated into a polymer, and according to Embodiment 1, OPPy / H 3 PO on an Au planar electrode Four films or OPPy / ATP films were formed to obtain electrodes each having a polymer having a molecular template of H 3 PO 4 or a molecular template of ATP.
得られたＯＰＰｙ／ＡＴＰ膜−Ａｕ平面電極、およびＯＰＰｙ／Ｈ３ＰＯ４膜−Ａｕ平面電極のそれぞれについて、試料液としてＡＴＰ溶液（１０−６ＭのＡＴＰ溶液）を用い、ＡＴＰが吸着されるか否かを調べた。各々の分子鋳型を有するポリマーを備えた各電極へのＡＴＰの吸着は、試料液と上記各電極とを接触させた後、塩溶液（１０ｍＭのＫＣｌ溶液）で各電極に吸着したＡＴＰを遊離させ、溶液中のＡＴＰ濃度を酵素基質法によって測定した。その結果、ＯＰＰｙ／ＡＴＰ膜−Ａｕ平面電極では、ＡＴＰの特異的捕捉が起こり、ＯＰＰｙ／Ｈ３ＰＯ４膜−Ａｕ平面電極では、ＡＴＰの特異的捕捉が起こらないことを示す結果を得た。 For each of the obtained OPPy / ATP film-Au planar electrode and OPPy / H 3 PO 4 film-Au planar electrode, ATP solution (10 −6 M ATP solution) is used as a sample solution, and ATP is adsorbed. I investigated whether or not. Adsorption of ATP to each electrode provided with a polymer having each molecular template is performed by bringing the sample solution into contact with each electrode and then releasing ATP adsorbed on each electrode with a salt solution (10 mM KCl solution). The ATP concentration in the solution was measured by the enzyme substrate method. As a result, it was found that specific capture of ATP occurred in the OPPy / ATP film-Au planar electrode, and specific capture of ATP did not occur in the OPPy / H 3 PO 4 film-Au planar electrode.
以下、発明を実施例によって説明する。以下の実施例は、本発明を例示するものであって、本発明を制限する意図はない。 The invention will now be described by way of examples. The following examples illustrate the invention and are not intended to limit the invention.
以下１〜３の実施例において、電気化学測定は電気化学測定システム（ＨＺ−３０００、北斗電工（株）製）を用いて行い、カーボン平面電極はグラッシーカーボン電極（電極面積７ｍｍ２、（株）ビー・エー・エス製（ＰＡＲＴＳ＃００２０１２））、参照電極をＡｇ／ＡｇＣｌ（飽和ＫＣｌ）、対極をＰｔ棒（直径１ｍｍ、長さ４ｃｍ、（株）ニラコ製）とし、電位はこの参照電極の電位に対する値を記載している。また１〜５の実施例において、使用したリン酸緩衝液（ｐＨ７．０）は、滅菌処理済純水中にＫＨ２ＰＯ４（特級、和光純薬工業（株）製）およびＮａ２ＨＰＯ４（特級、和光純薬工業（株）製）を溶解してｐＨ７．０に調整したものを用いた。 In Examples 1 to 3 below, electrochemical measurement is performed using an electrochemical measurement system (HZ-3000, manufactured by Hokuto Denko Co., Ltd.), and the carbon planar electrode is a glassy carbon electrode (electrode area 7 mm 2 , Co., Ltd.). BAS (PARTS # 002012)), the reference electrode is Ag / AgCl (saturated KCl), the counter electrode is a Pt bar (diameter 1 mm, length 4 cm, manufactured by Niraco Co., Ltd.), and the potential is the reference electrode Values for potential are listed. In Examples 1 to 5, the phosphate buffer (pH 7.0) used was sterilized pure water with KH 2 PO 4 (special grade, manufactured by Wako Pure Chemical Industries, Ltd.) and Na 2 HPO 4. (Special grade, manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in pH 7.0 was used.
（実施例１）ＡＴＰの分子鋳型をもつ過酸化ポリピロール膜の作製
上記実施の形態１に従い、詳細には以下の手順に従って、カーボン平面電極上に過酸化ポリピロール膜を作製した。
１）カーボン平面電極をコンパウンド（粒子サイズ０．１μｍ、（株）ソフト９９コーポレーション製）で５分間研磨した後、超音波洗浄を１分間行った。
２）純水を溶媒とし、１０ｍＭ濃度のＡＴＰ（アデノシン−５’−三リン酸二ナトリウム、生化学用、キシダ化学（株）製）および０．１Ｍ濃度のピロール（特級、和光純薬工業（株）製）を含む溶液を調製した。
３）この溶液中で、作用極をカーボン平面電極、参照電極をＡｇ／ＡｇＣｌ（飽和ＫＣｌ）、対極をＰｔ棒とし、電気化学測定システムにおいて作用極に定電位０．９８Ｖを３６０秒間加えることによってピロールを酸化重合し、作用極上にＡＴＰがドープされたポリピロール（ＰＰｙ／ＡＴＰ）膜を析出させた。
４）０．１ＭのＮａＯＨ（特級、和光純薬工業（株）製）溶液中で、作用極をＰＰｙ／ＡＴＰ電極とし、電気化学測定システムにおいて、サイクリックボルタンメトリを掃引速度５ｍＶｓ−１で作用極初期電位０．００Ｖ、折り返し電位１．２０Ｖとして２サイクル行い、カーボン平面電極上にＡＴＰ鋳型を有するポリピロール膜を過酸化ポリピロール（ＯＰＰｙ／ＡＴＰ）膜として作製した。
(Example 1) Production of a polypyrrole peroxide film having an ATP molecular template According to the first embodiment, in detail, a peroxide polypyrrole film was produced on a carbon planar electrode according to the following procedure.
1) The carbon planar electrode was polished for 5 minutes with a compound (particle size: 0.1 μm, manufactured by Soft 99 Corporation), and then subjected to ultrasonic cleaning for 1 minute.
2) Using pure water as a solvent, 10 mM ATP (adenosine-5′-triphosphate, biochemical, manufactured by Kishida Chemical Co., Ltd.) and 0.1 M pyrrole (special grade, Wako Pure Chemical Industries ( Solution) was prepared.
3) In this solution, the working electrode is a carbon plane electrode, the reference electrode is Ag / AgCl (saturated KCl), the counter electrode is a Pt rod, and a constant potential of 0.98 V is applied to the working electrode in an electrochemical measurement system for 360 seconds. Pyrrol was oxidatively polymerized to deposit a polypyrrole (PPy / ATP) film doped with ATP on the working electrode.
4) In a 0.1 M NaOH (special grade, manufactured by Wako Pure Chemical Industries, Ltd.) solution, the working electrode is a PPy / ATP electrode, and in an electrochemical measurement system, cyclic voltammetry at a sweep rate of 5 mVs −1 Two cycles were performed with an initial working electrode potential of 0.00 V and a folding potential of 1.20 V, and a polypyrrole film having an ATP template on a carbon planar electrode was produced as a polypyrrole peroxide (OPPy / ATP) film.
（実施例２）過酸化ポリピロール膜を用いたＡＴＰの検出
得られたカーボン平面電極上に作製したＯＰＰｙ／ＡＴＰ膜を用いて、測定対象試料として５．０ｍＭ濃度のＡＴＰ溶液を用いて以下の手順により酸化電流を測定した。
(Example 2) Detection of ATP using polypyrrole peroxide film Using the OPPy / ATP film prepared on the obtained carbon planar electrode, using a 5.0 mM ATP solution as a measurement target sample, the following procedure Was used to measure the oxidation current.
まず、上記実施例１で得られたＯＰＰｙ／ＡＴＰ膜を備えた電極を用い、
５）リン酸緩衝液（ｐＨ７．０）中で、作用極をＯＰＰｙ／ＡＴＰ電極、参照電極をＡｇ／ＡｇＣｌ（飽和ＫＣｌ）、対極をＰｔ棒とし、電気化学測定システムにおいて作用極に電位幅０.００〜２．００Ｖ（３サイクル、掃引速度５ｍＶｓ−１）の電位走査を行うことによりポリピロール膜の過酸化処理が確実に行われたことの確認を行った。
６）次に図３の電位プロファイルに従い電極のクリーニング、ＡＴＰの濃縮及び酸化的検出を行った。−０．５０Ｖを３０秒間電極に印加し、膜内に残存する可能性のあるＡＴＰを排除する。次に０．８０Ｖを１５秒間印加して被検溶液内のＡＴＰを膜内に濃縮する。その後、１．８０Ｖを１５秒間印加してＡＴＰの電解酸化検出を行う。まず、空試験として、図３のＢの電位プロファイルで実験を行った。リン酸緩衝液（ｐＨ７．０）中で、作用極をＯＰＰｙ／ＡＴＰ電極、参照電極をＡｇ/ＡｇＣｌ（飽和ＫＣｌ）、対極をＰｔ棒とし、電気化学測定システムにおいて作用極に−０．５０Ｖを１５秒間加えてＯＰＰｙ／ＡＴＰ電極をクリーニングし、そして作用極に０．８０Ｖを１５秒間加えて濃縮過程を行い、さらに１．８０Ｖを１５秒間加えて作用極に流れる電流を測定した。
７）ここで、被検体としてその濃度を５．０ｍＭとするようにＡＴＰを加え、作用極をＯＰＰｙ／ＡＴＰ電極、参照電極をＡｇ/ＡｇＣｌ（飽和ＫＣｌ）、対極をＰｔ棒とし、電気化学測定システムにおいて作用極に定電位−０．５０Ｖを１５秒間加えて作用極をクリーニングし、作用極に定電位０．８０Ｖを１５秒間加えて作用極上にＡＴＰを濃縮したのち、作用極に定電位１．８０Ｖを１５秒間加えて（図３のほぼＡで示される）電流値を測定した。
First, using the electrode provided with the OPPy / ATP film obtained in Example 1 above,
5) In phosphate buffer solution (pH 7.0), the working electrode is an OPPy / ATP electrode, the reference electrode is Ag / AgCl (saturated KCl), the counter electrode is a Pt rod, and the potential width is 0 at the working electrode in an electrochemical measurement system. It was confirmed that the peroxidation treatment of the polypyrrole film was reliably performed by performing a potential scan of 0.0 to 2.00 V (3 cycles, sweep speed 5 mVs −1 ).
6) Next, electrode cleaning, ATP concentration, and oxidative detection were performed according to the potential profile of FIG. -0.50 V is applied to the electrode for 30 seconds to eliminate any ATP that may remain in the membrane. Next, 0.80 V is applied for 15 seconds to concentrate ATP in the test solution into the membrane. Thereafter, 1.80 V is applied for 15 seconds to detect electrolytic oxidation of ATP. First, as a blank test, an experiment was performed with the potential profile of B in FIG. In phosphate buffer (pH 7.0), the working electrode is an OPPy / ATP electrode, the reference electrode is Ag / AgCl (saturated KCl), the counter electrode is a Pt rod, and -0.50 V is applied to the working electrode in the electrochemical measurement system. The OPPy / ATP electrode was cleaned by applying for 15 seconds, and 0.80 V was applied to the working electrode for 15 seconds to perform the concentration process, and then 1.80 V was applied for 15 seconds to measure the current flowing through the working electrode.
7) Here, ATP was added so that the concentration was 5.0 mM as an analyte, the working electrode was an OPPy / ATP electrode, the reference electrode was Ag / AgCl (saturated KCl), and the counter electrode was a Pt rod, and electrochemical measurement In the system, a constant potential of −0.50 V is applied to the working electrode for 15 seconds to clean the working electrode, a constant potential of 0.80 V is applied to the working electrode for 15 seconds to concentrate ATP on the working electrode, and then the constant potential of 1 is applied to the working electrode. .80 V was applied for 15 seconds and the current value was measured (shown as approximately A in FIG. 3).
図３に示される条件で測定した作用極電流値の変化を図４に示す。ここで、ＡＴＰ不在下の電位印加開始後４５秒における電流値をＩ１、ＡＴＰ存在下で測定された電位印加開始後１０５秒における電流値をＩ２とし、測定された電流値の差ΔＩ＝Ｉ２−Ｉ１としてＡＴＰの存在による電流値の変化を確認した。その結果、図４に示すように、５．０ｍＭ ＡＴＰでは７．２０×１０−６Ａ（＝ΔＩ）の電流値の変化が観察された。 FIG. 4 shows changes in the working electrode current value measured under the conditions shown in FIG. Here, the current value 45 seconds after the start of potential application in the absence of ATP is I1, the current value 105 seconds after the start of potential application measured in the presence of ATP is I2, and the difference ΔI = I2− The change in current value due to the presence of ATP was confirmed as I1. As a result, as shown in FIG. 4, a change in the current value of 7.20 × 10 −6 A (= ΔI) was observed at 5.0 mM ATP.
（比較例１）カーボン平面電極のみでの酸化電流測定
以下の手順に従って、カーボン平面電極（グラッシーカーボン電極、電極面積７ｍｍ２、（株）ビー・エー・エス製（ＰＡＲＴＳ＃００２０１２））を用いて、ＡＴＰ（生化学用、キシダ化学（株）製）の電気化学的挙動を測定した。
１）カーボン平面電極をコンパウンド（粒子サイズ０．１μｍ、（株）ソフト９９コーポレーション製）で５分間研磨した後、超音波洗浄を１分間行った。
２）リン酸緩衝液（ｐＨ７．０）中で、作用極をカーボン平面電極、参照電極をＡｇ/ＡｇＣｌ（飽和ＫＣｌ）、対極をＰｔ棒（直径１ｍｍ、（株）ニラコ製）とし、電気化学測定システムにおいて作用極に定電位１．８０Ｖ及び−１．００Ｖを各３分間加えて作用極に酸化還元処理を施した。
３）リン酸緩衝液（ｐＨ７．０）中で、作用極をカーボン平面電極、参照電極をＡｇ/ＡｇＣｌ（飽和ＫＣｌ）、対極をＰｔ棒とし、電気化学測定システムにおいて作用極に０．００〜２．００Ｖ（掃引速度５ｍＶｓ−１）の電位を印加し、ベースライン電流を測定した。
４）カーボン平面電極を用いたＡＴＰの酸化電流測定：リン酸緩衝液（ｐＨ７．０）２０ｍＬにＡＴＰ ０．１０ｇを加え、１０ｍＭのＡＴＰ溶液を調製した。また、同様に０．０１０〜１０ｍＭまでの様々な濃度のＡＴＰ溶液を調製した。
５）ＡＴＰ溶液中で、作用極をカーボン平面電極、参照電極をＡｇ/ＡｇＣｌ（飽和ＫＣｌ）、対極をＰｔ棒とし、電気化学測定システムにおいて作用極に０．００〜２．００Ｖ（掃引速度５ｍＶｓ−１）の電位を印加して掃引を行い、作用極に流れるＡＴＰによる酸化電流を測定した。
(Comparative example 1) Oxidation current measurement only with a carbon plane electrode According to the following procedures, a carbon plane electrode (glassy carbon electrode, electrode area 7 mm 2 , manufactured by BAS Co., Ltd. (PARTS # 002012)) was used. The electrochemical behavior of ATP (for biochemistry, manufactured by Kishida Chemical Co., Ltd.) was measured.
1) The carbon planar electrode was polished for 5 minutes with a compound (particle size: 0.1 μm, manufactured by Soft 99 Corporation), and then subjected to ultrasonic cleaning for 1 minute.
2) In phosphate buffer (pH 7.0), the working electrode was a carbon plane electrode, the reference electrode was Ag / AgCl (saturated KCl), and the counter electrode was a Pt rod (diameter 1 mm, manufactured by Nilaco Corporation). In the measurement system, constant potentials of 1.80 V and -1.00 V were applied to the working electrode for 3 minutes, respectively, and the working electrode was subjected to oxidation-reduction treatment.
3) In a phosphate buffer solution (pH 7.0), the working electrode is a carbon plane electrode, the reference electrode is Ag / AgCl (saturated KCl), the counter electrode is a Pt rod, and the working electrode in the electrochemical measurement system is 0.00 to A baseline current was measured by applying a potential of 2.00 V (sweep speed 5 mVs −1 ).
4) OTP oxidation current measurement using a carbon flat electrode: 0.10 g of ATP was added to 20 mL of phosphate buffer (pH 7.0) to prepare a 10 mM ATP solution. Similarly, ATP solutions having various concentrations from 0.010 to 10 mM were prepared.
5) In an ATP solution, the working electrode is a carbon plane electrode, the reference electrode is Ag / AgCl (saturated KCl), the counter electrode is a Pt rod, and the working electrode is 0.00 to 2.00 V (sweep speed 5 mVs) in the electrochemical measurement system. A potential of -1 ) was applied to sweep, and the oxidation current due to ATP flowing in the working electrode was measured.
この結果、０．１ｍＭ以上の濃度ではＡＴＰの酸化電流が観察できたが、０．１ｍＭ以下ではベースラインと比較して有意な酸化電流が観察できなかった
（実施例３）過酸化ポリピロール膜／カーボン平面電極を用いたＡＴＰの定量
実施例１に記載の方法で得られたカーボン平面電極上に作製したＯＰＰｙ／ＡＴＰ膜を用いて、実施例２と同様に、０．１０〜１０ｍＭの様々なＡＴＰ濃度における作用極と対極との間の酸化電流を測定し、ＡＴＰの定量を行った。各濃度における作用極電流差ΔＩ（ＡＴＰ存在下での酸化電流値−ベース電流値）を濃度に対してプロットした結果を図５に示す。図５に示されるように、ＡＴＰの濃度に依存して電流差が比例的に増大した。
As a result, ATP oxidation current could be observed at a concentration of 0.1 mM or more, but no significant oxidation current could be observed at a concentration of 0.1 mM or less compared to the baseline.
(Example 3) Quantification of ATP using polypyrrole peroxide film / carbon planar electrode Similar to Example 2 using an OPPy / ATP film prepared on a carbon planar electrode obtained by the method described in Example 1. In addition, ATP was quantified by measuring the oxidation current between the working electrode and the counter electrode at various ATP concentrations of 0.10 to 10 mM. FIG. 5 shows the results of plotting the working electrode current difference ΔI (oxidation current value in the presence of ATP−base current value) against the concentration at each concentration. As shown in FIG. 5, the current difference increased proportionally depending on the concentration of ATP.
（実施例４）過酸化ポリピロール膜／チップ電極を用いたＡＴＰの定量
実施例１に記載の方法と同様の方法を用い、チップ電極（ＡＣ１．Ｗ４．Ｒ１、プリントカーボン電極、作用電極面積２ｍｍ２、オンチップＡｇ／ＡｇＣｌ参照電極、オンチップＰｔ／Ａｕ（１５％／８５％）合金対極、ＢＶＴ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）上にＯＰＰｙ／ＡＴＰ膜を作製した。得られた分子鋳型を有するチップ電極を、デュアル電気化学アナライザー（ＡＬＳモデル８４２Ｂ、（株）ビー・エー・エス製）において、実施例２と同様に５．０ｍＭのＡＴＰ溶液を測定試料として、このチップ電極の酸化電流を測定した。電流値差ΔＩは実施例３での結果と同等の結果が得られた（実施例３：７．２×１０−６Ａ、実施例４：５．１×１０−６Ａ）。このことから、カーボン平面電極の代わりにチップ電極用いた場合においても同等の検出が可能であることが示された。チップ電極を用いることで、作用極・対極・参照電極の各電極を平面上に配置することが可能になるため一体型電気化学セルを形成でき、センサプローブ（検出部）の小型化が達成することができる。
(Example 4) Determination of ATP using polypyrrole peroxide film / tip electrode Using the same method as described in Example 1, a tip electrode (AC1.W4.R1, printed carbon electrode, working electrode area 2 mm 2) An OPPy / ATP film was prepared on an on-chip Ag / AgCl reference electrode, an on-chip Pt / Au (15% / 85%) alloy counter electrode, manufactured by BVT Technologies). In the dual electrochemical analyzer (ALS model 842B, manufactured by BAS Co., Ltd.), the chip electrode having the obtained molecular template was measured using a 5.0 mM ATP solution as a measurement sample in the same manner as in Example 2. The oxidation current of the chip electrode was measured. The current value difference ΔI was the same as the result in Example 3 (Example 3: 7.2 × 10 −6 A, Example 4: 5.1 × 10 −6 A). From this, it was shown that the same detection is possible even when the chip electrode is used instead of the carbon planar electrode. By using the tip electrode, the working electrode, the counter electrode, and the reference electrode can be arranged on a plane, so that an integrated electrochemical cell can be formed, and the sensor probe (detection unit) can be downsized. be able to.
図９は、実施例４および５で測定に用いたチップ電極と、実施例１〜３の電気化学的測定を行ったセルの写真である。図中Ｃは、対極（Ｐｔ棒）を、Ｗは作用極（カーボン平面電極）を、そしてＲは参照電極（Ａｇ／ＡｇＣｌ）を示す。 FIG. 9 is a photograph of the chip electrode used for the measurement in Examples 4 and 5 and the cell in which the electrochemical measurement of Examples 1 to 3 was performed. In the figure, C indicates a counter electrode (Pt bar), W indicates a working electrode (carbon planar electrode), and R indicates a reference electrode (Ag / AgCl).
（実施例５）トリプルパルスアンペロメトリー測定法を用いたＡＴＰの定量
実施例１に記載の方法と同様の方法により、実施例４に記載のチップ電極上に作製したＯＰＰｙ／ＡＴＰ膜を用いて、以下の手順に従って、トリプルパルスアンペロメトリー測定によりＡＴＰの定量を行った。
１）リン酸緩衝液（ｐＨ７．０）２０ｍＬにＡＴＰ（生化学用、キシダ化学（株）製）０．１０ｇを加え、１０ｍＭのＡＴＰ溶液を調製した。また、同様に１．０×１０−４ｍＭ〜１０ｍＭまで様々な濃度のＡＴＰ溶液を調製した。
２）デュアル電気化学アナライザー（ＡＬＳモデル８４２Ｂ、（株）ビー・エー・エス製）においてＥ１＝−０．５０Ｖ、Ｔ１＝０．５０秒、Ｅ２＝０．５０Ｖ、Ｔ２＝０．２０秒、Ｅ３＝１．８０Ｖ、Ｔ３＝０．０１０秒、サイクル数＝１０００の条件で、トリプルパルスアンペロメトリー測定を行った。用いたパルスプロファイルを図６に示す。なお、電流は各パルス後半半分の時間観測し、平均化している。下の実施例に示される電流は、この各電流の内、Ｅ３のパルスにおいて観測された電流からＥ２のパルスにおいて観測された電流の差をとったものとして示される。チップ電極にリン酸溶液を２０μＬ滴下し、ベース電流が安定していることを確認した後、さらにＡＴＰ溶液２０μＬを滴下した。このとき、
トリプルアンペロメトリー電流値が変化する様子を図７に示した。また、各ＡＴＰ濃度における電流値とＡＴＰ溶液濃度との関係を検量線として図８に示す。
(Example 5) Quantification of ATP using triple pulse amperometry measurement method Using the OPPy / ATP film prepared on the chip electrode described in Example 4 by the same method as described in Example 1. According to the following procedure, ATP was quantified by triple pulse amperometry measurement.
1) 0.10 g of ATP (for biochemistry, manufactured by Kishida Chemical Co., Ltd.) was added to 20 mL of a phosphate buffer (pH 7.0) to prepare a 10 mM ATP solution. Similarly, ATP solutions having various concentrations up to 1.0 × 10 −4 mM to 10 mM were prepared.
2) E1 = −0.50V, T1 = 0.50 seconds, E2 = 0.50V, T2 = 0.20 seconds, E3 in a dual electrochemical analyzer (ALS model 842B, manufactured by BAS) Triple pulse amperometry measurement was performed under the conditions of = 1.80 V, T3 = 0.010 seconds, and the number of cycles = 1000. The pulse profile used is shown in FIG. The current is averaged by observing the latter half of each pulse. The current shown in the example below is shown as the difference between the current observed in the E3 pulse and the current observed in the E2 pulse of each current. 20 μL of phosphoric acid solution was dropped onto the chip electrode, and after confirming that the base current was stable, 20 μL of ATP solution was further dropped. At this time,
FIG. 7 shows how the triple amperometry current value changes. Further, FIG. 8 shows the relationship between the current value and the ATP solution concentration at each ATP concentration as a calibration curve.
図８に示されるように、ＯＰＰｙ／ＡＴＰ膜を用いたトリプルパルスアンペロメトリー測定では、５００ｎＭ濃度程度までのＡＴＰを測定することが可能であることが示された。これは、上記比較例における０．１ｍＭ濃度以下のＡＴＰを測定できなかった結果と比較すると、１／１０００程度の薄い濃度のＡＴＰをも測定することを示している。 As shown in FIG. 8, triple pulse amperometry measurement using an OPPy / ATP film showed that ATP up to about 500 nM concentration can be measured. This indicates that ATP having a thin concentration of about 1/1000 is also measured as compared with the result in which ATP having a concentration of 0.1 mM or less in the comparative example cannot be measured.
なお、チップ電極（ＡＣ１．Ｗ４．Ｒ１、プリントカーボン電極、電極面積２ｍｍ２、ＢＶＴ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）を用いて、トリプルパルスアンペロメトリー測定法でＯＰＰｙ／ＡＴＰ膜を用いずにＡＴＰの検出を行ったところ、検出限界はカーボン平面電極の場合と同じく０．１ｍＭであった。これは、特定の理論に拘束されるのではなく、ＡＴＰが分子鋳型に入り込むのみならず、分子鋳型の近傍で複数のＡＴＰ分子がＯＰＰｙ／ＡＴＰ膜上に高度に濃縮されているためであると考えられた。 In addition, ATP is detected without using an OPPy / ATP film by a triple pulse amperometry measurement method using a chip electrode (AC1.W4.R1, printed carbon electrode, electrode area 2 mm 2 , manufactured by BVT Technologies). As a result, the detection limit was 0.1 mM as in the case of the carbon planar electrode. This is not due to a specific theory, but because ATP not only enters the molecular template, but also a plurality of ATP molecules are highly concentrated on the OPPy / ATP membrane in the vicinity of the molecular template. it was thought.
微量物質を分離、濃縮そして検出するための吸着剤、センサーおよびそれらを備える微量物質を分離、濃縮そして検出するためシステムが提供される。環境中に存在する微量の化学物質、農薬、および食品、医薬品中に存在する微量の微生物または微生物由来の物質を含む広範囲な分野で使用され得る。上記吸着剤、およびセンサーは、酵素などの生体由来物質を用いないので、取り扱いが簡便で、安定性に優れ、かつ広範囲な分野で使用され得るコストパーフォーマンスに優れた測定方法が提供される。 Adsorbents, sensors for separating, concentrating and detecting trace substances, sensors and systems for separating, concentrating and detecting trace substances comprising them are provided. It can be used in a wide range of fields, including trace amounts of chemicals present in the environment, pesticides, and foods, pharmaceuticals and trace amounts of microorganisms or microorganism-derived substances. Since the adsorbent and the sensor do not use a biological substance such as an enzyme, a measuring method that is easy to handle, excellent in stability, and excellent in cost performance that can be used in a wide range of fields is provided.
１ 電極
２ 未成長ポリピロール膜
３ ポリピロール膜
４ ケーブル
５ 感応部
６ 検知部
７ 容器
Ｓ 被検試料
Ｓ’ 綿棒
DESCRIPTION OF SYMBOLS 1 Electrode 2 Ungrown polypyrrole film | membrane 3 Polypyrrole film | membrane 4 Cable 5 Sensing part 6 Detection part 7 Container S Test sample S 'Cotton swab
Claims (9)
電極、
該電極上に配置されたポリマー層であって、該アニオン分子の立体構造に相補的な三次元構造を有する分子鋳型を備えたポリマー層、
該ポリマー層の導電性の変化を導出する導出部を備え、
該三次元構造が、該ポリマー層を形成する際に該ポリマーのモノマーを該標的分子の存在下で重合する工程、および得られた重合体を過酸化する工程によって形成される、センサー。 A sensor for detecting anionic molecules,
electrode,
A polymer layer disposed on the electrode, the polymer layer comprising a molecular template having a three-dimensional structure complementary to the three-dimensional structure of the anion molecule;
A derivation unit for deriving a change in conductivity of the polymer layer;
A sensor wherein the three-dimensional structure is formed by polymerizing monomers of the polymer in the presence of the target molecule and peroxidizing the resulting polymer in forming the polymer layer.
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