JP5429893B2 - Preparation of bioreactive carbon nanotubes by functionalizing redox proteins by non-covalent bonding - Google Patents
Preparation of bioreactive carbon nanotubes by functionalizing redox proteins by non-covalent bonding Download PDFInfo
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Description
本発明は、医学的検知のためのバイオセンサーの分野で役立つレドックスたんぱく質を（ポリマー結合材を使用することなく）非共有結合で結合させ機能化した生体反応性カーボンナノチューブの製法に関するものである。 The present invention relates to a process for producing bioreactive carbon nanotubes that are functionalized by binding non-covalently (without the use of a polymer binder) a redox protein useful in the field of biosensors for medical detection.
ナノスケールの物質に基づく酵素バイオセンサーについての多くの努力は、そのバイオ医学的な重要性のゆえに、グルコース・バイオセンサーの創作に向けられている。そのようなグルコース・バイオセンサーは、カーボンナノチューブ（ＣＮＴ）とグルコース・オキシダーゼ（ＧＯｘ）との組合せに基づくものであった。グルコース・オキシダーゼとカーボンナノチューブとを、例えば、テフロン（非特許文献１）、鉱油（M.D.Rubianes et al, 2003）、硬エポキシ（B.Perez et al, 2005）、ナフィオン（J.Wang et al, 2004）、ポリピロール（M.Gao et al, 2003; Y.C.Tsai et al, 2006; J.Wang et al, 2005）、キトサン（Y. Liu et al, 2006）、あるいはゾル−ゲル・マトリックス（A.Salimi et al, 2004）中で、物理的に結合（化学的結合ではない）させて、グルコースを検知するバイオ認識／電気化学的システムをつくり出すことにより、有効なグルコース・バイオセンサーが構築されうることが示された。一方、少数ながら、グルコースとカーボンナノチューブとを化学結合により結合させようとする研究もある（F.Patolsky et al, 2004;J.Liu et al, 2005; Y. Lin et al, 2004）。 Much effort for enzyme biosensors based on nanoscale materials has been devoted to the creation of glucose biosensors due to their biomedical importance. Such glucose biosensors were based on a combination of carbon nanotubes (CNT) and glucose oxidase (GOx). Glucose oxidase and carbon nanotubes can be used in, for example, Teflon (Non-patent Document 1), mineral oil (MDRubianes et al, 2003), hard epoxy (B. Perez et al, 2005), Nafion (J. Wang et al, 2004). ), Polypyrrole (M. Gao et al, 2003; YCTsai et al, 2006; J. Wang et al, 2005), chitosan (Y. Liu et al, 2006), or sol-gel matrix (A. Salimi et al, 2004) show that an effective glucose biosensor can be constructed by physically combining (not chemically bonding) to create a biorecognition / electrochemical system that detects glucose. It was done. On the other hand, there are a few studies that try to bind glucose and carbon nanotubes by chemical bonds (F. Patolsky et al, 2004; J. Liu et al, 2005; Y. Lin et al, 2004).
レドックス酵素バイオセンサーに基づくカーボンナノチューブの分野において公表された印刷物の多くにおいては、酵素とカーボンナノチューブ及び／又はポリマー・マトリックスとの間で何らの化学結合をさせることなく、酵素とカーボンナノチューブとを有機もしくは無機のポリマー結合材の中に取り込んでいることに注目するべきである。しかし、これらバイオセンサーの作製においては、酵素は電極物質（カーボンナノチューブでもポリマー結合材でもない）に共有結合で固定されていないので、結合材の使用が本当に必要であるのか、あるいは、グルコース・オキシダーゼをカーボンナノチューブの上に直接に固定させて、電気化学的グルコース・バイオセンサーをつくり出すことが可能かどうかを知ることは興味深いことであろう。 In many published printed material in the field of carbon nanotubes based on a redox enzyme biosensor without the any of the chemical bond between the enzyme and the carbon nanotubes and / or polymer matrix, the organic and an enzyme and the carbon nanotube Or it should be noted that it is incorporated into an inorganic polymer binder. However, in making these biosensors, the enzyme is not covalently fixed to the electrode material (not a carbon nanotube or polymer binder), so is it really necessary to use a binder or glucose oxidase? It would be interesting to know if it is possible to create an electrochemical glucose biosensor by directly immobilizing a carbon nanotube on the carbon nanotube.
高度に特異的な電気的バイオセンサーに向けて、非共有結合によってカーボンナノチューブを機能化する本質的・重要な仕事は、これまでにいくつかなされてきたが（M.Shim et al, 2002;R.J.Chenet al, 2004; R.J.Chen et al, 2003; D.-W. Park et al, 2006）、電気化学的バイオセンサーに向けての努力はなされていない。 There have been some essential and important work to functionalize carbon nanotubes by noncovalent bonds towards highly specific electrical biosensors (M. Shim et al, 2002; RJ Chenet al, 2004; RJChen et al, 2003; D.-W. Park et al, 2006), no effort has been made towards electrochemical biosensors.
本発明の目的は、レドックスたんぱく質を、ポリマー結合材を使用することなく非共有結合で結合させ機能化した生体反応性カーボンナノチューブの作製方法を提供することである。 An object of the present invention is to provide a method for producing a bioreactive carbon nanotube in which a redox protein is bonded by a non-covalent bond and functionalized without using a polymer binder.
〔発明の概要〕
上記目的を達成するため、どのようにすればポリマー結合材を要することなく、グルコース・オキシダーゼ（ＧＯｘ）がカーボンナノチューブのネットワークに固定されうるのか、我々は種々検討した。そして、カーボンナノチューブ上にレドックスたんぱく質ＧＯｘを非共有結合的に吸着させることに成功し、また、得られたＣＮＴ／ＧＯｘハイブリッドはグルコースの検出に好都合な性質を示すものであった。
[Summary of the Invention]
In order to achieve the above object, we have studied variously how glucose oxidase (GOx) can be immobilized on a network of carbon nanotubes without requiring a polymer binder. And it succeeded in adsorb | sucking redox protein GOx non-covalently on a carbon nanotube, and the obtained CNT / GOx hybrid showed the property convenient for the detection of glucose.
すなわち、本発明は、 ポリマー結合材を使用することなく、レドックスたんぱく質を非共有結合で結合させ機能化した生体反応性カーボンナノチューブ（すなわち、カーボンナノチューブ／レドックスたんぱく質のハイブリッド）を提供する。 That is, the present invention provides a bioreactive carbon nanotube (ie, a carbon nanotube / redox protein hybrid) in which a redox protein is bound by a non-covalent bond and functionalized without using a polymer binder.
また、本発明は、（ポリマー結合材を使用することなく）レドックスたんぱく質を非共有結合で結合させ機能化した前記生体反応性カーボンナノチューブの製法も提供するものであり、その製法は以下の工程を含んでいる：
(i) カーボンナノチューブを濃硝酸中で、７０〜９０℃の温度条件下に酸化する；
(ii) 得られた酸化型カーボンナノチューブを精製水に分散させ、０．１〜１．０ｍｇ／ｍＬの濃度にする；
(iii) レドックスたんぱく質を０．０４〜０．４ｍｇ／ｍＬの濃度に加え、混合する；
(iv) 得られた生体反応性カーボンナノチューブ（固体）を分離する。
The present invention also provides a method for producing the bioreactive carbon nanotube functionalized by binding a redox protein non-covalently (without using a polymer binder), and the method comprises the following steps: Contains:
(i) oxidizing carbon nanotubes in concentrated nitric acid at a temperature of 70-90 ° C ;
(ii) The obtained oxidized carbon nanotube is dispersed in purified water to a concentration of 0.1 to 1.0 mg / mL ;
(iii) Add redox protein to a concentration of 0.04-0.4 mg / mL and mix;
(iv) The obtained bioreactive carbon nanotube (solid) is separated.
＜略号＞
用いた略号の意味は次の通り。
ＣＮＴ：カーボンナノチューブ
ＤＷＣＮＴ：二層（double walled）カーボンナノチューブ
ＧＯｘ：グルコース・オキシダーゼ
<Abbreviation>
The meanings of the abbreviations used are as follows.
CNT: carbon nanotube DWCNT: double walled carbon nanotube GOx: glucose oxidase
本発明の、（ポリマー結合材なしで）レドックスたんぱく質を非共有結合で結合させ機能化した生体反応性カーボンナノチューブは、バイオセンサーを容易に構築する場合に扉を開くものである。これらは、例えば、電気化学的バイオセンサー（例：グルコースセンサー）として用いることができる。
[発明の実施の形態]
The bioreactive carbon nanotube functionalized by non-covalently binding a redox protein (without a polymer binder) of the present invention opens the door when a biosensor is easily constructed. These can be used, for example, as an electrochemical biosensor (eg, glucose sensor).
[Embodiment of the Invention]
〔発明の更に詳しい説明〕
次に、本発明を更に詳細に説明する。
先に述べたように、本発明は、ポリマー結合材を使用することなく、レドックスたんぱく質を非共有結合で結合させ機能化した生体反応性カーボンナノチューブを提供するものであり、言い換えれば、ポリマー結合材のない生体反応性カーボンナノチューブ／レドックスたんぱく質・ハイブリッドを提供するものである。
[Detailed description of the invention]
Next, the present invention will be described in more detail.
As described above, the present invention provides a bioreactive carbon nanotube in which a redox protein is non-covalently bonded and functionalized without using a polymer binder, in other words, a polymer binder. The present invention provides a bioreactive carbon nanotube / redox protein / hybrid having no odor.
ここで、本発明における「カーボンナノチューブ」は、炭素の異型（allotype）の一つである。これは、その直径が２ｎｍ〜１００ｎｍの柱状分子の形態を有しており、グラファイト・シートを丸めた形状をしている。そのカーボンナノチューブとして、単層カーボンナノチューブ、二層カーボンナノチューブ、多層カーボンナノチューブいずれも使用できるが、好ましくは二層カーボンナノチューブ又は多層カーボンナノチューブである。 Here, the “carbon nanotube” in the present invention is one of allotypes of carbon. This has a form of a columnar molecule having a diameter of 2 nm to 100 nm, and is formed by rounding a graphite sheet. As the carbon nanotube, any of a single-walled carbon nanotube, a double-walled carbon nanotube, and a multi-walled carbon nanotube can be used, but a double-walled carbon nanotube or a multi-walled carbon nanotube is preferable.
また、本発明における「非共有結合で結合させ機能化した」とは、“物理的に機能化した（させた）”ことを意味し、これには静電力、水素結合力、疎水性力あるいはπ−π相互作用力のような多くの型の非共有結合力（M. Shim et al, Nano Lett. 2, 285 (2002); R. J. Chen et al, Proc. Natl. Acad. Sci. 100, 4984 (2003); D. Cui, J. Nanosci. Nanotech. 7, 1298 (2007)）を含んでいる。 Further, in the present invention, “functionalized by non-covalent bonding” means “physically functionalized”, which includes electrostatic force, hydrogen bonding force, hydrophobic force or Many types of noncovalent forces such as π-π interaction forces (M. Shim et al, Nano Lett. 2, 285 (2002); RJ Chen et al, Proc. Natl. Acad. Sci. 100, 4984 (2003); D. Cui, J. Nanosci. Nanotech. 7, 1298 (2007)).
また、本発明における「レドックスたんぱく質」とは、基質から（又は基質へ）電子を交換（注入、又は引抜き）する生体認識の実行が可能なたんぱく質を意味する。上記レドックスたんぱく質として、基本的に種々のオキシダーゼ、パーオキシダーゼ、デヒドロゲナーゼ及びレダクターゼが使える。例えば、グルコース・オキシダーゼ、西洋ワサビ・パーオキシダーゼ、アルゼネート・レダクターゼ（アズリン）、アルコール・オキシダーゼ、アルコール・デヒドロゲナーゼ等である。中でも、グルコース・オキシダーゼが最も好ましく用いられる。 In addition, the “redox protein” in the present invention means a protein capable of performing biological recognition by exchanging (injecting or extracting) electrons from (or into) the substrate. As the redox protein, basically, various oxidases, peroxidases, dehydrogenases and reductases can be used. For example, glucose oxidase, horseradish peroxidase, arsenate reductase (azurin), alcohol oxidase, alcohol dehydrogenase and the like. Of these, glucose oxidase is most preferably used.
また、本発明における「ポリマー結合材」とは、ポリマー・マトリックスを意味する。 The “polymer binder” in the present invention means a polymer matrix.
先に述べたように、本発明は、また、前記生体反応性ハイブリッドの製法にも関係するものであり、その製法は以下の工程を含んでいる：
(i) カーボンナノチューブを濃硝酸中で、高温条件下に酸化する；
(ii) 得られた酸化型カーボンナノチューブを精製水に分散させ、所定濃度にする；
(iii) レドックスたんぱく質を所定濃度に加え、混合する；
(iv) 得られた生体反応性カーボンナノチューブ（固体）を分離する。
As mentioned above, the present invention also relates to a method for producing the bioreactive hybrid, which method comprises the following steps:
(i) oxidizing carbon nanotubes in concentrated nitric acid under high temperature conditions;
(ii) Disperse the obtained oxidized carbon nanotubes in purified water to a predetermined concentration;
(iii) Redox protein is added to the prescribed concentration and mixed;
(iv) The obtained bioreactive carbon nanotube (solid) is separated.
ここで、この方法の工程（ｉ）における「高温」とは、７０℃ないしは９０℃の範囲中の一定温度に維持することを意味する。また、この高温処理は反応が終わるまで、例えば、１２〜３６時間をかけて行なわれる。
工程（ｉｉ）における「酸化型ＣＮＴの所定濃度」とは、０．１−１．０ｍｇ／ｍＬの濃度を意味する。
工程（ｉｉｉ）における「レドックスたんぱく質の所定濃度」とは、０．０４−０．４ｍｇ／ｍＬの濃度を意味する。
また、我々は、本発明の（レドックスたんぱく質を非共有結合で結合させ機能化した）生体反応性カーボンナノチューブで修飾した電極を使用して「バイオセンサー」（生体物質を検出する装置）を構築することができる。
Here, “high temperature” in step (i) of this method means maintaining a constant temperature in the range of 70 ° C. to 90 ° C. Further, this high temperature treatment is performed, for example, over 12 to 36 hours until the reaction is completed.
The “predetermined concentration of oxidized CNT” in step (ii) means a concentration of 0.1 to 1.0 mg / mL.
The “predetermined concentration of redox protein” in step (iii) means a concentration of 0.04-0.4 mg / mL.
In addition, we construct “biosensors” (devices that detect biological materials) using the electrodes modified with bioreactive carbon nanotubes (functionalized by binding non-covalent redox proteins) of the present invention. be able to.
図１は、二層カーボンナノチューブ上に、非共有結合で固定化させたグルコース・オキシダーゼによるグルコースの酸化と、それに続くシグナル変換とを示す模式図である。グルコース・オキシダーゼ（たんぱく質）とカーボンナノチューブとの相互作用は複雑であり、例えば、上述したように、静電力、水素結合力、疎水性力、π−π相互作用等の種々の非共有結合力を含んでいる。 FIG. 1 is a schematic diagram showing the oxidation of glucose by glucose oxidase immobilized on a non-covalent bond on a double-walled carbon nanotube and the subsequent signal conversion. The interaction between glucose oxidase (protein) and carbon nanotubes is complicated. For example, as described above, various non-covalent bonding forces such as electrostatic force, hydrogen bonding force, hydrophobic force, and π-π interaction are exhibited. Contains.
本発明で用いた実験方法は、以下の通り。
＜装置＞
電圧電流実験はすべて、パソコンに接続され、一般目的電気化学システムｖ．４．９ソフトウェアで制御されたμオートラブIII（Ecochemie社, ユトレヒト、 オランダ）を用いて行なった。電気化学実験は、電圧電流セル（５ｍＬ）中、室温（２５℃）で三つの電極を配置した形で行なった。補助電極として白金電極が供され、参照電極として飽和Ａｇ／ＡｇＣｌが供された。電気化学的電位は全てＡｇ／ＡｇＣｌに対するものである。スキャンＴＥＭモード（ＳＴＥＭ；スポットサイズは０．７ｎｍ、加速電圧は２００ｋＶ）でＴＥＭイメージを得るためには、２００ｋＶで作動するJEM 2100F電界放出透過型電子顕微鏡（ＪＯＥＬ，東京，日本）を用いた。比表面積は多点ＢＥＴ法を用いたオートソーブ１装置（Quantachrome Instruments, Boynton beach, FL, USA）によって測定し、窒素が吸着物質に用いられた。試料は吸着実験の前に、真空で、２５０℃で１６ｈ処理して脱水した。
The experimental method used in the present invention is as follows.
<Device>
All voltage and current experiments are connected to a personal computer, and the general purpose electrochemical system v. 4.9 Software controlled μ Autolab III (Ecochemie, Utrecht, The Netherlands) was used. The electrochemical experiment was carried out in a voltage-current cell (5 mL) with three electrodes arranged at room temperature (25 ° C.). A platinum electrode was provided as an auxiliary electrode, and saturated Ag / AgCl was provided as a reference electrode. All electrochemical potentials are relative to Ag / AgCl. In order to obtain a TEM image in the scan TEM mode (STEM; spot size is 0.7 nm, acceleration voltage is 200 kV), a JEM 2100F field emission transmission electron microscope (JOEL, Tokyo, Japan) operating at 200 kV was used. The specific surface area was measured by an autosorb 1 apparatus (Quantachrome Instruments, Boynton beach, FL, USA) using a multipoint BET method, and nitrogen was used as the adsorbent. The sample was dehydrated by treatment at 250 ° C. for 16 h in vacuum before the adsorption experiment.
＜材料＞
二層カーボンナノチューブ（ＤＷＣＮＴ，カタログｎｏ．６３７３５１，純度９０％以上）、グラファイト粉末、過酸化水素（３０％水溶液）、グルコース、グルコース・オキシダーゼ、二塩基性リン酸カリウム及びリン酸は、シグマ−アルドリッチ社（日本）から購入した。
<Material>
Double-walled carbon nanotubes (DWCNT, catalog no. 637351, purity 90% or more), graphite powder, hydrogen peroxide (30% aqueous solution), glucose, glucose oxidase, dibasic potassium phosphate and phosphoric acid are Sigma-Aldrich Purchased from Japan (Japan).
実施例１
（ｉ）ポリマー結合材を使用せずに、レドックスたんぱく質を非共有結合で結合させ機能化した生体反応性カーボンナノチューブの調製
二層カーボンナノチューブ（ＤＷＣＮＴ）は、濃硝酸（６Ｍ）中、８０℃で、２４ｈ処理し、酸化させた。得られた酸／ＤＷＣＮＴ混合物は次に蒸留水で洗浄し、遠沈し、水溶液のｐＨが中性になるまでこの操作を数回繰り返した。次に、上記酸化カーボンナノチューブを、孔の大きさ０．２μｍの膜（Nuclepore Track-Etch Membrane, Whatman, UK）を用い、フィルム状(又はシート状）カーボンナノチューブを形成させつつ、真空で濾過した。
引き続いて、１ｍｌ当たりＧＯｘ０．２ｍｇ及び（上で得た）酸化ＤＷＣＮＴ０．５ｍｇ（対照として、同量のグラファイト・パウダーを使用）をバイアル中に分散させ、これを７５分間、かき混ぜた。このようにして、ポリマー結合材を使用せずに、グルコース・オキシダーゼを非共有結合で結合させて機能化した生体反応性二層カーボンナノチューブを得た。
Example 1
(I) Preparation of a bioreactive carbon nanotube functionalized by non-covalent bonding of a redox protein without using a polymer binder Double-walled carbon nanotube (DWCNT) is obtained at 80 ° C. in concentrated nitric acid (6M). , Treated for 24 h and oxidized. The resulting acid / DWCNT mixture was then washed with distilled water, spun down, and this operation was repeated several times until the pH of the aqueous solution became neutral. Next, the oxidized carbon nanotubes were filtered under vacuum using a membrane having a pore size of 0.2 μm (Nuclepore Track-Etch Membrane, Whatman, UK) while forming film-like (or sheet-like) carbon nanotubes. .
Subsequently, 0.2 mg of GOx per ml and 0.5 mg of oxidized DWCNT (obtained above) (using the same amount of graphite powder as a control) were dispersed in the vial and this was stirred for 75 minutes. In this way, a bioreactive double-walled carbon nanotube functionalized by binding glucose oxidase with a non-covalent bond without using a polymer binder was obtained.
（ｉｉ）評価
電気化学的測定のために、得られた上記ＤＷＣＮＴ／ＧＯｘ（対照：グラファイト／ＧＯｘ）を、研磨布に載せた０．０５μｍのアルミナ粒子を用いて予め研磨したガラス状カーボン電極（直径３ｍｍ）の表面上に落とした。このＤＷＣＮＴ／ＧＯｘ（対照：グラファイト／ＧＯｘ）を先ず、蒸留水で１ｍｇ／ｍｌの濃度に分散させ、次にその分散液をウルトラソニック・バスの中に１分間置き、その後、処理された分散液のうちの５μLをガラス状カーボン電極の表面から吸い取った。その分散液を室温に置き蒸発させると、ガラス状カーボン電極表面上でランダムな分布のフィルムとなった。５０ｍＭリン酸緩衝液（ｐＨ７．４）を用いて、適切な電圧範囲で毎秒５０ｍＶのスキャン速度で、サイクリック・ボルタメトリー実験を行なった。ＴＥＭの測定のためには、濃度１ｍｇ／ｍｌのＤＷＣＮＴ／ＧＯｘ（対照：グラファイト／ＧＯｘ）１μＬを銅のＴＥＭグリッド上に落とし、そのまま放置し空気中で乾燥させた。
(Ii) Evaluation
For the electrochemical measurement, the obtained DWCNT / GOx (control: graphite / GOx) was obtained from a glassy carbon electrode (3 mm in diameter) previously polished using 0.05 μm alumina particles placed on a polishing cloth. Dropped on the surface. This DWCNT / GOx (control: graphite / GOx) is first dispersed in distilled water to a concentration of 1 mg / ml, then the dispersion is placed in an ultrasonic bath for 1 minute, after which the treated dispersion Of this, 5 μL was blotted from the surface of the glassy carbon electrode. When the dispersion was evaporated at room temperature, a film with a random distribution on the surface of the glassy carbon electrode was obtained. Cyclic voltammetry experiments were performed using a 50 mM phosphate buffer (pH 7.4) at a scan rate of 50 mV / sec over the appropriate voltage range. For the measurement of TEM, 1 μL of DWCNT / GOx (control: graphite / GOx) having a concentration of 1 mg / ml was dropped on a copper TEM grid and left to dry in the air.
図２は、得られたＤＷＣＮＴ／ＧＯｘハイブリッドのＴＥＭ像を示すものである。このＴＥＭによってはＧＯｘ分子を直接に観察することは難しいけれども、ＤＷＣＮＴは相互に連絡された濃いナノチューブ・ネットワークを形成していることは明らかである（図２の右側部分では明らかに視認できる）。他方、グラファイト粉は粒構造である（図示せず）。ＤＷＣＮＴとグラファイトの形態上の相違は、電気化学的測定から分かるように、ＧＯｘ分子の物理的捕捉に深く影響する（図３参照）。 FIG. 2 shows a TEM image of the obtained DWCNT / GOx hybrid. Although it is difficult to directly observe GOx molecules with this TEM, it is clear that DWCNTs form a dense nanotube network interconnected (visible on the right side of FIG. 2). On the other hand, the graphite powder has a grain structure (not shown). The morphological difference between DWCNT and graphite has a profound effect on the physical trapping of GOx molecules, as can be seen from the electrochemical measurements (see FIG. 3).
図３Ａは５ｍＭ過酸化水素に対するサイクリックボルタモグラムで、（ａ）がグラファイト／ＧＯｘ膜電極を用いた場合、（ｂ）がＤＷＣＮＴ／ＧＯｘ膜電極を用いた場合である。この図から、ＤＷＣＮＴ／ＧＯｘ膜電極を用いた場合、過酸化水素の酸化は約＋０．６Ｖで始まるが、グラファイト／ＧＯｘ膜電極を用いた場合は過酸化水素の酸化は約＋０．８Ｖで始まることは明かである。この電気化学的測定の結果から、ＤＷＣＮＴを基礎とする電極はグラファイトを基礎とする電極に比べて表面積が大きく、過酸化水素（グルコース・オキシダーゼ酵素によるグルコース酸化の副生物）の低濃度検出に有利であることを示している（ＤＷＣＮＴの表面積／電気化学的容量は、M. Pumera, Nanoscale Res. Lett. 2, 87 (2007)参照)。この観察は、ＢＥＴ法によって測定されたＤＷＣＮＴ及びグラファイトの各々の比表面積（ＤＷＣＮＴ：58.55 m2／g 、グラファイト：11.07 m2／g）と整合している。 FIG. 3A is a cyclic voltammogram for 5 mM hydrogen peroxide, where (a) is a graphite / GOx membrane electrode and (b) is a DWCNT / GOx membrane electrode. From this figure, when the DWCNT / GOx film electrode is used, the oxidation of hydrogen peroxide starts at about + 0.6V, but when the graphite / GOx film electrode is used, the oxidation of hydrogen peroxide starts at about + 0.8V. It is clear. From the results of this electrochemical measurement, the electrode based on DWCNT has a larger surface area than the electrode based on graphite, which is advantageous for detecting low concentrations of hydrogen peroxide (byproduct of glucose oxidation by glucose oxidase enzyme). (For the surface area / electrochemical capacity of DWCNT, see M. Pumera, Nanoscale Res. Lett. 2, 87 (2007)). This observation is consistent with the specific surface areas of DWCNT and graphite measured by the BET method (DWCNT: 58.55 m 2 / g, graphite: 11.07 m 2 / g).
グルコースに対するＤＷＣＮＴ／ＧＯｘ膜電極（対照：グラファイト／ＧＯｘ膜電極）の応答を調べた。図３Ｂは１０ｍＭグルコースに対するハイドロダイナミックボルタモグラムである。グラファイト／ＧＯｘ膜電極では、グルコース・オキシダーゼ酵素がグラファイトのミクロ粒子に有意に捕捉若しくは吸着されなかったためか、グルコースに対するレドックス反応が何ら観察されなかった。ＤＷＣＮＴ／ＧＯｘ膜電極の場合は、状況は劇的に異なっている。グルコースに対し、約＋０．６Ｖで始まるかなりの酸化電流がＤＷＣＮＴ／ＧＯｘ膜電極で観察される。図３の結果は、ＧＯｘレドックス酵素がＤＷＣＮＴを、優れた信号変換器として作用するＤＷＣＮＴへと機能付与させたことを示している。 The response of the DWCNT / GOx membrane electrode (control: graphite / GOx membrane electrode) to glucose was examined. FIG. 3B is a hydrodynamic voltammogram for 10 mM glucose. At the graphite / GOx membrane electrode, no redox reaction to glucose was observed because the glucose oxidase enzyme was not significantly captured or adsorbed on the graphite microparticles. In the case of DWCNT / GOx membrane electrodes, the situation is dramatically different. For glucose, a significant oxidation current starting at about +0.6 V is observed at the DWCNT / GOx membrane electrode. The results in FIG. 3 show that the GOx redox enzyme functionalizes DWCNT to DWCNT that acts as an excellent signal converter.
次に、ＤＷＣＮＴネットワークに固定されたＧＯｘの安定性を調べた。図４は、２．５ｍＭグルコースに対するＤＷＣＮＴ／ＧＯｘ電極の電流応答を連続的に約１５分間記録したものである。図示されるように、この時間における応答電流の減少は観察されていない。ＤＷＣＮＴ／ＧＯｘ電極の長時間の安定性を見るため、ＤＷＣＮＴ／ＧＯｘ電極を４℃、乾燥状態で一ヶ月保存した場合には、応答電流は４０％減少していた。この数字は、報告されたカーボンナノチューブ／ポリマー複合物／ＧＯｘ電極における数字（J. Wang, Electroanalysis13, 983 (2001); A. Salimi et al, Anal. Biochem. 333, 49 (2004)）よりも少し低いが、この相違の理由は現在のところ不明である。 Next, the stability of GOx fixed to the DWCNT network was examined. FIG. 4 is a continuous recording of the current response of the DWCNT / GOx electrode to 2.5 mM glucose for about 15 minutes. As shown, no decrease in response current at this time has been observed. In order to see the long-term stability of the DWCNT / GOx electrode, when the DWCNT / GOx electrode was stored at 4 ° C. in a dry state for one month, the response current was reduced by 40%. This figure is slightly less than that reported for carbon nanotube / polymer composite / GOx electrodes (J. Wang, Electroanalysis 13, 983 (2001); A. Salimi et al, Anal. Biochem. 333, 49 (2004)). Although low, the reason for this difference is currently unknown.
図５は、グルコースの添加（濃度範囲は１．４〜１８．５ｍＭ）に対するグラファイト／ＧＯｘ電極（ａ）及びＤＷＣＮＴ／ＧＯｘ電極（ｂ）の電流応答を示すものである。濃度と応答の間の関係は、酵素系についての典型的ミカエリスーメンテン曲線である。見かけのミカエリスーメンテン定数（KM app）は、ラインウィーバー−バーク・プロットから１４．１４ｍＭと計算された。この値は、文献値（H. Zhou et al, Sens. Actuators B 101, 224 (2004)）と整合する。 FIG. 5 shows the current response of the graphite / GOx electrode (a) and the DWCNT / GOx electrode (b) to the addition of glucose (concentration range is 1.4 to 18.5 mM). The relationship between concentration and response is a typical Michaelis-Menten curve for enzyme systems. The apparent Michaelis-Menten constant (K M app ) was calculated to be 14.14 mM from the Lineweaver-Burk plot. This value is consistent with literature values (H. Zhou et al, Sens. Actuators B 101, 224 (2004)).
Claims (1)
(i) カーボンナノチューブを濃硝酸中で、７０〜９０℃の温度条件下に酸化する；
(ii) 得られた酸化型カーボンナノチューブを精製水に分散させ、０．１〜１．０ｍｇ／ｍＬの濃度にする；
(iii) レドックスたんぱく質を０．０４〜０．４ｍｇ／ｍＬの濃度に加え、混合する；
(iv) 得られた生体反応性カーボンナノチューブを分離する。
A process for producing bioreactive carbon nanotubes that are functionalized by binding a redox protein with a non-covalent bond without using a polymer binder, the process comprising the following steps.
(i) oxidizing carbon nanotubes in concentrated nitric acid at a temperature of 70-90 ° C ;
(ii) The obtained oxidized carbon nanotube is dispersed in purified water to a concentration of 0.1 to 1.0 mg / mL ;
(iii) Add redox protein to a concentration of 0.04-0.4 mg / mL and mix;
(iv) The obtained bioreactive carbon nanotube is separated.
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