JP6124327B2 - Metal detection sensor and metal detection method and apparatus - Google Patents
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Description
本発明は非常に微量の水銀等の金属を特別な前処理等を行わずに簡単に検出可能な金属検出センサー並びに金属検出方法及び装置に関する。 The present invention relates to a metal detection sensor, a metal detection method, and an apparatus that can easily detect a very small amount of metal such as mercury without performing special pretreatment.
世界各地の自然環境への水銀放出は、数１０年もの間、緊急を要する問題であり続けてきた。水銀放出は、鉱業、化石燃料の燃焼、産業廃棄物、火山活動を含む多くの放出源に由来する。揮発性や長期間の大気滞留のため、水銀は人間社会にとって最も深刻かつ普遍的な危険の一つである。とりわけ、Ｈｇ２＋イオンは環境水における最も深刻な水銀汚染物質の一つであり、生命体や人間の健康に有害な物質に変化するため、規制する必要がある。水銀は対流圏での循環（tropospheric cycling）の間に生態系に蓄積されるので、潜在的な危険を評価するために汚染の初期段階での非常に低い水銀濃度をモニターすることが強く求められる。現在のところ、ガスクロマトグラフィー（ＧＣ）と原子吸光分光（atomic absorption spectroscopy、ＡＡＳ）が水銀検出のための最も高感度の方法である。その検出限界はｐｐｂ（１０−７重量％）レベルを優に超えるが、これらの方法はその目的のための専用の機器を必要とし、更にはサンプルとして取り出された水から水銀を抽出するための化学的な処理も必要とされる。電気化学的インピーダンス分光法（electrochemical impedance spectroscopy）、核磁気共鳴、フォトルミネッセンス、比色センサーのような多様な方法も報告されたが、それらは、多くの場合、特定物質の選択的な検出のために何段階かのステップを必要とし、また感度もＧＣやＡＡＳに比べて満足できるものではなかった。 The release of mercury into the natural environment around the world has been an urgent issue for decades. Mercury emissions come from many sources, including mining, fossil fuel combustion, industrial waste, and volcanic activity. Mercury is one of the most serious and universal dangers to human society because of its volatility and long-term atmospheric retention. In particular, Hg 2+ ions are one of the most serious mercury pollutants in environmental water, and change into substances that are harmful to living organisms and human health. Since mercury accumulates in ecosystems during tropospheric cycling, it is strongly required to monitor very low mercury concentrations in the early stages of pollution to assess potential hazards. At present, gas chromatography (GC) and atomic absorption spectroscopy (AAS) are the most sensitive methods for mercury detection. Although its detection limit is well above the ppb (10 −7 wt%) level, these methods require dedicated equipment for that purpose, and also for extracting mercury from the water sampled. Chemical treatment is also required. Various methods have also been reported, such as electrochemical impedance spectroscopy, nuclear magnetic resonance, photoluminescence, and colorimetric sensors, which are often used for selective detection of specific substances. In addition, several steps were required, and the sensitivity was not satisfactory as compared with GC and AAS.
この点に関して、表面プラズモンを利用した光学センシングは最も見込みのある技術である。というのも、これにより迅速、簡単、低価格でかつ高感度の検出が可能になるからである。この種のセンシングのうちでも、表面プラズモン共鳴（surface plasmon resonance、ＳＰＲ）誘電体センサーによれば、簡単で素早い検出を行うことができるが、このセンサーはＨｇ２＋のような金属種の場合には十分に高い感度を示さない（非特許文献７、８）。 In this regard, optical sensing using surface plasmons is the most promising technology. This is because it allows for rapid, simple, inexpensive and highly sensitive detection. Among these types of sensing, a surface plasmon resonance (SPR) dielectric sensor can perform simple and quick detection, but this sensor can be used in the case of a metal species such as Hg 2+. It does not show sufficiently high sensitivity (Non-Patent Documents 7 and 8).
一方、プラズモン増強振動分光法（plasmon enhanced vibrational spectroscopy）は、リンカー分子（linker molecule）（あるいはホスト材料）の振動状態をモニターすることによって、目的とする種（あるいはゲスト材料）を高い感度で検出できるようにする別の方法である（非特許文献９、１０）。この方法は従来はもっぱら有機分子やセラミクスに対して適用されており、金属原子やイオンの検出に採用された例は見当たらない。しかし、この方法を用いて水銀との結合による分子の結合状態の変化を振動分光を通してモニターすることができれば、新しい水銀検出の方法として利用できることが期待できる。 On the other hand, plasmon enhanced vibrational spectroscopy can detect the target species (or guest material) with high sensitivity by monitoring the vibrational state of the linker molecule (or host material). This is another method (Non-Patent Documents 9 and 10). Conventionally, this method has been applied exclusively to organic molecules and ceramics, and no examples have been found for the detection of metal atoms and ions. However, if this method can be used to monitor changes in the molecular binding state due to binding to mercury through vibrational spectroscopy, it can be expected to be used as a new mercury detection method.
しかしながら、環境水中の水銀を検出しようとすると、通常の環境水は多くの不純物、とりわけ、生物由来の各種の有機物が溶解、分散しており、また場所や季節により、不純物の種類や量が大きく変動する。従って、単に高感度であるだけではなく、このような環境水の多様性の影響を最小化し、かつ水銀を選択的に検出できるセンサー及び水銀検出方法が求められている。また、環境水などに含まれる水銀以外の各種の金属も問題とされる場合が多々あるため、水銀以外の微量金属用のセンサー及び検出方法も求められている（例えばカドミウムやヒ素など）。 However, when trying to detect mercury in environmental water, normal environmental water has many impurities, especially various organic substances of biological origin, dissolved and dispersed, and the type and amount of impurities vary depending on the location and season. fluctuate. Accordingly, there is a need for a sensor and a mercury detection method that are not only highly sensitive, but that can minimize the influence of the diversity of environmental water and can selectively detect mercury. In addition, since various metals other than mercury contained in environmental water and the like are often problematic, sensors and detection methods for trace metals other than mercury are also required (for example, cadmium and arsenic).
本発明の課題は、ＧＣやＡＡＳに匹敵するような高感度であって、かつ特殊な前処理を行うことなく比較的一般的な機器を使用して水銀等の金属種の原子やイオンの検出を行うことができる、環境水中の微量な元素の原子やイオンの検出に適したセンサーを提供し、また当該センサーを使用した金属検出方法及び装置を提供することにある。 The object of the present invention is to detect atoms and ions of metal species such as mercury using a relatively common device with high sensitivity comparable to that of GC and AAS and without any special pretreatment. It is an object of the present invention to provide a sensor suitable for detecting a small amount of element atoms and ions in environmental water, and to provide a metal detection method and apparatus using the sensor.
本発明の一側面によれば、導電性材料で構成したナノ構造体の少なくともプラズモン共鳴が生起する領域上に検出対象の金属と結合するアプタマーを設けた、金属検出センサーが与えられる。
ここで、前記導電性材料は金属または導電性金属酸化物であってよい。
また、前記金属はＡｕ、Ｃｕ、Ｐｔ及びＡｇからなる群から選択されてよい。
また、前記導電性材料はＡｕであり、前記アプタマーにチオール基を導入することにより前記アプタマーが前記導電性材料に吸着するようにしてよい。
また、前記導電性金属酸化物はＩｎＳｎ２Ｏ３及びＲＯ２からなる群から選択されてよい。
また、前記ナノ構造体は、前記導電性材料からなり相互に絶縁された複数の領域を含む構造を有してよい。
また、前記複数の領域は５ｎｍ〜２０ｎｍのナノギャップで相互に電気的に絶縁されていてよい。
また、前記アプタマーは、チオール基を導入した、１５個のチミン塩基を有するシングルストランドＤＮＡオリゴヌクレオチド（５’−ＳＨ−（ＣＨ２）６−ＴＴＴ ＴＴＴ ＴＴＴ ＴＴＴ ＴＴＴ−３’）であり、前記検出対象の金属は水銀であってよい。
また、ＡＴＲ結晶上に前記ナノ構造体を形成してよい。
また、前記ＡＴＲ結晶はシリコン結晶であってよい。
本発明の他の側面によれば、前記何れかの金属検出センサーに検出対象の液体を付与し、前記金属検出センサーに照射された光の反射光中の前記検出対象金属に対応する吸収を測定する金属検出方法が与えられる。
ここで、前記照射された光は赤外線であってよい。
また、前記吸収の測定は前記反射光のスペクトルを測定することによってよい。
また、前記スペクトルの測定はフーリエ変換赤外分光器を使用して行ってよい。
また、前記検出対象の液体は有機物を含んでよい。
また、前記検出対象の液体は環境水であってよい。
本発明の更に他の側面によれば、前記何れかの金属検出センサーを含み、測定対象の液体が導入されるフローセルと、前記フローセルに赤外ビームを照射する光源と、前記フローセルからの前記赤外ビームの反射光が導入される分光計とを設けた、金属検出装置が与えられる。
According to one aspect of the present invention, a metal detection sensor is provided in which an aptamer that binds to a metal to be detected is provided on at least a region where plasmon resonance occurs in a nanostructure formed of a conductive material.
Here, the conductive material may be a metal or a conductive metal oxide.
The metal may be selected from the group consisting of Au, Cu, Pt, and Ag.
The conductive material may be Au, and the aptamer may be adsorbed to the conductive material by introducing a thiol group into the aptamer.
The conductive metal oxide may be selected from the group consisting of InSn 2 O 3 and RO 2 .
The nanostructure may have a structure including a plurality of regions made of the conductive material and insulated from each other.
The plurality of regions may be electrically insulated from each other with a nanogap of 5 nm to 20 nm.
The aptamer is a single-stranded DNA oligonucleotide (5′-SH— (CH 2 ) 6 -TTT TTT TTT TTT TTT-3 ′) having 15 thymine bases into which a thiol group has been introduced, The target metal may be mercury.
Further, the nanostructure may be formed on the ATR crystal.
The ATR crystal may be a silicon crystal.
According to another aspect of the present invention, the liquid to be detected is applied to any one of the metal detection sensors, and the absorption corresponding to the detection target metal in the reflected light of the light irradiated to the metal detection sensor is measured. A metal detection method is provided.
Here, the irradiated light may be infrared rays.
The absorption may be measured by measuring a spectrum of the reflected light.
The spectrum may be measured using a Fourier transform infrared spectrometer.
The liquid to be detected may include an organic substance.
The detection target liquid may be environmental water.
According to still another aspect of the present invention, a flow cell including any one of the metal detection sensors, into which a liquid to be measured is introduced, a light source that irradiates the flow cell with an infrared beam, and the red light from the flow cell. A metal detection device is provided that includes a spectrometer into which the reflected light of the outer beam is introduced.
本発明によれば、化学的な前処理や特殊な専用装置を使用せずに従来の高感度測定に匹敵する金属イオン検出感度を有する金属検出センサー並びにこの金属検出センサーを利用して金属原子やイオンを検出する金属検出方法及び装置が与えられる。 According to the present invention, a metal detection sensor having a metal ion detection sensitivity comparable to a conventional high-sensitivity measurement without using chemical pretreatment or a special dedicated device, and a metal atom or A metal detection method and apparatus for detecting ions is provided.
本発明の一実施例においては、水銀と結合したアプタマー（aptamer）の振動状態の変化を高感度で検出する高効率の「プラズモン増幅器」、つまりプラズモンを利用した高効率の電磁（ＥＭ）増幅器を使用した水銀センサーが提供される。さらに具体的に説明すれば、以下で説明する実施例においては、アプタマーとして特定のＤＮＡを使用し、またプラズモン増幅器としては、例えば特許文献１に示されるようなナノギャップを有する金のナノ構造を使用して、実際の湖水から採取したままで化学的前処理を行っていない微量の水銀を含む試料中でＨｇ２＋と結合したＤＮＡアプタマーの変化を光学的に検知する。 In one embodiment of the present invention, a high-efficiency “plasmon amplifier” that detects a change in vibration state of an aptamer combined with mercury with high sensitivity, that is, a high-efficiency electromagnetic (EM) amplifier using plasmon is provided. Used mercury sensor is provided. More specifically, in the examples described below, a specific DNA is used as an aptamer, and as a plasmon amplifier, for example, a gold nanostructure having a nanogap as shown in Patent Document 1 is used. Used to optically detect changes in DNA aptamers bound to Hg 2+ in samples containing trace amounts of mercury as collected from actual lake water but not chemically pretreated.
本発明の一実施例の水銀センサーでは、自由空間からの電界はシリコンなどの基板を通過し、水−固体界面で反射して、減衰長がサブμｍレンジのいわゆるエバネセント場をもたらす。金ナノ構造が存在すると、この減衰長はナノメートルにまで縮小し、光の強度は金表面、特にそのナノギャップを覆う分子に集中する（非特許文献１１）。この現象を利用し、赤外線分光にとって最も深刻な問題であるところのバルクの水からの巨大な信号を排除することによって、微量のターゲットとなる物質を検出する。 In the mercury sensor of one embodiment of the present invention, the electric field from free space passes through a substrate such as silicon and is reflected at the water-solid interface, resulting in a so-called evanescent field with an attenuation length in the sub-μm range. In the presence of gold nanostructures, this attenuation length is reduced to nanometers, and the intensity of light is concentrated on the gold surface, particularly on the molecules covering the nanogap (Non-Patent Document 11). By utilizing this phenomenon, a very small amount of target substance is detected by eliminating a huge signal from bulk water, which is the most serious problem for infrared spectroscopy.
図１（ａ）は金表面に固定されたＤＮＡアプタマー（１５個のチミン塩基からなる）による水銀の結合を示す概念図である（非特許文献３、４、８）。先ずＨｇ２＋イオンが負に帯電したＤＮＡに引き寄せられ、シングルストランド（一本鎖）ＤＮＡの直線状構造（図１（ａ）左側）をヘアピン状に折り曲げられた構造（図１（ａ）右側）に変化させることにより、このＨｇ２＋イオンが２つのチミン塩基の間に入る。また、両チミン塩基と結合する水銀近傍の原子における化学的／電気的状態が変化することにより、Ｈｇ−Ｎ結合の双極子モーメントなどに大きな変化を生じ、その結果、振動スペクトルの信号強度に変化が現れる（非特許文献４）。 FIG. 1 (a) is a conceptual diagram showing the binding of mercury by a DNA aptamer (consisting of 15 thymine bases) immobilized on the gold surface (Non-patent Documents 3, 4, and 8). First, Hg 2+ ions are attracted to the negatively charged DNA, and the linear structure of the single-stranded (single-stranded) DNA (left side of FIG. 1 (a)) is bent into a hairpin shape (right side of FIG. 1 (a)). This Hg 2+ ion falls between the two thymine bases. In addition, a change in the chemical / electrical state of the atoms in the vicinity of mercury that binds to both thymine bases causes a large change in the dipole moment of the Hg-N bond, resulting in a change in the signal intensity of the vibration spectrum. Appears (Non-Patent Document 4).
本発明のプラズモン増強赤外センサー材料を全反射減衰（ＡＴＲ）液体フローセルに取り付けた赤外分光装置を使用して得られたデータから、Ｈｇ２＋に関連する鋭い赤外信号がｐｐｔレベル（１０−１０ｗ％）の感度で、湖水の水中に含まれている生体由来の分子に基づく信号から分離して検出されることが示された。従って、本発明のセンサー材料は水中の金属種をモニターする高い能力を持つことが分かった。 From the data obtained using an infrared spectroscopic device in which the plasmon enhanced infrared sensor material of the present invention is attached to an attenuated total reflection (ATR) liquid flow cell, a sharp infrared signal associated with Hg 2+ is found at the ppt level (10 − 10 w%) with a sensitivity of 10 % by weight), and was detected separately from the signal based on biological molecules contained in the water of the lake water. Therefore, it was found that the sensor material of the present invention has a high ability to monitor metal species in water.
なお、以下の実施例ではモニター対象の金属種としてＨｇ２＋を例に挙げて説明したが、それ以外の金属種についても、その金属種と特異的に結合するアプタマーを適宜使用することによって、Ｈｇ２＋の場合と同様な原理に基づいて微量の当該金属種を検出することが可能となることは言うまでもない。 In the following examples, Hg 2+ was described as an example of the metal species to be monitored. However, for other metal species, Hg can be obtained by appropriately using an aptamer that specifically binds to the metal species. It goes without saying that a trace amount of the metal species can be detected based on the same principle as in the case of 2+ .
また、以下の実施例ではエバネセント場をもたらすために金のナノ構造を使用したが、この構造には他の材料も利用することができる。例を挙げれば、Ｃｕ、Ｐｔ、Ａｇなどの金属、更にはＩｎＳｎ２Ｏ３（ＩＴＯ）やＲｕＯ２などの導電性酸化物も利用可能である。また、ナノ構造とは、より具体的には幅が５ｎｍ〜２０ｎｍのナノギャップで相互に絶縁された上記金属その他の導電性材料の複数の島状体から構成される構造である。ここで島状体とは特定の形状を指すものではなく、任意の形状を取ることが可能な、他から電気的に切り離された上記材料からなる小領域を意味する。 Also, although the following examples used gold nanostructures to provide an evanescent field, other materials can be utilized for this structure. For example, metals such as Cu, Pt, and Ag, and conductive oxides such as InSn 2 O 3 (ITO) and RuO 2 can also be used. The nanostructure is more specifically a structure composed of a plurality of islands of the above metal or other conductive material insulated from each other by a nanogap having a width of 5 nm to 20 nm. Here, the island-shaped body does not indicate a specific shape, but means a small region made of the above-mentioned material electrically separated from others, which can take any shape.
以下では、本発明の一実施例の水銀センサーを使用した測定系で実際の湖水の水、及びその水にごく微量の水銀を添加した試料を測定した結果を説明する。 Below, the result of having measured the actual lake water and the sample which added the trace amount mercury to the water by the measuring system using the mercury sensor of one Example of this invention is demonstrated.
［光学系の設定］
本実施例の測定は、ＭＣＴ検出器（mercury cadmium telluride detector）を装着した標準的なフーリエ変換赤外（ＦＴＩＲ）分光計（Nicolet-Japan NEXUS-670）、ＫＢｒビームスプリッター、及び特注のフローセル（非特許文献１１）を使用して行った。赤外線測定は、分解能４ｃｍ−１で適切な（ｓ−あるいはｐ−）偏光条件の下で、全反射減衰（ＡＴＲ）構成で行った。赤外ビームの入射角はシリコンＡＴＲ結晶の表面垂直方向に対して３０度傾けた。
[Optical system settings]
The measurement of this example consists of a standard Fourier transform infrared (FTIR) spectrometer (Nicolet-Japan NEXUS-670) equipped with an MCT detector (mercury cadmium telluride detector), a KBr beam splitter, and a custom flow cell (non- Patent Document 11) was used. Infrared measurements were performed in an attenuated total reflection (ATR) configuration under appropriate (s- or p-) polarization conditions with a resolution of 4 cm- 1 . The incident angle of the infrared beam was inclined by 30 degrees with respect to the direction perpendicular to the surface of the silicon ATR crystal.
［ＤＮＡアプタマーで被覆された赤外活性金ナノ構造の作成］
自然酸化層ＳｉＯ２を有するシリコンＡＴＲ結晶を１０％（アミノプロピル）トリエトキシシラン／メタノール溶液（ＡＰＴＥＳ）に３０分間暴露した。次に、直径１１ｎｍの金ナノ粒子（ＡｕＮＰ）懸濁液の液滴をＡＰＴＥＳ表面に置いて、ＡｕＮＰを単層以下で吸着させた。次に、このＡｕＮＰ層を３ｍｌ／分の一定流量の水中でＡｕ３＋／ＮＨ２ＯＨ溶液（０．３ｍＭのＨＡｕＣＬ４（Aldrich）及び０．４ｍＭのＮＨ２ＯＨ（Acros Organics））に暴露することによって、成長させた。ＡｕＮＰ膜の成長の間、赤外線スペクトルを記録し、膜の光学的な変化をその場で調べ、制御した。膜形成の最終段階は、ナノ構造金基板中での水の振動（ωＨ２Ｏ＝３４００ｃｍ−１）とプラズモン励起（図２（ａ）参照）の混成したスペクトルの形状・強度を観察しながら制御した。なお、このような赤外線金ナノ構造とその製造方法については特許文献１において詳細に説明し、既に当業者に周知の事項となっているため、必要であれば当該文献を参照されたい。
[Preparation of infrared active gold nanostructures coated with DNA aptamer]
Silicon ATR crystals with a native oxide layer SiO 2 were exposed to a 10% (aminopropyl) triethoxysilane / methanol solution (APTES) for 30 minutes. Next, a droplet of a gold nanoparticle (AuNP) suspension having a diameter of 11 nm was placed on the APTES surface to adsorb AuNP in a single layer or less. Next, this AuNP layer is exposed to an Au 3+ / NH 2 OH solution (0.3 mM HAuCL 4 (Aldrich) and 0.4 mM NH 2 OH (Acros Organics)) in water at a constant flow rate of 3 ml / min. , Grew. During the growth of the AuNP film, an infrared spectrum was recorded and the optical changes of the film were examined and controlled in situ. The final stage of film formation was controlled while observing the shape and intensity of the hybrid spectrum of water vibration (ω H2O = 3400 cm −1 ) and plasmon excitation (see FIG. 2A) in the nanostructured gold substrate. . Such an infrared gold nanostructure and its manufacturing method will be described in detail in Patent Document 1 and are already well known to those skilled in the art, so please refer to that document if necessary.
チオール基を導入した、１５個のチミン塩基を有するシングルストランド（一本鎖）ＤＮＡオリゴヌクレオチド（５’−ＳＨ−（ＣＨ２）６−ＴＴＴ ＴＴＴ ＴＴＴ ＴＴＴ ＴＴＴ−３’）を脱イオン処理した純水（抵抗率ρ＞１８．４ＭΩ・ｃｍ）に溶解し１０ｍｌの１μＭ溶液を得た。このＤＮＡ溶液を金ナノ構造を有する上記ＡＴＲフローセル中に導入して、その金表面に単分子層のＤＮＡを固定した。 Pure deionized treatment of a single-stranded DNA oligonucleotide (5′-SH— (CH 2 ) 6 -TTT TTT TTT TTT TTT-3 ′) having 15 thymine bases introduced with a thiol group Dissolved in water (resistivity ρ> 18.4 MΩ · cm) to obtain 10 ml of 1 μM solution. This DNA solution was introduced into the ATR flow cell having a gold nanostructure, and the monolayer DNA was immobilized on the gold surface.
［検出対象の試料である、制御されたＨｇ２＋濃度を有する環境水の準備］
環境水として、霞ケ浦から採取した水を濾紙（細孔径５μｍ）でろ過することによってミクロンスケール以上の微粒子を除去したものを用いた。水銀検出実験は、Ｈｇ２＋（ＨｇＣｌ２，Sigma Aldrich）を環境水で希釈しＤＮＡ／金ナノ構造を有するＡＴＲフローセル中に導入して行った。水溶液中におけるＨｇ２＋の他の水銀成分（ＨｇＣｌ２及びＨｇＣｌ＋）の割合は電離平衡によって変化する。本実験における濃度領域（ｐｐｔ領域）では、Ｈｇ２＋イオンは最大濃度を持つ水銀成分である。
[Preparation of environmental water with controlled Hg 2+ concentration, which is a sample to be detected]
As the environmental water, water collected from Kasumigaura was filtered with a filter paper (pore diameter: 5 μm) to remove fine particles of micron scale or larger. The mercury detection experiment was performed by diluting Hg 2+ (HgCl 2 , Sigma Aldrich) with environmental water and introducing it into an ATR flow cell having a DNA / gold nanostructure. The proportion of Hg 2+ other mercury components (HgCl 2 and HgCl + ) in the aqueous solution varies with ionization equilibrium. In the concentration region (ppt region) in this experiment, Hg 2+ ions are mercury components having the maximum concentration.
［環境水中の水銀イオンの光学的検出］
環境水中における微量のＨｇ２＋イオンの検出は以下のようにして行った。先ず、最適化された金ナノ構造を作製した後、上述したようにチオール基を導入したＤＮＡアプタマーの１μＭ脱イオン水溶液をフローセル中に導入して、このＤＮＡアプタマーを金表面に吸着させた。このＤＮＡアプタマーは１５個のチミン塩基からできていて、他のイオンに比べてＨｇ２＋に対して大きな選択性を有している（非特許文献３、８）。チオール末端基と金との強い結合により、３０分間の吸着処理を行うことで、ＤＮＡアプタマーによるほぼ全面的な被覆が達成される。その後、環境水を導入して脱イオン水と置き換えた。水銀によって汚染された湖水環境汚染を模擬するため、環境水をフローセルに導入する前に、これに多様な濃度の希釈ＨｇＣｌ２溶液を僅かに混合した。
[Optical detection of mercury ions in environmental water]
Detection of trace amounts of Hg 2+ ions in environmental water was performed as follows. First, after preparing an optimized gold nanostructure, a 1 μM deionized aqueous solution of a DNA aptamer into which a thiol group was introduced as described above was introduced into a flow cell, and this DNA aptamer was adsorbed on the gold surface. This DNA aptamer is made of 15 thymine bases and has a high selectivity for Hg 2+ compared to other ions (Non-patent Documents 3 and 8). By performing the adsorption treatment for 30 minutes due to the strong bond between the thiol end group and gold, almost the entire covering with the DNA aptamer is achieved. Thereafter, environmental water was introduced to replace deionized water. In order to simulate the environmental pollution of the lake contaminated by mercury, it was mixed slightly with various concentrations of diluted HgCl 2 solution before introducing the environmental water into the flow cell.
ナノ構造の材料が金である場合には、上述したようにＤＮＡアプタマーにチオール基を導入することでＤＮＡアプタマーをナノ構造の表面に良好に吸着させて固定することができる。しかし、ナノ構造に他の材料を使用した場合には、当該材料への吸着等に好適な基を適宜選択してアプタマーに導入するなど、別の手段でアプタマーをナノ構造に固定するようにしてもよい。 When the nanostructure material is gold, the DNA aptamer can be favorably adsorbed and immobilized on the surface of the nanostructure by introducing a thiol group into the DNA aptamer as described above. However, when another material is used for the nanostructure, the aptamer is fixed to the nanostructure by another means, such as appropriately selecting a group suitable for adsorption to the material and introducing it into the aptamer. Also good.
図２（ａ）及び（ｂ）に、金ナノ構造上に単層ＤＮＡアプタマーを吸着させた後、２つの異なる偏光の下で記録した脱イオン水中での相対赤外線反射スペクトルを示す。何れの偏光状態でもωがほぼ１３８０ｃｍ−１及び３４００ｃｍ−１の２か所（図中で縦方向の破線で示す）で吸収パターンがあることがわかる。１３８０ｃｍ−１における反吸収シグナルは、金ナノ構造の作製後に残留した過剰の分子（ＡＰＴＥＳやクエン酸三ナトリウム等）の脱着に対応する。この種のパターンは各種のチオール分子の吸着の間によく見られるものであり、強固な金−硫黄結合に比較するとこれら過剰分子の結合が弱く脱離しやすいことに起因し得る。もう一つのブロードな吸収シグナルはωＨ２Ｏ＝３４００ｃｍ−１であるが、これは水のＯ−Ｈの伸縮運動に対応するものであり、ＤＮＡアプタマーの吸着につれて次第にこれの吸収と置き換わる。 FIGS. 2 (a) and 2 (b) show relative infrared reflectance spectra in deionized water recorded under two different polarizations after adsorbing a single layer DNA aptamer on a gold nanostructure. Any two polarized in polarization state ω approximately 1380 cm -1 and 3400 cm -1 (shown by vertical broken lines in the figure) in it can be seen that an absorption pattern. The anti-absorption signal at 1380 cm −1 corresponds to the desorption of excess molecules (APTES, trisodium citrate, etc.) remaining after the gold nanostructures are made. This type of pattern is often seen during the adsorption of various thiol molecules, and can be attributed to the weak binding of these excess molecules and the ease of desorption compared to strong gold-sulfur bonds. Another broad absorption signal is ω H2O = 3400 cm −1 , which corresponds to the stretching motion of water O—H and gradually replaces this absorption as the DNA aptamer adsorbs.
ＤＮＡアプタマーのいくつかの特徴的な吸収ピークが、波数１４００ｃｍ−１、１６５６ｃｍ−１及び１７２４ｃｍ−１における縦方向の実線で示される位置で明確に観察された。ここで、これらの位置はω＝１４００〜１７３０ｃｍ−１の範囲にあり、また偏光方向の影響を強く受けている。ｓ偏光の場合（図２（ａ））には、ω＝１６５６ｃｍ−１及び１７２４ｃｍ−１を中心としたチミンのＣ＝０及びＣ＝Ｃ伸縮に対応するピーク（信号強度０．２％）が明確に観察された（非特許文献１６〜１９）。ｐ偏光（図２（ｂ））では、ωＴ＝１４００ｃｍ−１において、もっと強い吸収線（強度１．８％）が観察された。水中における遊離状態のチミンＤＮＡの場合のもっと弱い信号に関する報告（非特許文献２１）があるが、これとは対照的に、強い異方性を持つこの非常に大きなピークは、ＤＮＡが金表面に対して良好に整列していることを強く示唆するものである。このことは、このＤＮＡアプタマーの塩基がチミンのみで構成されていて、ＤＮＡが直線的で異方的な構造を簡単にとれるというユニークな塩基配列によるものである。飽和吸着に近い被覆状態では、高い充填率及び負に帯電したＤＮＡ同士の静電的反発により、ＤＮＡの整列が起こる。 Several characteristic absorption peaks of the DNA aptamer were clearly observed at the positions indicated by the vertical solid lines at wave numbers 1400 cm −1 , 1656 cm −1 and 1724 cm −1 . Here, these positions are in the range of ω = 1400 to 1730 cm −1 and are strongly influenced by the polarization direction. In the case of s-polarized light (FIG. 2A), peaks (signal intensity of 0.2%) corresponding to C = 0 and C = C stretching of thymine centered at ω = 1656 cm −1 and 1724 cm −1 are obtained. It was clearly observed (Non-Patent Documents 16 to 19). In p-polarized light (FIG. 2B), a stronger absorption line (intensity 1.8%) was observed at ω T = 1400 cm −1 . In contrast to this, there is a report on a weaker signal in the case of free thymine DNA in water (Non-Patent Document 21), but in contrast to this very large peak with strong anisotropy, the DNA is on the gold surface. It strongly suggests that it is well aligned. This is due to the unique base sequence in which the base of the DNA aptamer is composed only of thymine and the DNA can easily take a linear and anisotropic structure. In the coating state close to saturation adsorption, DNA alignment occurs due to high packing rate and electrostatic repulsion between negatively charged DNAs.
赤外光による電磁界は金ナノギャップに強く集中するので（非特許文献２０）、観測された分子振動シグナルはほとんどがナノギャップに存在していてその側壁に垂直な方向であるＤＮＡアプタマーからのものである。ｓ偏光の場合には、電界はナノギャップの表面に垂直に向いていて、ギャップ中のＤＮＡアプタマーもその長手方向軸に沿って励起される。これと対照的に、ｐ偏光された光の電界はＳｉ表面の垂直方向から３０度傾いていて、ｓ偏光された光とは異なる対称性でＤＮＡアプタマーを励起することができる。この異方性により、図２（ａ）と図２（ｂ）で対比できるような、スペクトル上で異なる吸収特性がもたらされる。 Since the electromagnetic field due to infrared light is strongly concentrated in the gold nanogap (Non-patent Document 20), most of the observed molecular vibration signals are from DNA aptamers that are present in the nanogap and are perpendicular to the sidewalls. Is. In the case of s-polarized light, the electric field is oriented perpendicular to the surface of the nanogap and the DNA aptamer in the gap is also excited along its longitudinal axis. In contrast, the electric field of p-polarized light is tilted 30 degrees from the vertical direction of the Si surface, and can excite DNA aptamers with a different symmetry than s-polarized light. This anisotropy results in different absorption characteristics on the spectrum that can be contrasted in FIGS. 2 (a) and 2 (b).
図２（ｃ）は、純粋な脱イオン水中のＤＮＡアプタマーの初期スペクトル（偏光なし）を参照スペクトルとして規格化した環境水の相対赤外吸収スペクトルを示す。１６時間経過しても、ωＴ＝１４００ｃｍ−１におけるチミンに関連するピークには何の変化も認められなかった。このことは、このωＴ＝１４００ｃｍ−１の振動に関連する構造的な要素は非常に安定していて、湖水から採取されたままの（つまり何も前処理していない）自然状態のままの水に曝した後であっても変化しないことを示している。 FIG. 2 (c) shows the relative infrared absorption spectrum of environmental water normalized with the initial spectrum (no polarization) of the DNA aptamer in pure deionized water as the reference spectrum. Even after 16 hours, no change was observed in the peak associated with thymine at ω T = 1400 cm −1 . This means that the structural elements associated with this vibration of ω T = 1400 cm −1 are very stable and remain in their natural state as they are collected from the lake water (ie nothing has been pretreated). It shows no change even after exposure to water.
ＤＮＡアプタマーに対応するスペクトルがこのように不変であることとは対照的に、生体由来分子に関連する振動バンドは湖水環境中のこれらの分子の存在を反映しスペクトル中に現れる。このような振動バンドは以下の通りである：１５５８ｃｍ−１近傍に存在する２つのブロードなピーク（α：Ｃ＝Ｏ、Ｎ−Ｈ、アミドＩＩ）、１６５６ｃｍ−１近傍に存在するショルダー（β：Ｃ＝Ｏ、Ｎ−Ｈ、アミドＩ）、及び３３００ｃｍ−１近傍に存在するピーク（γ：Ｎ−Ｈの伸縮）。これらのスペクトルのパターンはアミノ酸（あるいは、脂肪酸及びアルドース）から期待される位置に一致している。これらの物質は微生物の分解によって生成される主要な成分として湖水中に存在している（非特許文献２１）。 In contrast to the spectrum corresponding to DNA aptamers being invariant in this way, vibrational bands associated with biological molecules appear in the spectrum reflecting the presence of these molecules in the lake environment. Such vibration bands are as follows: two broad peaks existing near 1558 cm −1 (α: C═O, N—H, amide II), a shoulder existing near 1656 cm −1 (β: C = O, N—H, amide I), and a peak existing in the vicinity of 3300 cm −1 (γ: N—H stretching). These spectral patterns are consistent with the positions expected from amino acids (or fatty acids and aldoses). These substances exist in lake water as main components produced by the decomposition of microorganisms (Non-patent Document 21).
図３（ａ）は、Ｈｇ２＋の濃度を３．６８ｐｐｂから３６．８ｐｐｔまで変化させたときの環境水からの相対赤外線スペクトルの変化を示すグラフである。この測定における基準スペクトルとして、Ｈｇ２＋の添加前の湖水から採取したままの環境水から得られたものを使用した。各スペクトルは吸着がＨｇ２＋の濃度によって定まる平衡状態にほぼ到達するように、３０分間累算した。Ｈｇ２＋を含まない元の環境水のスペクトル（図２（ｃ））とは異なり、ωＴ＝１４００ｃｍ−１にピーク（反吸収シグナル）がはっきりと現れることを観測できた。１４００ｃｍ−１におけるこのピークはＨｇ２＋濃度に対応してその強度が変化した。この変化はＨｇ２＋を含まない水からは観測できず、Ｈｇ含有溶液からだけ観測できた。 FIG. 3A is a graph showing a change in relative infrared spectrum from the environmental water when the concentration of Hg 2+ is changed from 3.68 ppb to 36.8 ppt. As a reference spectrum in this measurement, a spectrum obtained from environmental water as collected from the lake water before the addition of Hg 2+ was used. Each spectrum was accumulated for 30 minutes so that adsorption almost reached an equilibrium state determined by the concentration of Hg 2+ . Unlike the original ambient water spectrum not containing Hg 2+ (FIG. 2C), it was observed that a peak (anti-absorption signal) clearly appeared at ωT = 1400 cm −1 . This peak at 1400 cm −1 changed in intensity corresponding to the Hg 2+ concentration. This change could not be observed from water not containing Hg 2+ , but only from a solution containing Hg.
図３（ｂ）は非特許文献３、４、８で認められているモデルに基づく、Ｈｇ２＋吸着後のＤＮＡアプタマーの概念図である。Ｈｇ２＋の吸着により、ＤＮＡアプタマーはその形状が直線状からヘアピン状へと折れ曲がることができ、これによりその直線状かつ高度の異方性を有するという特徴が減殺される。この変化を起こす微視的なレベルでの駆動力は、２つのチミン塩基がＨｇ２＋イオンによって結合されて対になることである。図３（ｂ）の右側部分に示すように、２つのイミノプロトンを溶液中に放出することにより、新たなＮ−ＨｇＩＩ−Ｎ結合が形成される（非特許文献４）。このプロセスでは、イミド構造中のＮ原子近傍（図３（ｂ）右側の破線長方形で示す）におけるかなりの電荷再配分をともなう。１３９３（±１３）ｃｍ−１付近にある振動モードはイミド構造中のＮ原子の大きな原子移動に関係することが知られている（非特許文献１６〜１８）ので、ωＴ＝１４００ｃｍ−１において観察された反吸収ピークは、ほぼ間違いなく、Ｎ−ＨｇＩＩ−Ｎ結合による双極子モーメント変化を高感度で反映するところのこのＮ原子にかかわる振動モードに対応する。一方、ω＝１７２４ｃｍ−１における同じイミド構造中のＣ＝Ｏに関係する帯域（図３（ａ）で下向きの小さな矢印で示す）は吸収強度がわずかに増加するだけである。これは、ＨｇＩＩ原子から２番目に近い箇所以遠の原子について起こる電荷再配分は非常に小さいことを示唆している。 FIG. 3 (b) is a conceptual diagram of a DNA aptamer after Hg 2+ adsorption based on the models recognized in Non-Patent Documents 3, 4, and 8. Adsorption of Hg 2+ allows the DNA aptamer to bend from a linear shape to a hairpin shape, thereby diminishing its linear and highly anisotropic characteristics. The driving force at the microscopic level that causes this change is that the two thymine bases are combined by Hg 2+ ions to form a pair. As shown in the right part of FIG. 3B, a new N—Hg II— N bond is formed by releasing two imino protons into the solution (Non-patent Document 4). This process involves significant charge redistribution near the N atom in the imide structure (shown by the dashed rectangle on the right side of FIG. 3 (b)). It is known that the vibration mode in the vicinity of 1393 (± 13) cm −1 is related to the large atomic migration of N atoms in the imide structure (Non-Patent Documents 16 to 18), so at ω T = 1400 cm −1 The observed anti-absorption peak almost certainly corresponds to the vibrational mode involving this N atom, which reflects the dipole moment change due to the N—Hg II— N bond with high sensitivity. On the other hand, the band related to C = O in the same imide structure at ω = 1724 cm −1 (indicated by a small downward arrow in FIG. 3A) has only a slight increase in absorption intensity. This suggests that the charge redistribution that occurs for atoms farthest away from the Hg II atom is very small.
その一方では、Ｈｇの吸着と同期して生物由来分子に関係するα：１５５８ｃｍ−１、β：１６５６ｃｍ−１及びγ：３３００ｃｍ−１に赤外線吸収シグナルが増加することも、観測される。Ｈｇ２＋濃度の関数としてプロットされたこれらの振動スペクトルについての規格化された信号強度を図３（ｃ）に示す。これら４種類の振動モード全てについての信号強度は同じ挙動を示し、これらの成分の吸着機構は皆同じであることがわかる。これは、生物由来分子はＨｇ２＋吸着プロセスの進行と同時に金ナノギャップに入ることを意味する。つまり、ＤＮＡアプタマー構造が折りたたまれることで、生物由来分子が金ギャップにより多く入るための場所を与え、これによってこれら分子の吸着信号がかなり大きくなる。ωＴ＝１４００ｃｍ−１におけるチミン−ＨｇＩＩ関連の信号は上述の生物由来分子によるいずれの信号ともエネルギー的に十分に分離されており、湖水から採取した水などの環境水を化学的な前処理なしで直接測定することによってｐｐｔレベルのスペクトル応答を簡単に得ることができる。これはいくつかの化学的処理ステップを測定前に行うことが通常必要とされる多くの他の技法（非特許文献２）に比べて明確な利点となる。 On the other hand it is related to the biological molecule in synchronism with the adsorption of Hg α: 1558cm -1, β: 1656cm -1 and gamma: also infrared absorption signal increases to 3300 cm -1, are observed. The normalized signal intensity for these vibrational spectra plotted as a function of Hg 2+ concentration is shown in FIG. 3 (c). The signal intensities for all these four vibration modes show the same behavior, and it can be seen that the adsorption mechanisms of these components are all the same. This means that biological molecules enter the gold nanogap simultaneously with the progress of the Hg 2+ adsorption process. That is, the folded DNA aptamer structure provides a place for more biological molecules to enter the gold gap, thereby significantly increasing the adsorption signal of these molecules. The signal related to thymine-Hg II at ω T = 1400 cm −1 is sufficiently energetically separated from any of the above-mentioned biological molecules, and chemical pretreatment of environmental water such as water collected from lake water A spectral response at the ppt level can be easily obtained by direct measurement without. This is a distinct advantage over many other techniques (Non-Patent Document 2) that usually require several chemical processing steps to be performed prior to measurement.
光を利用した従来技術と比べて、本発明で使用したランダムなナノギャップ構造は高い感度と迅速な検出をもたらす。湖水から採取したままの水に添加した非常に低い水銀濃度（３７ｐｐｔ）を標準的なＦＴ−ＩＲ分光計を使用して光学的に検出できたことは驚くべきことである。すなわち、本発明には、水銀を他のバックグラウンド成分から抽出するための専用装置も化学的前処理も必要ないという大きな利点がある。振動スペクトルにおける水銀吸着に関連した信号と生物由来分子からの信号との間のエネルギーを利用することによって、水銀イオンを環境水から選択的に検出することが可能である。この簡潔な方法は水銀検出に限らず、金属イオンを補足するアプタマーの振動が、スペクトル中のバックグラウンド信号となる水中の生体分子の振動と分離できるという条件を満たせば多様な金属種において適用可能であり、大きな可能性をもっていることを示すものである。 Compared with the prior art using light, the random nanogap structure used in the present invention provides high sensitivity and rapid detection. It is surprising that a very low mercury concentration (37 ppt) added to as-collected water from the lake water could be detected optically using a standard FT-IR spectrometer. That is, the present invention has the great advantage that no specialized equipment or chemical pretreatment is required to extract mercury from other background components. Mercury ions can be selectively detected from environmental water by using the energy between the signal associated with mercury adsorption in the vibrational spectrum and the signal from biological molecules. This simple method is not limited to mercury detection, but can be applied to various metal species as long as the vibration of the aptamer that captures metal ions can be separated from the vibration of biomolecules in water, which is the background signal in the spectrum. It shows that it has great potential.
以上説明したように、本発明によれば、対費用効果が高く、単純であり、また水銀イオンの環境モニターを現場で行うことができる可能性のある、その場プラズモンセンシングが与えられる。本発明のセンサー材料のキーとなる特徴は、高密度のナノギャップであり、これにより、金ナノ構造が中赤外域の電磁場増幅器として動作し、水銀と結合したＤＮＡアプタマーの分子振動シグナルを増幅できるようになる。Ｈｇ２＋に対応する信号は、湖水などからの環境水中に主要な成分として存在する残留生体由来分子の信号から区別して選択的に抽出することができる。上述の実施例においては、既存の標準的なＦＴＩＲ分光器を使用したが、このような通常の測定系構成でさえも、ｐｐｔレベル（１０−１０％レベル）に至るまでの極めて高い感度を示した。別の金属を選択吸着するアプタマーと組み合わせたり、また携帯型の赤外線分光計を採用すれば、本発明の適用範囲がさらに広がり、また環境モニタリング、実験室での化学研究、産業分野での品質管理などの現場での応用にも適用できるようになる。 As described above, the present invention provides in-situ plasmon sensing that is cost-effective and simple, and that may be capable of environmental monitoring of mercury ions in the field. A key feature of the sensor material of the present invention is the high density nanogap, which allows the gold nanostructure to operate as a mid-infrared electromagnetic field amplifier and amplify the molecular vibration signal of the DNA aptamer bound to mercury. It becomes like this. A signal corresponding to Hg 2+ can be selectively extracted by distinguishing from a signal of a residual biological molecule existing as a main component in environmental water such as lake water. In the above-described embodiment, an existing standard FTIR spectrometer is used, but even such a normal measurement system configuration exhibits extremely high sensitivity up to the ppt level (10 −10 % level). It was. Combined with aptamers that selectively adsorb other metals or adopt a portable infrared spectrometer, the scope of the present invention can be expanded further, and environmental monitoring, chemical research in the laboratory, quality control in the industrial field It can also be applied to on-site applications such as.
以上説明したように、本発明によれば生物由来の分子などのバックグラウンド成分を多く含む環境水中のごく微量の水銀イオンを、専用に設計された装置や特別な化学的前処理を必要とせずに簡単に検出することができる。さらには、この特定の応用以外にも、検出対象の物質の拡大や現場測定への利用も容易なので、広い産業分野への貢献が大きいと期待される。 As described above, according to the present invention, a very small amount of mercury ions in the environmental water containing a large amount of background components such as biological molecules can be obtained without specially designed equipment or special chemical pretreatment. Can be easily detected. Furthermore, in addition to this specific application, it is easy to expand the substance to be detected and to be used for on-site measurement, so it is expected to contribute greatly to a wide range of industrial fields.
Claims (18)
前記アプタマーにチオール基を導入することにより前記アプタマーが前記導電性材料に吸着するようにした、
請求項３に記載の金属検出センサー。 The conductive material is Au;
The aptamer was adsorbed on the conductive material by introducing a thiol group into the aptamer.
The metal detection sensor according to claim 3.
前記検出対象の金属は水銀である、
請求項１から８の何れかに記載の金属検出センサー。 The aptamer is a single-strand DNA oligonucleotide (5′-SH— (CH 2) 6 -TTT TTT TTTTTTT TTT-3 ′) having 15 thymine bases into which a thiol group is introduced,
The metal to be detected is mercury;
Metal detection sensor according to any one of claims 1 to 8.
前記金属検出センサーに照射された光の反射光中の前記検出対象金属に対応する吸収を測定することにより、プラズモン増強振動分光法による測定を行う
金属検出方法。 Any metal detection sensor according to claim 1 to 11 to impart a liquid to be detected,
A metal detection method for performing measurement by plasmon enhanced vibrational spectroscopy by measuring absorption corresponding to the detection target metal in reflected light of light irradiated on the metal detection sensor.
前記フローセルに赤外ビームを照射する光源と、
前記フローセルからの前記赤外ビームの反射光が導入される分光計と
を設け、
プラズモン増強振動分光法により金属の検出を行う金属検出装置。
A flow cell comprising the metal detection sensor according to claim 10 or 11 , into which a liquid to be measured is introduced,
A light source for irradiating the flow cell with an infrared beam;
Setting a spectrometer reflected light is introduced in the infrared beam from the flow cell,
A metal detector that detects metals by plasmon enhanced vibrational spectroscopy .
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