JP2008032393A - Detection element having nano-gap electrode and detection method using it - Google Patents
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Abstract
Description
本発明は、ナノギャップ電極を有する検出素子及びこれを用いたチミンの検出方法に関する。詳しくは、分子認識結果を直接電流変化として捉える事によって化学物質を検出する検出素子及び検出方法である。 The present invention relates to a detection element having a nanogap electrode and a thymine detection method using the same. Specifically, the present invention relates to a detection element and a detection method for detecting a chemical substance by directly capturing a molecular recognition result as a current change.
化学物質のセンシング技術は従来の分析化学の延長線を超えて急速に発達している。
特に、DNAなどの特定の物質を高感度化には遺伝子工学や生命化学分野では重要であり、１ｆｍｏｌ（１×１０−１５モル）のペプチドが検出できる質量分析装置や、一分子からの蛍光検出も可能な単分子蛍光計測装置が市販されている。（例えばScientific Analysis Instruments社製 MALDITof/Tof-MS/MS質量分析器、オリンパス社製単分子蛍光計測装置MF２０型（商品名））。
このほかに、核酸塩基の検出については、塩基と特異的に結合する機能性分子のスペクトル変化、電気化学的変化として捉える提案が学術雑誌に報告されている。しかし、上記の従来技術では、大がかりな装置や高価な装置を必要とし、目的の塩基類の検出には多くの時間を要し、迅速、高感度、簡便な目的物質のセンシングには適していなかった。
Chemical sensing technology is rapidly developing beyond the extension of conventional analytical chemistry.
In particular, it is important in the field of genetic engineering and biochemistry to increase the sensitivity of specific substances such as DNA, and is a mass spectrometer capable of detecting 1 fmol (1 × 10-15 mol) peptide, or fluorescence detection from a single molecule. Single-molecule fluorescence measuring devices that can be used are commercially available. (For example, MALDITof / Tof-MS / MS mass spectrometer manufactured by Scientific Analysis Instruments, single molecule fluorescence measurement device MF20 (trade name) manufactured by Olympus).
In addition to this, with regard to detection of nucleobases, proposals have been reported in academic journals that are regarded as spectral changes and electrochemical changes of functional molecules that specifically bind to bases. However, the above-described conventional technology requires a large-scale device or an expensive device, requires a lot of time to detect the target bases, and is not suitable for rapid, high-sensitivity and simple sensing of the target substance. It was.
また、ナノギヤップ電極問にアフィニティーセンサーを配置し、これと相補的に結合可能なパートナー化合物を導電性微粒子上に修飾した仲介粒子が特異的に電極間に結合配置されることにより、導電性変化が起こることを利用する新しいセンサーも提案されている（特許文献１参照）。 In addition, an affinity sensor is placed on the nanogap electrode, and intermediary particles in which a partner compound that can be complementarily bound to the nanogap electrode is modified on the conductive fine particles are specifically bound and arranged between the electrodes. A new sensor utilizing what happens has also been proposed (see Patent Document 1).
しかし、上記導電性微粒子を仲介して目的物を検出する方法は、比較的簡便な方法に近づいているが、ホスト分子あるいはゲスト分子のいずれかを金等の導電性微粒子に前もって結合して置く必要があり、迅速な方法とは言い難かった。 However, the method for detecting the target substance through the conductive fine particles is approaching a relatively simple method, but either the host molecule or the guest molecule is previously bonded to the conductive fine particles such as gold. It was necessary and it was hard to say that it was a quick method.
さらに、数ナノメートルサイズの物質を直接検出する方法としては、スキャニング型のトンネル電流顕微鏡がある。このタイプの顕微鏡は高感度、高解像度で分子を観測できるが、単結晶や単分子膜等の規則性構造のある表面上にある物質の検出に限定されることや探針をミクロンメートル単位の面積範囲をスキャニングし無ければならない等、迅速な方法と言い難かった。 Further, as a method for directly detecting a substance having a size of several nanometers, there is a scanning tunneling current microscope. Although this type of microscope can observe molecules with high sensitivity and high resolution, it is limited to the detection of substances on the surface with a regular structure such as a single crystal or a monomolecular film, and the probe is in the order of micrometers. It was difficult to say that it was a quick method because the area range had to be scanned.
本発明者らは１０ナノメートル以下のナノギャップ電極間に核酸塩基化合物と水素結合可能な部位を持つ機能性センサー分子類（特許文献２参照）をナノギャップ電極に架橋した素子を用いて形成結果を直接電気伝導度の変化として捉えることのできることを見いだしている。しかしながら、数ナノメートルから１０ナノメートル程度の間隔を持つ電極に架橋できる３ナノメートルから１０ナノメートル程度センサー分子の合成は多段階反応を必要とし、多大な労力を必要とした。
本発明は、上記のような問題点を解消するため、合成が簡単な２ナノメートル程度のセンサー化合物を用いて極微量の化学物質、特にDNAの構成物質である核酸化合物との水素結合相互作用を直接電流変化として捉えることを可能とするナノギャップ電極を有する検出素子及び検出方法を提供することを目的とする。 In order to solve the above problems, the present invention uses a sensor compound of about 2 nanometers that is easy to synthesize, and uses hydrogen bonding interaction with a very small amount of chemical substances, particularly nucleic acid compounds that are constituents of DNA. An object of the present invention is to provide a detection element and a detection method having a nanogap electrode that can be directly regarded as a current change.
本発明者らは、上記課題を解決するために鋭意検討を重ねた結果、１０ナノメートル以下のギャップを持つ電極に２ナノメートル以下のセンサー分子を非架橋状態で表面修飾し、検出対象物質が結合した時、非結合状態で流れていたトンネル電流が増加する現象を見いだし、本発明に至ったものである。すなわち、本発明は、
検出対象物の化学物質と、結合する部位を有するセンサー分子を、ナノギャップ電極表面に結合させた化学物質の検出素子であって、センサー分子はナノギャップ電極表面に固定化するための部分構造及び検出対象物の化学物質と結合するレセプター部分構造からなり、かつ、分子長が２ナノメートル以下であって、前記電極のギャップ巾が最大１０ナノメートル以下であることを特徴とする化学物質の検出素子である。
また、本発明においては、センサー分子を、式（１）
さらに、本発明においては、センサー分子を、式（２）
A detection element of a chemical substance in which a chemical substance to be detected and a sensor molecule having a binding site are bonded to the surface of the nanogap electrode, the sensor molecule being immobilized on the nanogap electrode surface, Detection of a chemical substance comprising a receptor partial structure that binds to a chemical substance to be detected, a molecular length of 2 nanometers or less, and a gap width of the electrodes of 10 nanometers or less It is an element.
In the present invention, the sensor molecule is represented by the formula (1)
Furthermore, in the present invention, the sensor molecule is represented by the formula (2)
また、本発明においては、ナノギャップ電極を、金、銀、白金、パラジウム、ニッケルから選ばれる金属の一つとすることができる。
またさらに、本発明は、前記電極の検出対象物質をチミンとすることができる。
さらに、本発明は、検出対象物の化学物質と、結合する部位を有するセンサー分子を、ナノギャップ電極表面に結合させた化学物質の検出素子であって、センサー分子はナノギャップ電極表面に固定化するための部分構造及び検出対象物の化学物質と結合するレセプター部分構造からなり、前記電極のギャップ巾が最大１０ナノメートル以下であることを特徴とする化学物質の検出素子を用いて、検出対象物の化学物質を捕捉し、捕捉前と捕捉後のギャップ電極間の電気抵抗値変化として検出する化学物質の検出方法である。
In the present invention, the nanogap electrode can be one of metals selected from gold, silver, platinum, palladium, and nickel.
Furthermore, in the present invention, the detection target substance of the electrode can be thymine.
Furthermore, the present invention provides a chemical substance detection element in which a chemical substance to be detected and a sensor molecule having a binding site are bonded to the nanogap electrode surface, and the sensor molecule is immobilized on the nanogap electrode surface. A detection target of a chemical substance comprising a partial structure for the detection and a receptor partial structure that binds to a chemical substance of the detection target, wherein the gap width of the electrode is 10 nanometers or less at maximum This is a method for detecting a chemical substance, in which a chemical substance is captured and detected as a change in electric resistance between the gap electrodes before and after capture.
本発明の新規なセンサー素子構造は、ギャップ電極間の電気抵抗値変化を直接検出することにより、検出対象物の化学物質をダイレクトに検知することができる。代表的な検出対象物の化学物質はチミンであり、核酸塩基の１つであるチミン化合物と特異的に水素結合し、かつギャップ電極に容易に合成できるセンサー化合物を非架橋構造で結合させる事により、トンネル電流型顕微鏡では必須なスキャニングを省くことにより、従来の検出法よりも迅速かつ鋭敏に標的物質を検出しうる核酸塩基センサーを提供できる。 The novel sensor element structure of the present invention can directly detect a chemical substance to be detected by directly detecting a change in electrical resistance value between gap electrodes. A typical chemical substance to be detected is thymine, which is specifically bonded to a thymine compound, which is one of the nucleobases, and bonded to the gap electrode in a non-crosslinked structure. By omitting the essential scanning in the tunneling current microscope, it is possible to provide a nucleobase sensor capable of detecting a target substance more rapidly and sensitively than the conventional detection method.
図１は本発明の化学物質センサーの原理図である。
図１−ａは検出前の図である。Ａは導電性の電極である。その間隔はトンネル電流が測定できる程度の間隔を持つ。電極表面には導電性を持つ分子（Ｂ）が化学的に結合している。センサー分子の末端には検出対象と特異的に結合可能なレセプター部位を持つ（Ｃ）。
図１−ｂは検出対象物質（Ｄ）が図１−ａのセンサー電極に結合した状態を示している。図１−ａの電極はナノメートルスケールの間隔を持つことから、ＳＴＭの原理に使用されているトンネル電流が観測されている。トンネル電流量は測定物質間の距離の関数であり、短いほど多く流れる。図１−ｂの状態、すなわち検出対象化合物（Ｄ）がセンサー部位に結合するとセンサー部位を含めた電極間の距離が短くなる。その結果、図１−ａの状態より図１−ｂの状態がより多くのトンネル電流が流れることになり、特定の化学物質（Ｄ）を検出できる事になる。
このセンサー構造を使用すれば、今までの探針でトンネル電流の変化の分布図を作成する必要が無く、高感度、迅速に化学物質を検出できることが可能である。
FIG. 1 is a principle diagram of a chemical substance sensor of the present invention.
FIG. 1A is a diagram before detection. A is a conductive electrode. The interval is such that the tunnel current can be measured. A molecule (B) having conductivity is chemically bonded to the electrode surface. The end of the sensor molecule has a receptor site that can specifically bind to the detection target (C).
FIG. 1B shows a state in which the detection target substance (D) is bound to the sensor electrode of FIG. Since the electrode of FIG. 1-a has a nanometer-scale interval, a tunnel current used in the STM principle is observed. The amount of tunnel current is a function of the distance between the measured substances, and the shorter the amount, the more current flows. When the detection target compound (D) binds to the sensor site in the state of FIG. 1-b, the distance between the electrodes including the sensor site is shortened. As a result, more tunnel current flows in the state of FIG. 1-b than in the state of FIG. 1-a, and a specific chemical substance (D) can be detected.
If this sensor structure is used, it is not necessary to create a distribution map of changes in tunnel current with a conventional probe, and it is possible to detect chemical substances with high sensitivity and speed.
ギャップ電極は分子サイズオーダーのギャップを持つ電極であり、センサー化合物との共有結合形成を可能とする材料ならば使用可能であるが、金、銀、白金、パラジウム、ニッケルから選ばれる金属の一つが良く、金電極が最も望ましい。すなわち、本発明者らにより既に分子サイズのギャップを持つ電極構造の製造方法および該方法により製造された電極構造を有する分子素子に関する特許文献（特許文献３〜５参照）のなかで述べられたナノギャップ電極構造が望ましい。 A gap electrode is an electrode having a gap of molecular size order, and any material that can form a covalent bond with a sensor compound can be used, but one of metals selected from gold, silver, platinum, palladium, and nickel is used. Good, gold electrodes are most desirable. That is, the nanometers described in the patent documents (see Patent Documents 3 to 5) related to the manufacturing method of the electrode structure having a molecular size gap and the molecular element having the electrode structure manufactured by the method by the present inventors. A gap electrode structure is desirable.
使用する電極のギャップ巾は、使用するセンサー分子と検出対象とする化学物質の分子サイズで最適巾が決定されるが、架橋型のセンサー系とは異なり、トンネル電流が測定可能な範囲に近接すればよいのでその融通性は高い。実際に使用される巾としてはギャップ電極の作成歩留まりおよびセンサー分子の合成の容易さから、最大１０ｎｍ以下の金製のギャップ電極が望ましい。 The gap width of the electrode to be used is determined by the molecular size of the sensor molecule to be used and the chemical substance to be detected, but unlike the cross-linked sensor system, it is close to the range where the tunnel current can be measured. The flexibility is high. As a width actually used, a gold gap electrode having a maximum of 10 nm or less is desirable from the viewpoint of the yield of gap electrodes and the ease of synthesis of sensor molecules.
前記ナノギャップ電極に化学結合可能なセンサー分子の構造は式（１）又は式（２）で表される。
式中の（アセチル基で保護されたチオール基（アセチルチオ基）は取扱中に酸化によりジスルフィド誘導体に変化を防ぐためにアセチル基で保護してある。この保護基は使用直前もしくはin situで脱保護して用いることができる。式（１）又は式（２）のフェニレンエチニレンユニットは電極に固定化した時、導電性をできるだけ高める、剛直な鎖であることから電極間の距離をせばめて導電性の向上に寄与できることから選ばれた。さらに、ここで使用されている２，６−ジアミドピリジン誘導体はチミン類の人工レセプターとして広く利用されている化合物である。なお、式（１）は公知の化合物であり、式（２）の化合物合成法は発明者らが7th International Synposium on Polymers and Advanced Technorojies(2003, Florida, USA) にて発表済みであり、既知化合物である。 In the formula, the thiol group protected with an acetyl group (acetylthio group) is protected with an acetyl group to prevent the disulfide derivative from being changed by oxidation during handling. This protective group is deprotected immediately before use or in situ. The phenylene ethynylene unit of formula (1) or formula (2) is a rigid chain that increases the conductivity as much as possible when it is fixed to the electrode. In addition, the 2,6-diamidepyridine derivative used here is a compound that is widely used as an artificial receptor for thymines. The compound synthesis method of formula (2) has been published by the inventors at 7th International Synposium on Polymers and Advanced Technorojies (2003, Florida, USA). It is.
以下に本発明を実施例に基づき詳細に説明するが、本発明はこれらの実施例に限定されるものでない。
（式（２）の化合物のギャップ電極への修飾およびブチルチミンによる導電性変化の測定）
＜１＞式（２）の化合物およびピロリジンを塩化メチレン溶液（０．１ｍＭ／Ｌ）を調製し、この溶液にナノギャップ電極を室温、２４時間、浸積した。修飾反応後、電極はエタノールにて良く洗浄して電気測定に使用した。
＜２＞この修飾済電極を１．０ｍＭ／ＬのＮ−ブチルチミン溶液に１時間、浸積し、
＜３＞余分のブチルチミンを除く目的でエタノール中、１時間浸積した。
＜２＞と＜３＞の操作を繰り返し、各々の段階でＩ−Ｖ曲線を測定し、Ｖ＝０付近での抵抗値を算出した。
図２に得られた結果の一例を示す。図２のＢｕＴ＋およびＢｕＴ−は、それぞれブルチミンを結合させた状態およびブチルチミンを洗浄した状態を示している。
図２から、ブチルチミンを結合させるとギャップ電極の抵抗が低くなり、ブチルチミンを取ると抵抗値が上昇した。すなわち、検出対象のブチルチミンの存在に対応した電気的応答が得られた。
Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
(Modification of the compound of formula (2) to the gap electrode and measurement of conductivity change by butylthymine)
<1> A methylene chloride solution (0.1 mM / L) of the compound of formula (2) and pyrrolidine was prepared, and a nanogap electrode was immersed in this solution at room temperature for 24 hours. After the modification reaction, the electrode was thoroughly washed with ethanol and used for electrical measurement.
<2> The modified electrode is immersed in a 1.0 mM / L N-butylthymine solution for 1 hour,
<3> It was immersed in ethanol for 1 hour in order to remove excess butylthymine.
The operations of <2> and <3> were repeated, an IV curve was measured at each stage, and a resistance value near V = 0 was calculated.
An example of the results obtained is shown in FIG. In FIG. 2, BuT + and BuT− indicate a state in which bruthymine is bound and a state in which butylthymine is washed, respectively.
From FIG. 2, when butylthymine was bonded, the resistance of the gap electrode was lowered, and when butylthymine was taken, the resistance value was increased. That is, an electrical response corresponding to the presence of the detection target butylthymine was obtained.
（アミノピリジンを有する分子ワイヤ（新規化合物I）の製造）
市販の２−アミノ−５−ヨードピリジン（０．１００ｇ）とアセチル基で保護したフェニレンエチニレン化合物（０．１３８ｇ）、
を２ｍＬのトルエンに溶解後、触媒としてテトラキス（トリフェニルフォスフィン）パラジウム（０．０２６３ｇ）およびトリフェニルフォスフィン（２３．８ｍｇ）ヨウ化銅（８．６６ｍｇ）、２ｍＬのトリエチルアミンを加え、窒素下で３日、４０℃加熱撹拌する。反応終了後、シリカゲル−塩化メチレン−酢酸エチルのカラムクロマトグラフィーにて精製を行い、０．１１７ｇ（収率７０．３％）で化合物１を得た。
構造確認はＥＳＩ質量分析（アセトニトリル溶液）および１Ｈ−ＮＭＲ測定より行った。
ＥＳＩ質量分析： 計算値 ｍ／ｚ ＝ ３６８．１０［Ｍ］＋
実測値 ｍ／ｚ ＝４１０．２［Ｍ＋ＣＨ３ＣＮ＋Ｈ］＋
１Ｈ−ＮＭＲ（重クロロホルム） δ：8.28(d, J=1.7Hz, 1H), 7.57(dd, J=8.5Hzと1.7Hz, 1H) 7.55(d, J=8.4Hz, 2H), 7.51-7.46(m, 4H), 7.40(d, J=8.4Hz, 2H), 6.46(d, J=8.5Hz, 1H), 4.61(br, 2H), 2.44(s, 3H)
(Production of molecular wire with aminopyridine (novel compound I))
Commercially available 2-amino-5-iodopyridine (0.100 g) and an acetyl-protected phenyleneethynylene compound (0.138 g),
Was dissolved in 2 mL of toluene, tetrakis (triphenylphosphine) palladium (0.0263 g) and triphenylphosphine (23.8 mg) copper iodide (8.66 mg) were added as catalysts, and 2 mL of triethylamine was added under nitrogen. And stir at 40 ° C. for 3 days. After completion of the reaction, purification was performed by silica gel-methylene chloride-ethyl acetate column chromatography to obtain Compound 1 at 0.117 g (yield 70.3%).
The structure was confirmed by ESI mass spectrometry (acetonitrile solution) and 1 H-NMR measurement.
ESI mass spectrometry: Calculated value m / z = 368.10 [M] +
Actual value m / z = 410.2 [M + CH 3 CN + H] +
1 H-NMR (deuterated chloroform) δ: 8.28 (d, J = 1.7 Hz, 1H), 7.57 (dd, J = 8.5 Hz and 1.7 Hz, 1H) 7.55 (d, J = 8.4 Hz, 2H), 7.51- 7.46 (m, 4H), 7.40 (d, J = 8.4Hz, 2H), 6.46 (d, J = 8.5Hz, 1H), 4.61 (br, 2H), 2.44 (s, 3H)
新規化合物Iの導電性測定結果
＜１＞新規化合物Iおよびピロリジンの塩化メチレン溶液（０．１ｍＭ／Ｌ）を調製し、この溶液にナノギャップ電極を室温、２４時間、浸積した。修飾反応後、電極はエタノールにて良く洗浄して電気測定に使用した。
＜２＞この修飾済電極を１．０ｍＭ／ＬのＮ−ブチルチミン溶液に１時間、浸積後、余分のブチルチミンを除く目的でジクロロエタンにて洗浄した。
＜３＞Ｎ−ブチルチミンを脱離させる目的で、エタノール中、１時間浸積した。
＜２＞と＜３＞の操作を繰り返し、各々の段階でＩ−Ｖ曲線を測定し、Ｖ＝０付近での抵抗値を算出した。図３に得られた結果の一例を示す。図３の＋ＢｕＴおよび−ＢｕＴは、それぞれＮ−ブルチミンを結合させた状態およびＮ−ブチルチミンを洗浄した状態を示している。
図３から、Ｎ−ブチルチミンを結合させるとギャップ電極の抵抗が低くなり、Ｎ−ブチルチミンを取ると抵抗値が上昇した。すなわち、検出対象のブチルチミンの存在に対応した電気的応答が得られた。
Measurement Results of Conductivity of Novel Compound I <1> A methylene chloride solution (0.1 mM / L) of novel compound I and pyrrolidine was prepared, and a nanogap electrode was immersed in this solution at room temperature for 24 hours. After the modification reaction, the electrode was thoroughly washed with ethanol and used for electrical measurement.
<2> This modified electrode was immersed in a 1.0 mM / L N-butylthymine solution for 1 hour and then washed with dichloroethane for the purpose of removing excess butylthymine.
<3> It was immersed in ethanol for 1 hour for the purpose of eliminating N-butylthymine.
The operations of <2> and <3> were repeated, an IV curve was measured at each stage, and a resistance value near V = 0 was calculated. An example of the results obtained is shown in FIG. In FIG. 3, + BuT and -BuT indicate a state in which N-burtimine is bound and a state in which N-butylthymine is washed, respectively.
From FIG. 3, when N-butylthymine was bonded, the resistance of the gap electrode was lowered, and when N-butylthymine was taken, the resistance value was increased. That is, an electrical response corresponding to the presence of the detection target butylthymine was obtained.
（チミンと等価なウラシル基を有する分子ワイヤ（新規化合物II）の製造）
市販の５−ブロモウラシル（０．３１４ｇ）とアセチル基で保護したフェニレンエチニレン化合物（０．５００ｇ）、
構造確認はＥＳＩ質量分析（アセトニトリル溶液）および１Ｈ−ＮＭＲ測定より行った。
ＥＳＩ質量分析： 計算値 ｍ／ｚ ＝３８４．０８［Ｍ］＋
実測値 ｍ／ｚ ＝４２７．０［Ｍ＋Ｎａ＋Ｈ２Ｏ］＋
１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ６） δ：11.40(br, 2Ｈ), 7.95(s, 1H), 7.65(d, J=8.6Hz, 2H), 7.60(d, J= 8.6, 2H), 7.51-7.48(m, 4H), 2.47(s, 3H)
(Production of molecular wire having a uracil group equivalent to thymine (new compound II))
Commercially available 5-bromouracil (0.314 g) and an acetyl-protected phenyleneethynylene compound (0.500 g),
The structure was confirmed by ESI mass spectrometry (acetonitrile solution) and 1 H-NMR measurement.
ESI mass spectrometry: calculated m / z = 384.08 [M] +
Found m / z = 427.0 [M + Na + H 2 O] +
1 H-NMR (DMSO-d 6 ) δ: 11.40 (br, 2H), 7.95 (s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.60 (d, J = 8.6, 2H), 7.51 -7.48 (m, 4H), 2.47 (s, 3H)
新規化合物IIの導電性測定結果
＜１＞新規化合物IIおよびピロリジンのジクロロエタン−エタノール（１：１）混合溶液（０．１ｍＭ／Ｌ）を調製し、この溶液にナノギャップ電極を室温、２日間、浸積した。修飾反応後、電極はエタノールにて良く洗浄して電気測定に使用した。
＜２＞この修飾済電極を０．５ｍＭ／Ｌのアデニン溶液に１時間、浸積後、余分のアデニンを除く目的でジクロロエタンにて洗浄した。
＜３＞アデニンを脱離させる目的で、エタノール中、１時間浸積した。
＜２＞と＜３＞の操作を繰り返し、各々の段階でＩ−Ｖ曲線を測定し、Ｖ＝０付近での抵抗値を算出した。図４に得られた結果の一例を示す。図４の＋adenineおよび−adenineは、それぞれアデニンを結合させた状態およびアデニンを洗浄した状態を示している。
図４から、アデニンを結合させるとギャップ電極の抵抗が低くなり、アデニンを取ると抵抗値が上昇した。すなわち、検出対象のアデニンの存在に対応した電気的応答が得られた。
Conductivity measurement results of novel compound II <1> A dichloroethane-ethanol (1: 1) mixed solution (0.1 mM / L) of novel compound II and pyrrolidine was prepared, and a nanogap electrode was added to this solution at room temperature for 2 days. Soaked. After the modification reaction, the electrode was thoroughly washed with ethanol and used for electrical measurement.
<2> This modified electrode was immersed in a 0.5 mM / L adenine solution for 1 hour, and then washed with dichloroethane for the purpose of removing excess adenine.
<3> For the purpose of desorbing adenine, it was immersed in ethanol for 1 hour.
The operations of <2> and <3> were repeated, an IV curve was measured at each stage, and a resistance value near V = 0 was calculated. An example of the results obtained is shown in FIG. + Adenine and −adenine in FIG. 4 indicate a state where adenine is bound and a state where adenine is washed, respectively.
From FIG. 4, when adenine was bonded, the resistance of the gap electrode was lowered, and when adenine was removed, the resistance value was increased. That is, an electrical response corresponding to the presence of adenine to be detected was obtained.
シトシンを有する分子ワイヤ（新規化合物III）の製造
新規化合物IIIは次の行程に従い製造した。各工程の実施例を示す。
（行程１ 中間体（３ａ）の製造）
3-ヨード安息香酸クロリド
(５．００ｇ)、シトシン (０．６９５ｇ) を脱水ピリジン（１５ｍＬ）中、室温・窒素下で、１時間攪拌した。同量のシトシンを追加した後、5日間反応を続けた。氷冷下、１N-HCl(５０ｍＬ)をゆっくり加え1時間攪拌、生じた固体を濾過し５０ｍＬの熱エタノール-水（50/50＝v/v）で洗浄した。固体を８０ｍＬの熱エタノール-水に分散・攪拌・濾過の過程を数回繰り返した後、真空乾燥した（６０℃、１２時間）。収量３．８３ｇ(収率約９０%)。構造確認はＥＳＩ質量分析（エタノール溶液）および１Ｈ−ＮＭＲ測定より行った。
ＥＳＩ質量分析： 計算値 ｍ／ｚ ＝３４０．９７［Ｍ］＋
実測値 ｍ／ｚ ＝３６４．０６［Ｍ＋Ｎａ］＋
１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ６） δ：〜11.5(br,, 2H), 8.35(s, 1H), 7.99(d, 8.4Hz, 1H), 7.97(d, J=8Hz, 1H), 7.88(d, J=6.8Hz, 1H), 7.31(dd, J=7.6Hz, 8Hz, 1H), 7.12(br, 1H),
(Process 1 Production of intermediate (3a))
3-iodobenzoic acid chloride
(5.00 g) and cytosine (0.695 g) were stirred in dehydrated pyridine (15 mL) at room temperature under nitrogen for 1 hour. The reaction was continued for 5 days after the same amount of cytosine was added. Under ice-cooling, 1N-HCl (50 mL) was slowly added and stirred for 1 hour. The resulting solid was filtered and washed with 50 mL of hot ethanol-water (50/50 = v / v). The process of dispersing, stirring and filtering the solid in 80 mL of hot ethanol-water was repeated several times, and then dried in vacuo (60 ° C., 12 hours). Yield 3.83 g (about 90% yield). The structure was confirmed by ESI mass spectrometry (ethanol solution) and 1 H-NMR measurement.
ESI mass spectrometry: calculated m / z = 340.97 [M] +
Actual value m / z = 364.06 [M + Na] +
1 H-NMR (DMSO-d 6 ) δ: ˜11.5 (br ,, 2H), 8.35 (s, 1H), 7.99 (d, 8.4 Hz, 1H), 7.97 (d, J = 8 Hz, 1H), 7.88 (d, J = 6.8Hz, 1H), 7.31 (dd, J = 7.6Hz, 8Hz, 1H), 7.12 (br, 1H),
（行程２ 中間体（３ｂ）の製造）
３ａ（３．４０ｇ）、トリメチルシリルアセチレン(１０ｍＬ)、テトラキストリフェニルホスフィンパラジウム(0)（０．６ｇ）、トリフェニルホスフィン(０．２５ｇ)、ジイソプロピルエチルアミン（１０ｍＬ）を５０ｍＬのN,N-ジメチルアセタミド中で混合し、窒素下で攪拌しながらヨウ化銅(I)(１g)を投入した（反応温度６０℃）。反応液が赤茶透明状態を経て、黄色固体が生成したところで２０ｍＬのN,N-ジメチルアセタミドを加え、温度を５０℃としてさらに５時間反応させた。生じた固体を減圧濾過して集め、５０ｍＬのクロロホルムで洗浄・乾燥し、白色固体１．４８ｇを得た。先の濾液を減圧蒸留により濃縮し、１５０ｍＬのクロロホルムを加えることにより固体を析出した。固体を濾別、クロロホルム５０ｍＬで洗浄・乾燥し、目的物の３ｂ、０．９４ｇを得た。合計収量 ２．４２ｇ（収率約７８％）。構造確認はＥＳＩ質量分析（エタノール溶液）および１Ｈ−ＮＭＲ測定より行った。
ＥＳＩ質量分析： 計算値 ｍ／ｚ ＝３１１．１１［Ｍ］＋
実測値 ｍ／ｚ ＝３３４．９４［Ｍ＋Ｎａ］＋
１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ６） δ：〜11.5(br, 2H), 8.09(s, , 1H), 8.00(d, 8.0Hz, 1H), 7.88(d, J=6.8Hz, 1H), 7.67(d, J=7.6Hz, 1H), 7.51(dd, J=7.6Hz, 8.0Hz, 1H), 7.10(br, 1H), 0.25(s, 9H)
(
3a (3.40 g), trimethylsilylacetylene (10 mL), tetrakistriphenylphosphine palladium (0) (0.6 g), triphenylphosphine (0.25 g), diisopropylethylamine (10 mL) were added to 50 mL of N, N-dimethylamine. The mixture was mixed in cetamide, and copper (I) iodide (1 g) was added while stirring under nitrogen (reaction temperature 60 ° C.). When the reaction liquid passed through a red brown transparent state and a yellow solid was formed, 20 mL of N, N-dimethylacetamide was added, and the temperature was set to 50 ° C., and the reaction was further continued for 5 hours. The resulting solid was collected by filtration under reduced pressure, washed and dried with 50 mL of chloroform, and 1.48 g of a white solid was obtained. The previous filtrate was concentrated by distillation under reduced pressure, and 150 mL of chloroform was added to precipitate a solid. The solid was separated by filtration, washed with 50 mL of chloroform and dried to obtain the target product 3b, 0.94 g. Total yield 2.42 g (yield approximately 78%). The structure was confirmed by ESI mass spectrometry (ethanol solution) and 1 H-NMR measurement.
ESI mass spectrometry: calculated m / z = 311.11 [M] +
Actual value m / z = 334.94 [M + Na] +
1 H-NMR (DMSO-d 6 ) δ: ˜11.5 (br, 2H), 8.09 (s,, 1H), 8.00 (d, 8.0 Hz, 1H), 7.88 (d, J = 6.8 Hz, 1H), 7.67 (d, J = 7.6Hz, 1H), 7.51 (dd, J = 7.6Hz, 8.0Hz, 1H), 7.10 (br, 1H), 0.25 (s, 9H)
（行程３ 中間体（３ｃ）の製造）
３ｂ（１．４７ｇ）を乾燥テトラヒドロフラン１５０ｍＬに分散し、バス温度を−１０℃に下げ、穏やかに攪拌しながら４．５ｍＬの１Mフッ化テトラ-n-ブチルアンモニウムテトラヒドロン溶液を、シリンジで注入した。温度を保ちながら６時間反応した後、水１．０ｍＬ（過剰量）を加えさらに３時間攪拌を続けた。生じた白色固体を濾別、３０ｍＬの冷メタノールで洗浄、真空下乾燥し、０．３２３ｇの目的物を得た。先の濾液を３０ｍＬまで濃縮、水３０ｍＬを加えた後、メタノール分を留去することによりさらに固体を得た。固体を濾別、冷水で洗浄後、真空乾燥し目的物０．６７ｇを得た。合計収量０．９９３ｇ （収率約８３％）。構造確認はＥＳＩ質量分析（エタノール溶液）および１Ｈ−ＮＭＲ測定より行った。
ＥＳＩ質量分析： 計算値 ｍ／ｚ ＝２３９．０７［Ｍ］＋
実測値 ｍ／ｚ ＝２４２．３０［Ｍ＋Ｈ］＋
１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ６） δ：〜11.5(br, 2H), 8.09(s, 1H), 8.01(d, 8.0Hz, 1H), 7.88(d, 6.8Hz, 1H), 7.70(d, J=7.6Hz, 1H), 7.52(dd, J=7.6Hz, 8.0Hz, 1H), 4.32(s, 1H),
(Process 3 Production of intermediate (3c))
3b (1.47 g) was dispersed in 150 mL of dry tetrahydrofuran, the bath temperature was lowered to −10 ° C., and 4.5 mL of 1M tetra-n-butylammonium fluoride tetrahydrone solution was injected with a syringe with gentle stirring. . After reacting for 6 hours while maintaining the temperature, 1.0 mL (excess amount) of water was added and stirring was continued for another 3 hours. The resulting white solid was filtered off, washed with 30 mL of cold methanol, and dried under vacuum to obtain 0.323 g of the desired product. The previous filtrate was concentrated to 30 mL, 30 mL of water was added, and then methanol was distilled off to obtain a solid. The solid was separated by filtration, washed with cold water, and then vacuum-dried to obtain 0.67 g of the desired product. Total yield 0.993 g (yield about 83%). The structure was confirmed by ESI mass spectrometry (ethanol solution) and 1 H-NMR measurement.
ESI mass spectrometry: calculated value m / z = 239.07 [M] +
Actual measurement value m / z = 242.30 [M + H] +
1 H-NMR (DMSO-d 6 ) δ: ˜11.5 (br, 2H), 8.09 (s, 1H), 8.01 (d, 8.0 Hz, 1H), 7.88 (d, 6.8 Hz, 1H), 7.70 (d , J = 7.6Hz, 1H), 7.52 (dd, J = 7.6Hz, 8.0Hz, 1H), 4.32 (s, 1H),
（行程４ 新規化合物IIIの製造）
３ｃ（０．９８９ｇ)、パラアセチルチオヨードベンゼン（１．１５ｇ,等モル量）、テトラキストリフェニルホスフィンパラジウム（０．２１２ｇ）、トリフェニルホスフィン（０．０５５ｇ）、ジイソプロピルエチルアミン(４ｍＬ)を、２０ｍＬのN,N-ジメチルアセタミド中で混合し、窒素置換後攪拌しながらヨウ化銅(I)(０．２０２ｇ)を投入し、温度４０℃で２４時間反応を行った。生成した茶色固体を濾過により取り除き、濾液を１５ｍＬまで濃縮、クロロホルム５０ｍＬを投入して新たな固体を析出した。これをクロロホルム洗浄・乾燥し、３ｃの粗製物０．４６ｇを得た。粗製物５０ｍｇをソックスレー抽出器におき、テトラヒドロフラン５０ｍＬで１０時間抽出、冷却後生じた白色固体を濾別、最少量のテトラヒドロフランで洗浄後、真空乾燥した。このソックスレー抽出操作を繰り返すことにより、粗製物０．３２ｇから精製物０．１５２ｇを得た。収率９％以上。構造確認はＥＳＩ質量分析（エタノール溶液）および１Ｈ−ＮＭＲ測定より行った。
ＥＳＩ質量分析： 計算値 ｍ／ｚ ＝３８９．０８［Ｍ］＋
実測値 ｍ／ｚ ＝４１２．４９［Ｍ＋Ｎａ］＋
１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ６） δ：〜11.5(br, 2H), 8.22(s, 1H), 8.04(d, 8.0Hz, 1H), 7.80(d, J=7.6Hz, 1H), 7.68(d, J=8.4Hz, 2H), 7.58(dd, J=7.6Hz, 8.0Hz, 1H), 7.50(d, J=8.8Hz, 1H), 7.10(br, 1H), 2.47(s, 3H)
(Process 4 Production of new compound III)
3 mL (0.989 g), paraacetylthioiodobenzene (1.15 g, equimolar amount), tetrakistriphenylphosphine palladium (0.212 g), triphenylphosphine (0.055 g), diisopropylethylamine (4 mL) in 20 mL In N, N-dimethylacetamide, copper (I) iodide (0.202 g) was added with stirring after nitrogen substitution, and the reaction was performed at a temperature of 40 ° C. for 24 hours. The generated brown solid was removed by filtration, the filtrate was concentrated to 15 mL, and 50 mL of chloroform was added to precipitate a new solid. This was washed with chloroform and dried to obtain 0.46 g of a crude product of 3c. 50 mg of the crude product was placed in a Soxhlet extractor and extracted with 50 mL of tetrahydrofuran for 10 hours. After cooling, the resulting white solid was filtered off, washed with a minimum amount of tetrahydrofuran, and then dried in vacuo. By repeating this Soxhlet extraction operation, 0.152 g of a purified product was obtained from 0.32 g of the crude product. Yield 9% or more. The structure was confirmed by ESI mass spectrometry (ethanol solution) and 1 H-NMR measurement.
ESI mass spectrometry: calculated value m / z = 389.08 [M] +
Actual value m / z = 412.49 [M + Na] +
1 H-NMR (DMSO-d 6 ) δ: ˜11.5 (br, 2H), 8.22 (s, 1H), 8.04 (d, 8.0 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 8.4Hz, 2H), 7.58 (dd, J = 7.6Hz, 8.0Hz, 1H), 7.50 (d, J = 8.8Hz, 1H), 7.10 (br, 1H), 2.47 (s, 3H )
新規化合物IIIの導電性測定結果
＜１＞新規化合物IIIおよびピロリジンのジクロロエタン−ＤＭＳＯ（４：１）混合溶液（０．２ｍＭ／Ｌ）を調製し、この溶液にナノギャップ電極を室温、３時間、浸積した。修飾反応後、電極はエタノールにて良く洗浄して電気測定に使用した。
＜２＞この修飾済電極を０．５ｍＭ／Ｌのアシクロビル溶液に１時間、浸積後、余分のアシクロビルを除く目的でジクロロエタンにて洗浄した。
＜３＞アシクロビルを脱離させる目的で、エタノール中、１時間浸積した。
＜２＞と＜３＞の操作を繰り返し、各々の段階でＩ−Ｖ曲線を測定し、Ｖ＝０付近での抵抗値を算出した。図５に得られた結果の一例を示す。図５の＋アシクロビルおよび−アシクロビルは、それぞれアシクロビルを結合させた状態およびアシクロビルを洗浄した状態を示している。
図５から、アシクロビルを結合させるとギャップ電極の抵抗が低くなり、アシクロビルを取ると抵抗値が上昇した。すなわち、検出対象のアシクロビルの存在に対応した電気的応答が得られた。
Conductivity measurement results of novel compound III <1> A mixed solution (0.2 mM / L) of dichloroethane-DMSO (4: 1) of novel compound III and pyrrolidine was prepared, and a nanogap electrode was added to this solution at room temperature for 3 hours. Soaked. After the modification reaction, the electrode was thoroughly washed with ethanol and used for electrical measurement.
<2> This modified electrode was immersed in a 0.5 mM / L acyclovir solution for 1 hour and then washed with dichloroethane for the purpose of removing excess acyclovir.
<3> For the purpose of eliminating acyclovir, it was immersed in ethanol for 1 hour.
The operations of <2> and <3> were repeated, an IV curve was measured at each stage, and a resistance value near V = 0 was calculated. An example of the results obtained is shown in FIG. + Acyclovir and -Acyclovir in FIG. 5 indicate the state in which acyclovir is bound and the state in which acyclovir is washed, respectively.
From FIG. 5, when acyclovir was bonded, the resistance of the gap electrode was lowered, and when acyclovir was taken, the resistance value was increased. That is, an electrical response corresponding to the presence of acyclovir to be detected was obtained.
（３−アミノシクロヘキサノンを有する分子ワイヤ（新規化合物IＶ）の製造）
４−アミノチオフェノール（１．３５ｇ）とジメドン（１．５０ｇ）の混合物にトルエン（２００ｍＬ）とパラトルエンスルホン酸（０．０４ｇ）を加え、１３０℃、８時間撹拌した。１００℃、減圧下でトルエン（５０ｍＬ）を留去後、ヘキサン（２００ｍＬ）を加え、析出した固体を集めた。これをエタノール（５０ｍＬ）に溶解し、シクロヘキサン（４００ｍＬ）に注ぎ、析出した淡黄色固体、化合物４を１．４０ｇ（収率５３％）を得た。
構造確認はＥＳＩ質量分析（エタノール溶液）および１Ｈ−ＮＭＲ測定より行った。
ＥＳＩ質量分析： 計算値 ｍ／ｚ ＝ ２４７．１０［Ｍ］＋、
実測値 ｍ／ｚ ＝２４８．０［Ｍ＋Ｈ］＋、
３１６．０［Ｍ＋Ｎａ＋ＥｔＯＨ］＋
１Ｈ−ＮＭＲ（ＣＤＣｌ３） δ：ca.11.5(br, 2H), 8.22(s, 1H), 8.04(d, 8.0Hz, 1H), 7.80(d, J=7.6Hz, 1H), 7.68(d, J=8.4Hz, 2H), 7.58(dd, J=7.6Hz, 8.0Hz, 1H), 7.50(d, J=8.8Hz, 2H), 2.47(s, 3H)
(Production of molecular wire having 3-aminocyclohexanone (new compound IV))
Toluene (200 mL) and paratoluenesulfonic acid (0.04 g) were added to a mixture of 4-aminothiophenol (1.35 g) and dimedone (1.50 g), and the mixture was stirred at 130 ° C. for 8 hours. Toluene (50 mL) was distilled off at 100 ° C. under reduced pressure, hexane (200 mL) was added, and the precipitated solid was collected. This was dissolved in ethanol (50 mL) and poured into cyclohexane (400 mL) to obtain 1.40 g (yield 53%) of a precipitated pale yellow solid, compound 4.
The structure was confirmed by ESI mass spectrometry (ethanol solution) and 1 H-NMR measurement.
ESI mass spectrometry: Calculated value m / z = 247.10 [M] + ,
Actual value m / z = 248.0 [M + H] + ,
316.0 [M + Na + EtOH] +
1 H-NMR (CDCl 3 ) δ: ca.11.5 (br, 2H), 8.22 (s, 1H), 8.04 (d, 8.0 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.68 ( d, J = 8.4Hz, 2H), 7.58 (dd, J = 7.6Hz, 8.0Hz, 1H), 7.50 (d, J = 8.8Hz, 2H), 2.47 (s, 3H)
化合物４導電性測定結果
＜１＞化合物４のジクロロエタン溶液（０．２ｍＭ／Ｌ）を調製し、この溶液にナノギャップ電極を室温、１６時間、浸積した。修飾反応後、電極はエタノールにて良く洗浄して電気測定に使用した。
＜２＞この修飾済電極を０．５ｍＭ／Ｌの５−フルオロシトシン（Ｆ−Ｃｙｔ）溶液に１時間、浸積後、余分のＦ−Ｃｙｔを除く目的でジクロロエタンにて洗浄した。
＜３＞Ｆ−Ｃｙｔを脱離させる目的で、エタノール中、１時間浸積した。
＜２＞と＜３＞の操作を繰り返し、各々の段階でＩ−Ｖ曲線を測定し、Ｖ＝０付近での抵抗値を算出した。図４に得られた結果の一例を示す。図４の＋Ｆ−Ｃｙｔおよび−Ｆ−Ｃｙｔは、それぞれＦ−Ｃｙｔを結合させた状態およびＦ−Ｃｙｔを洗浄した状態を示している。
図４から、Ｆ−Ｃｙｔを結合させるとギャップ電極の抵抗が低くなり、Ｆ−Ｃｙｔを取ると抵抗値が上昇した。すなわち、検出対象のＦ−Ｃｙｔの存在に対応した電気的応答が得られた。
Compound 4 conductivity measurement results <1> A dichloroethane solution (0.2 mM / L) of compound 4 was prepared, and a nanogap electrode was immersed in this solution at room temperature for 16 hours. After the modification reaction, the electrode was thoroughly washed with ethanol and used for electrical measurement.
<2> This modified electrode was immersed in a 0.5 mM / L 5-fluorocytosine (F-Cyt) solution for 1 hour and then washed with dichloroethane for the purpose of removing excess F-Cyt.
<3> For the purpose of desorbing F-Cyt, it was immersed in ethanol for 1 hour.
The operations of <2> and <3> were repeated, an IV curve was measured at each stage, and a resistance value near V = 0 was calculated. An example of the results obtained is shown in FIG. + F-Cyt and -F-Cyt in FIG. 4 indicate a state where F-Cyt is bound and a state where F-Cyt is washed, respectively.
From FIG. 4, when F-Cyt was coupled, the resistance of the gap electrode was lowered, and when F-Cyt was taken, the resistance value was increased. That is, an electrical response corresponding to the presence of F-Cyt to be detected was obtained.
本発明のセンサー素子構造、化学物質の分子認識センサー、分子素子などナノテクノロジー分野およびバイオ・環境分野における高感度分子認識センサーとして有用性は極めて高い。 The sensor element structure of the present invention, a chemical substance molecular recognition sensor, a molecular element, etc. are extremely useful as a highly sensitive molecular recognition sensor in the fields of nanotechnology and bio / environment.
Claims (11)
A detection element of a detection target chemical substance in which a detection target chemical substance and a sensor molecule having a binding site are bonded to the nanogap electrode surface, and the sensor molecule is fixed to the nanogap electrode surface at one end. A wire-like molecule having a partial structure to be converted and a receptor partial structure that binds to a chemical substance to be detected at the other end, the molecular length is 2 nanometers or less, and the gap width of the electrode is maximum A chemical substance detection element characterized by being 10 nanometers or less.
General formula
General formula
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2009210272A (en) * | 2008-02-29 | 2009-09-17 | Nippon Telegr & Teleph Corp <Ntt> | Molecule analysis method and molecule analysis element |
| US8410144B2 (en) | 2009-03-31 | 2013-04-02 | Arqule, Inc. | Substituted indolo-pyridinone compounds |
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| KR101927415B1 (en) | 2012-11-05 | 2019-03-07 | 삼성전자주식회사 | Nanogap device and signal processing method from the same |
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| JP2002533698A (en) * | 1998-12-23 | 2002-10-08 | インスティトゥート フュア フィジカーリッシェ ホッホテヒノロギー エー．ファウ． | Affinity sensors for detecting the formation of specific binding and uses thereof |
| JP2003185662A (en) * | 1992-04-23 | 2003-07-03 | Massachusetts Inst Of Technol <Mit> | Optical and electric method and apparatus for detecting molecule |
| JP2005079335A (en) * | 2003-08-29 | 2005-03-24 | National Institute Of Advanced Industrial & Technology | Method for manufacturing nano gap electrode and element having the nano gap electrode manufactured by this method |
| JP2006248934A (en) * | 2005-03-09 | 2006-09-21 | National Institute Of Advanced Industrial & Technology | Conductive 2,6-diamidopyridine compound |
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| JP2003185662A (en) * | 1992-04-23 | 2003-07-03 | Massachusetts Inst Of Technol <Mit> | Optical and electric method and apparatus for detecting molecule |
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| JP2002533698A (en) * | 1998-12-23 | 2002-10-08 | インスティトゥート フュア フィジカーリッシェ ホッホテヒノロギー エー．ファウ． | Affinity sensors for detecting the formation of specific binding and uses thereof |
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| JP2009210272A (en) * | 2008-02-29 | 2009-09-17 | Nippon Telegr & Teleph Corp <Ntt> | Molecule analysis method and molecule analysis element |
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