JP6573817B2 - Method and apparatus for detecting a target substance contained in a gaseous test sample - Google Patents
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Description
本発明は、気体状の被検試料中に含まれる標的物質の検出方法及び装置に関する。本発明は、例えば大気中に含まれる残留農薬や環境物質等の、種々の物質の検出に有用である。 The present invention relates to a method and apparatus for detecting a target substance contained in a gaseous test sample. The present invention is useful for detecting various substances such as residual agricultural chemicals and environmental substances contained in the atmosphere.
従来、例えば大気中に含まれる残留農薬等の検出は、ガスクロマトグラフィー、ガス検知管、酸化物半導体表面への吸着による導電性や共振周波数の変化の検出等により行われている。 Conventionally, for example, residual agricultural chemicals contained in the atmosphere are detected by gas chromatography, a gas detector tube, detection of changes in conductivity or resonance frequency due to adsorption to the surface of an oxide semiconductor, and the like.
これらの方法のうち、ガスクロマトグラフィーや酸化物半導体を利用する方法は、定量的な感度は比較的高いが、装置が大型となり携帯はできない。一方、ガス検知管は携帯型ではあるものの、物質特異性及び定量的感度が共に十分ではない。 Among these methods, the method using gas chromatography or an oxide semiconductor has relatively high quantitative sensitivity, but the apparatus is large and cannot be carried. On the other hand, although the gas detection tube is portable, both substance specificity and quantitative sensitivity are not sufficient.
一方、本願発明者らは先に、脂質二重膜中にチャネルタンパク質を保持し、膜を介するイオンチャネルを形成することに成功しており（非特許文献１）、この脂質二重膜中に保持されるチャネルタンパク質のイオンチャネルの開放及び閉塞を電気的に測定することにより、微量のコカイン等の麻薬を検出することに成功し、特許出願している（特許文献１）。 On the other hand, the inventors of the present application have previously held a channel protein in a lipid bilayer membrane and succeeded in forming an ion channel via the membrane (Non-patent Document 1). We succeeded in detecting trace amounts of narcotics such as cocaine by electrically measuring the opening and closing of the ion channel of the retained channel protein (Patent Document 1).
本願発明者らが先に特許出願した、特許文献１に記載された方法は、携帯可能な簡便な装置を用いて微量のコカイン等を高い特異性と感度をもって検出することが可能である。しかしながら、この方法を、大気中に含まれる残留農薬等の検出に適用しようとすると、残留農薬等を高感度に検出できないことがわかった。 The method described in Patent Document 1 previously filed by the inventors of the present application can detect a small amount of cocaine or the like with high specificity and sensitivity using a portable and simple device. However, it has been found that if this method is applied to detection of residual agricultural chemicals contained in the atmosphere, residual agricultural chemicals cannot be detected with high sensitivity.
したがって、本発明の目的は、気体状の被検試料中に含まれる、検出すべき標的物質を簡便、迅速、高感度に検出することができる、気体状の被検試料中に含まれる標的物質の検出方法及びそのための装置を提供することである。 Accordingly, an object of the present invention is to provide a target substance contained in a gaseous test sample, which can detect a target substance contained in the gaseous test sample in a simple, rapid and sensitive manner. And a device for the same.
本願発明者らは、特許文献１記載の方法により気体状の被検試料中に含まれる標的物質の高感度検出が困難である理由について検討した。特許文献１記載の方法では、ダブルウェルチャンバーの各ウェルには、脂質溶液の膜で被覆される緩衝液が含まれる。本願発明者らは、気体状の被検試料中に含まれる標的物質は、この脂質溶液の膜を通過することができないので検出できないのではないかと考えた。そして、この問題を解決すべく鋭意検討の結果、一方のウェルにヒドロゲルを配置し、このヒドロゲルに気体状の被検試料を接触させて標的物質をヒドロゲルに吸収させれば、気体状の被検試料中の標的物質を高感度に検出可能ではないかと考えた。そして、一方のウェルにヒドロゲルを配置した場合であっても、ヒドロゲルと脂質溶液との界面に脂質二重膜が形成され、この脂質二重膜にチャネルタンパク質が保持されることを見出し、かつ、ヒドロゲルに気体状の被検試料を直接接触させることにより、高感度に被検試料中の標的物質を検出可能であることを実験的に確認して本発明を完成した。 The inventors of the present application examined the reason why it is difficult to detect the target substance contained in the gaseous test sample with high sensitivity by the method described in Patent Document 1. In the method described in Patent Document 1, each well of the double well chamber contains a buffer solution that is coated with a lipid solution membrane. The inventors of the present application thought that the target substance contained in the gaseous test sample cannot be detected because it cannot pass through the membrane of the lipid solution. As a result of intensive studies to solve this problem, if a hydrogel is placed in one well and a gaseous test sample is brought into contact with this hydrogel to absorb the target substance into the hydrogel, the gaseous test is performed. We thought that the target substance in the sample could be detected with high sensitivity. And even when a hydrogel is arranged in one well, a lipid bilayer is formed at the interface between the hydrogel and the lipid solution, and channel lipids are retained in this lipid bilayer, and The present invention was completed by experimentally confirming that the target substance in the test sample can be detected with high sensitivity by bringing the gaseous test sample into direct contact with the hydrogel.
すなわち、本発明は、気体状の被検試料中に含まれる標的物質の検出装置であって、前記被検試料と直接接触する、第１の領域に配置されたヒドロゲルと、該ヒドロゲルと脂質二重膜を介して隣接する、第２の領域に配置された脂質二重膜形成性の脂質溶液／水系媒体と、これらの領域の間に電圧を印加してこれらの領域間に流れる電流を測定する手段とを具備し、
前記ヒドロゲルと前記脂質溶液との界面に存在する前記脂質二重膜は透孔を有し、前記電流は該透孔を介して流れ、該電流の流れ方が、前記被検試料中に前記標的物質が含まれる場合と含まれない場合とで異なる、気体状の被検試料中に含まれる標的物質の検出装置を提供する。
That is, the present invention is an apparatus for detecting a target substance contained in a gaseous test sample, the hydrogel disposed in a first region that is in direct contact with the test sample, and the hydrogel and lipid. A lipid bilayer-forming lipid solution / aqueous medium placed in a second region adjacent through the bilayer and a voltage applied between these regions to measure the current flowing between these regions And means for
The lipid bilayer membrane present at the interface between the hydrogel and the lipid solution has a through-hole, and the current flows through the through-hole, and the current flows through the target in the test sample. Provided is a device for detecting a target substance contained in a gaseous test sample, which is different depending on whether or not the substance is contained.
また、本発明は、気体状の被検試料中に含まれる標的物質の検出装置の部品であって、前記被検試料と直接接触する、第１の領域に配置されたヒドロゲルと、該ヒドロゲルと脂質二重膜を介して隣接する、第２の領域に配置された脂質二重膜形成性の脂質溶液／水系媒体とを具備する、気体状の被検試料中に含まれる標的物質の検出装置部品を提供する。 Further, the present invention is a component of a detection device for a target substance contained in a gaseous test sample, the hydrogel disposed in a first region that is in direct contact with the test sample, and the hydrogel An apparatus for detecting a target substance contained in a gaseous test sample, comprising a lipid bilayer-forming lipid solution / aqueous medium disposed in a second region adjacent to each other via a lipid bilayer membrane Provide parts.
さらに、本発明は、気体状の被検試料中に含まれる標的物質の検出方法であって、
前記被検試料との接触の結果前記標的物質を含む、第１の領域に配置されたヒドロゲルと、該ヒドロゲルと脂質二重膜を介して隣接する、第２の領域に配置された脂質二重膜形成性の脂質溶液／水系媒体との間に電圧を印加してこれらの領域間に流れる電流を測定することを含み、
前記ヒドロゲルと前記脂質溶液との界面に存在する前記脂質二重膜は、透孔を有しており、該透孔を介して前記第１の領域と第２の領域は連通しており、前記電流は該透孔を介して流れ、該電流の流れ方が、前記被検試料中に前記標的物質が含まれる場合と含まれない場合とで異なり、測定した電流に基づき標的物質を検出する、気体状の被検試料中に含まれる標的物質の検出方法を提供する。
Furthermore, the present invention is a method for detecting a target substance contained in a gaseous test sample,
As a result of contact with the test sample, the hydrogel disposed in the first region containing the target substance, and the lipid duplex disposed in the second region adjacent to the hydrogel via the lipid bilayer membrane Applying a voltage between the membrane-forming lipid solution / aqueous medium and measuring the current flowing between these regions,
The lipid bilayer membrane present at the interface between the hydrogel and the lipid solution has a pore, and the first region and the second region communicate with each other through the pore, An electric current flows through the through-hole, and the flow of the electric current is different depending on whether or not the target substance is included in the test sample, and the target substance is detected based on the measured current. Provided is a method for detecting a target substance contained in a gaseous test sample.
本発明の装置及び方法によれば、大気等の気体状の被検試料中に含まれる、残留農薬や環境物質等の、広範囲の所望の物質を簡便、迅速、高感度に検出することができる。 According to the apparatus and method of the present invention, a wide range of desired substances such as residual agricultural chemicals and environmental substances contained in a gaseous test sample such as the atmosphere can be detected simply, rapidly, and with high sensitivity. .
本発明の装置及び方法に供される気体状の被検試料は、検出すべき標的物質を含む気体であれば何ら限定されるものではなく、代表的な例として大気や室内の空気等を挙げることができるがこれらに限定されるものではない。 The gaseous test sample used in the apparatus and method of the present invention is not limited as long as it contains a target substance to be detected, and representative examples include air and indoor air. However, the present invention is not limited to these.
本発明の装置及び方法により検出される標的物質としては、これと特異的に結合できるアプタマーのような特異結合性物質を作出可能な物質であれば何ら限定されるものではなく、例えば大気中に含まれる、例えば、オメトエート等の有機リン系農薬のような残留農薬や、大気や室内空気に含まれる様々な臭気物質、大気中に含まれる種々の病原体等を例示することができるがこれらに限定されるものではない。また、後述のように、チャネルタンパク質と特異的に結合してチャネルタンパク質のチャネルを開閉可能な、該チャネルタンパク質のリガンドは、特異的結合性物質がなくても検出が可能であるので、このようなリガンドも標的物質とすることができる。 The target substance detected by the apparatus and method of the present invention is not limited as long as it is a substance capable of producing a specific binding substance such as an aptamer that can specifically bind to the target substance. For example, in the atmosphere Examples include residual pesticides such as organophosphorus pesticides such as ometoate, various odorous substances contained in the atmosphere and indoor air, and various pathogens contained in the atmosphere. Is not to be done. In addition, as described later, since the ligand of the channel protein that can specifically open and close the channel of the channel protein by binding with the channel protein can be detected without a specific binding substance, Other ligands can also be targeted.
本発明の装置及び方法では、ヒドロゲルを用いる。ヒドロゲルとしては、アガロース、寒天、ゼラチン等を用いることができるが、これらに限定されるものではない。 The apparatus and method of the present invention uses a hydrogel. As the hydrogel, agarose, agar, gelatin and the like can be used, but the hydrogel is not limited thereto.
本発明の装置及び方法の一実施形態では、標的物質と特異的に結合する物質（本発明において「特異結合性物質」と呼ぶ）が用いられる。ここで、「特異的に結合する」とは、標的物質とは結合するが、標的物質以外の物質とは結合しないか、又は少なくとも標的物質以外の物質であって被検試料中に存在する可能性がある他の物質とは結合しないことを意味する。なお、標的物質は、互いに区別することを意図しない一群の類似物質である場合も包含される。 In one embodiment of the apparatus and method of the present invention, a substance that specifically binds to a target substance (referred to as “specific binding substance” in the present invention) is used. Here, “specifically binds” means that it binds to a target substance but does not bind to a substance other than the target substance, or at least a substance other than the target substance and exists in the test sample. This means that it does not bind to other substances that have sex. The target substance includes a group of similar substances that are not intended to be distinguished from each other.
特異結合性物質は、標的物質と特異的に結合でき、結合後の結合物のサイズが結合前の特異結合性物質のサイズよりも大きくなる物質であり、アプタマー並びに抗体及びその抗原結合性断片等を挙げることができるがこれらに限定されるものではない。 A specific binding substance is a substance that can specifically bind to a target substance, and the size of the bound substance after binding is larger than the size of the specific binding substance before binding, such as aptamers, antibodies, and antigen-binding fragments thereof, etc. However, it is not limited to these.
特異結合性物質としては、アプタマーが好ましい。アプタマーは、DNAやRNA等のポリヌクレオチド（安定性の観点から好ましくはDNA）から成るものであり、特定の物質と特異的に結合するものである。アプタマーは、通常、数十〜百数十のヌクレオチドから成るポリヌクレオチドであり、市販の核酸合成機を用いて任意の塩基配列を有するものを容易に化学合成できるので、抗体よりも容易、安価、迅速に製造することができる。このため、種々の免疫測定における抗体に代わるものとして近年盛んに研究されている。 As the specific binding substance, an aptamer is preferable. The aptamer is composed of a polynucleotide (preferably DNA from the viewpoint of stability) such as DNA or RNA, and specifically binds to a specific substance. Aptamers are usually polynucleotides consisting of several tens to one hundred and several tens of nucleotides, and those having an arbitrary base sequence can be easily chemically synthesized using a commercially available nucleic acid synthesizer. It can be manufactured quickly. For this reason, research has been actively conducted in recent years as an alternative to antibodies in various immunoassays.
任意の物質と特異的に結合するアプタマーは、SELEX (Systematic Evolution of Ligands by EXponential Enrichment)と呼ばれる方法により作出可能である。SELEX法自体は既に周知であり、これに基づき、様々な物質と特異的に結合するアプタマーが既に得られている。また、所望の物質と特異的に結合するアプタマーを効率良く作出する、SELEX法の改良法も種々提案されている。SELEX法は、核酸の自動合成装置を用いてランダムな塩基配列を有する非常に多数のポリヌクレオチドのライブラリーを形成し、固相に結合した標的物質とこのライブラリーを反応させ、標的物質に結合したポリヌクレオチドを回収し、これをPCRにより増幅して再び標的物質を固定化した固相に添加するという工程を１０回〜数十回程度繰り返して標的物質との結合力が高いポリヌクレオチドを濃縮していく方法であり、偶然を積極的に利用する方法であるので、ほとんど全ての物質に対して特異的に結合可能なアプタマーを作出することができると考えられている。標的物質に特異的に結合するアプタマーが得られ、その塩基配列を決定した後には、その塩基配列を持つポリヌクレオチドは、自動合成装置により容易に化学合成することができる。また、SELEX法によれば、所望の物質と特異的に結合するアプタマーは、通常、複数種類得られるので、その中から、標的物質と未結合の状態で後述する透孔を通過できるものを選択することも可能である。 Aptamers that specifically bind to any substance can be produced by a method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment). The SELEX method itself is already well known, and based on this, aptamers that specifically bind to various substances have already been obtained. In addition, various improved methods of the SELEX method have been proposed for efficiently producing an aptamer that specifically binds to a desired substance. The SELEX method uses a nucleic acid automatic synthesizer to form a library of a large number of polynucleotides having random base sequences, reacts the target substance bound to a solid phase with this library, and binds to the target substance. The process of collecting the obtained polynucleotide, amplifying it by PCR and adding it again to the solid phase on which the target substance is immobilized is repeated about 10 to several tens of times to concentrate the polynucleotide having a high binding force to the target substance. Since this is a method that actively uses chance, it is considered that aptamers that can specifically bind to almost all substances can be produced. After an aptamer that specifically binds to a target substance is obtained and its base sequence is determined, a polynucleotide having the base sequence can be easily chemically synthesized by an automatic synthesizer. In addition, according to the SELEX method, a plurality of aptamers that specifically bind to a desired substance are usually obtained. From these, select one that can pass through a through-hole described later in an unbound state with a target substance. It is also possible to do.
特異結合性物質として用いられるアプタマーは、その一端に、同一の塩基が１５個〜５０個、さらに好ましくは２０個〜４０個連続する同一塩基の繰り返し領域を有することが好ましい。このような同一塩基の繰り返し領域は、アプタマー分子内の他の領域や他のアプタマー分子とハイブリダイズすることがほとんどなく、標的物質と結合後も直線状で存在するので、標的物質と結合後、この領域が後述する透孔に突き刺さって、透孔が標的物質とアプタマーの結合物により閉塞されやすくなるので好ましい。同一塩基としては特にシトシン(c)が好ましい。これは、ポリグアニン(g)は化学合成されないので、同一塩基がシトシンであれば、繰り返し領域が同一分子内又は他分子内の領域とハイブリダイズする可能性を排除できるからである。一端にこのような繰り返し領域を持つアプタマーは、標的物質と特異的に結合するアプタマーの一端にこのような繰り返し領域を単に付加することにより通常得ることができるし、上記したSELEX法に用いられるライブラリーとして、一端に繰り返し領域を有するものを用いることによっても作出することができる。 The aptamer used as the specific binding substance preferably has a repeating region of the same base having 15 to 50, more preferably 20 to 40, identical bases at one end. Such repeated regions of the same base rarely hybridize with other regions in the aptamer molecule or other aptamer molecules, and exist linearly after binding to the target substance. This region is preferable because it pierces a later-described through-hole, and the through-hole is likely to be blocked by a combination of the target substance and the aptamer. As the same base, cytosine (c) is particularly preferable. This is because polyguanine (g) is not chemically synthesized, so if the same base is cytosine, it is possible to eliminate the possibility that the repeated region hybridizes with the region in the same molecule or in another molecule. An aptamer having such a repeating region at one end can be usually obtained by simply adding such a repeating region to one end of an aptamer that specifically binds to the target substance, and can be used in the live SELEX method described above. It can also be created by using a rally having a repeating region at one end.
特異的結合物質は、前記ヒドロゲル中に含まれる。ヒドロゲル中の特異結合性物質の終濃度は、特に限定されないが、標的物質が存在する場合にチャネルタンパク質のチャネルが閉塞されることを促進するために、想定される標的物質の濃度範囲の上限値の全量と結合できる量であることが好ましい。特異結合性物質の終濃度の具体的な濃度範囲は、ケースバイケースで適宜設定されるが、通常、1nM〜1mM程度である。なお、特異結合性物質と、前記標的物質の結合は、通常、室温において速やかに起きる。 A specific binding substance is included in the hydrogel. Although the final concentration of the specific binding substance in the hydrogel is not particularly limited, in order to promote the blockage of the channel of the channel protein in the presence of the target substance, the upper limit value of the concentration range of the target substance to be assumed An amount that can be combined with the total amount of is preferable. The specific concentration range of the final concentration of the specific binding substance is appropriately set on a case-by-case basis, but is usually about 1 nM to 1 mM. The binding between the specific binding substance and the target substance usually occurs rapidly at room temperature.
本発明の装置及び方法においては、脂質二重膜に形成する透孔であって、標的物質と結合していない前記特異結合性物質は通過できるが、前記標的物質と結合した前記特異結合性物質は通過できないサイズの透孔としては、このようなサイズのチャネルを持つチャネルタンパク質を用いることができる。チャネルタンパク質は、分子内にチャネルと呼ばれる透孔を有するタンパク質であり、生体内ではこのチャネルを介して各種イオン等の輸送が行われる。チャネルタンパク質としては、α−ヘモリシン、外膜タンパク質(Outer membrane protein) F (OmpF)、マイコバクテリウム・スメグマチスポリン(Mycobacterium smegmatis porin) A （MspA）、ストレプトリジンO等を挙げることができるがこれらに限定されるものではない。また、脂質二重膜に形成することが可能な、このような透孔として、金属錯体や人工ペプチドも利用することができる。ここで、利用可能な金属錯体としては、MOP(Metal organic polyhedra)を挙げることができる。また、利用可能な人工ペプチドとしては、相互に架橋を施し透孔を安定化した人工アラメチシンを挙げることができる。これらは、脂質二重膜に保持可能であることが報告されている。 In the apparatus and method of the present invention, the specific binding substance that is a through-hole formed in the lipid bilayer membrane and can pass through the specific binding substance that is not bound to the target substance, but is bound to the target substance. A channel protein having a channel of such a size can be used as a through-hole having a size that cannot pass through. A channel protein is a protein having a pore called a channel in a molecule, and various ions and the like are transported through the channel in a living body. Examples of channel proteins include α-hemolysin, Outer membrane protein F (OmpF), Mycobacterium smegmatis porin A (MspA), and streptolysin O. It is not limited to. Moreover, a metal complex and an artificial peptide can also be utilized as such a through-hole which can be formed in a lipid bilayer membrane. Here, examples of usable metal complexes include MOP (Metal organic polyhedra). In addition, examples of usable artificial peptides include artificial alamethicins that are cross-linked with each other to stabilize the pores. These have been reported to be retained in lipid bilayers.
チャネルタンパク質は、ヒドロゲルと、該ヒドロゲルに隣接する脂質溶液／水系媒体（後述）との界面に形成される脂質二重膜中に保持される。そして、この脂質二重膜は、自己支持性フィルムに設けられた微小な貫通孔を塞ぐ形で形成することが安定性の観点から好ましい。すなわち、自己支持性フィルムの微小な貫通孔（好ましくは直径が200μｍ未満、特に100nm〜100μｍ）を塞ぐ形で脂質二重膜を形成し、この脂質二重膜にチャネルタンパク質を保持し、このチャネルタンパク質のチャネルを透孔として利用することが好ましい。 The channel protein is retained in a lipid bilayer formed at the interface between the hydrogel and a lipid solution / aqueous medium (described below) adjacent to the hydrogel. And it is preferable from a stability viewpoint that this lipid bilayer membrane is formed in the form which plugs up the fine through-hole provided in the self-supporting film. That is, a lipid bilayer is formed so as to block a minute through-hole (preferably less than 200 μm, particularly 100 nm to 100 μm) in the self-supporting film, and the channel protein is held in the lipid bilayer, It is preferable to use protein channels as pores.
自己支持性フィルムの微小な貫通孔を塞ぐ形で脂質二重膜を形成し、この脂質二重膜にチャネルタンパク質を保持する方法は、特許文献１等に記載されており、公知である。本発明においても、特許文献１等に記載された、微小な貫通孔を持つ自己支持性フィルムを、ヒドロゲルと脂質溶液／水系媒体との境界面に配置することが好ましい。なお、自己支持性フィルムを構成する物質としては、特許文献１等に記載のとおり、パラキシレン系ポリマー（商品名パリレン）を好ましく用いることができる。 A method of forming a lipid bilayer membrane so as to block minute through-holes in a self-supporting film and retaining a channel protein in the lipid bilayer membrane is described in Patent Document 1 and the like, and is well known. Also in the present invention, it is preferable to dispose a self-supporting film having fine through-holes described in Patent Document 1 or the like at the interface between the hydrogel and the lipid solution / aqueous medium. In addition, as a substance which comprises a self-supporting film, a paraxylene-type polymer (brand name parylene) can be preferably used as it describes in patent document 1 grade | etc.,.
本発明の方法に用いられる検出装置の模式断面図を図１に示す。図１中、１０は、第１の領域である、ダブルウェルチャンバー（２個のウェルを持つ基板）の一方のウェルに配置されたヒドロゲルを示す。ヒドロゲル１０には、上記した特異的結合物質（図示せず）が含まれる。さらに、ヒドロゲルには、気体状の被検試料中に含まれる標的物質１２が、ヒドロゲルと被検試料の接触の結果として含まれる。なお、図１中、標的物質１２は理解のために図示されているが、縮尺は実物とは全く異なっており、実際には標的物質は分子であるから目に見えず、自己支持性フィルムの透孔よりも遥かに小さいことはいうまでもない。第２の領域１１を構成する、ダブルウェルチャンバーのもう一方のウェルには、脂質溶液／水系媒体が含まれる。図１中、脂質溶液は１４で示され、水系媒体は１６で示される。水系媒体は、好ましくは水系緩衝液又は水である。図示のように、水系媒体１６の周囲を脂質溶液１４の膜が被覆している。第１の領域と第２の領域は、上記した、透孔を有する自己支持性フィルム１８により隔てられている。自己支持性フィルム１８の透孔部分には脂質二重膜２０が形成されている。各ウェルの底部にはそれぞれ電極２１が配置され、これらの電極の間に電圧を印加して流れる電流を測定する回路が接続されている。なお、電圧の印加と電流の測定のための回路は、図１に示されるものに限定されるものではなく、電極間に電圧を印加して、電極間に流れる電流を測定可能な回路であればいずれのものでもよい。 A schematic cross-sectional view of a detection apparatus used in the method of the present invention is shown in FIG. In FIG. 1, 10 indicates a hydrogel disposed in one well of a double well chamber (substrate having two wells), which is the first region. The hydrogel 10 includes the specific binding substance (not shown) described above. Further, the hydrogel contains the target substance 12 contained in the gaseous test sample as a result of contact between the hydrogel and the test sample. In FIG. 1, the target substance 12 is illustrated for the sake of understanding, but the scale is completely different from the actual substance, and since the target substance is actually a molecule, it is not visible. Needless to say, it is much smaller than the through-hole. The other well of the double well chamber constituting the second region 11 contains a lipid solution / aqueous medium. In FIG. 1, the lipid solution is indicated by 14 and the aqueous medium is indicated by 16. The aqueous medium is preferably an aqueous buffer or water. As shown in the figure, the membrane of the lipid solution 14 covers the periphery of the aqueous medium 16. The first region and the second region are separated by the above-described self-supporting film 18 having a through hole. A lipid bilayer membrane 20 is formed in the through-hole portion of the self-supporting film 18. An electrode 21 is disposed at the bottom of each well, and a circuit for measuring a flowing current by applying a voltage is connected between these electrodes. The circuit for applying voltage and measuring current is not limited to that shown in FIG. 1, and any circuit that can measure the current flowing between the electrodes by applying a voltage between the electrodes. Any of them may be used.
脂質二重膜２０の模式拡大図を図２に示す。図２中、２０が脂質二重膜である。脂質二重膜２０には、チャネルタンパク質２２が保持され、チャネルタンパク質２２のチャネルが透孔となって脂質二重膜２０を貫通している。２４は、特異結合性物質であるアプタマーであり、図２は、アプタマー２４が標的物質１２と結合してチャネルタンパク質２２のチャネルを閉塞している状態を模式的に示している。 A schematic enlarged view of the lipid bilayer membrane 20 is shown in FIG. In FIG. 2, 20 is a lipid bilayer membrane. The lipid bilayer membrane 20 holds a channel protein 22, and the channel of the channel protein 22 becomes a through hole and penetrates the lipid bilayer membrane 20. Reference numeral 24 denotes an aptamer that is a specific binding substance, and FIG. 2 schematically shows a state in which the aptamer 24 binds to the target substance 12 and blocks the channel of the channel protein 22.
次に、図１及び図２に示す装置の作製方法を説明する。 Next, a method for manufacturing the device illustrated in FIGS. 1 and 2 will be described.
まず、ダブルウェルチャンバーの一方のウェル（第１の領域）に、加熱溶融したヒドロゲルを入れる。次に、溶融状態のヒドロゲルに上記した特異結合物質とチャネルタンパク質を添加する。ここで、チャネルタンパク質の濃度は、特に限定されるものではなく、適宜選択することができるが、通常、1pM〜1μM程度、好ましくは0.1nM〜100nM程度である。また、特異結合物質の濃度は、上記のとおり、通常、1nM〜1mM程度である。 First, the heated and melted hydrogel is put into one well (first region) of the double well chamber. Next, the above-mentioned specific binding substance and channel protein are added to the molten hydrogel. Here, the concentration of the channel protein is not particularly limited and can be appropriately selected, but is usually about 1 pM to 1 μM, preferably about 0.1 nM to 100 nM. Further, the concentration of the specific binding substance is usually about 1 nM to 1 mM as described above.
次に、ヒドロゲルを室温に放置する等して固化させた後、気体状の被検試料と接触させる。ここで、接触時間は、特に限定されないが、通常、１分間〜３時間程度、好ましくは１０分間〜２時間程度である。この接触により、被検試料中に標的物質が含まれる場合には、標的物質がヒドロゲル内に吸収され、特異結合性物質と結合する。 Next, after allowing the hydrogel to solidify, for example, by leaving it at room temperature, it is brought into contact with a gaseous test sample. Here, the contact time is not particularly limited, but is usually about 1 minute to 3 hours, preferably about 10 minutes to 2 hours. By this contact, when the target substance is contained in the test sample, the target substance is absorbed into the hydrogel and binds to the specific binding substance.
次に、他方のウェル（第２の領域）に脂質溶液を入れる。ここで、脂質としては、脂質二重膜、すなわち、親水性領域と疎水性領域を１分子中に有する脂質分子が、疎水性領域を内側、親水性領域を外側に向けて２層に並んだ膜を形成できる脂質であれば特に限定されないが、生体膜における反応を模するためには、生体膜と同じか類似したものが好ましく、この分野において従来から広く用いられているリン脂質、例えば、ジフィタノイルフォスファチジルコリン(diphytanoyl phosphatidylcholine, DPhPC)、ジパルミトイルフォスファチジルコリン(dipalmytoyl phosphatidylcholine)、パルミトイルオレオイルフォスファチジルコリン(1-Palmitoyl 2-Oleoyl phosphatidylcholine, POPC)、ジオレオイルフォスファチジルコリン(Dioleoyl phosphatidylcholine, DOPC)等を好ましい例として挙げることができる。これらの多くは市販されているので、市販品を好ましく用いることができる。 Next, the lipid solution is placed in the other well (second region). Here, as lipids, lipid bilayer membranes, that is, lipid molecules having a hydrophilic region and a hydrophobic region in one molecule are arranged in two layers with the hydrophobic region on the inside and the hydrophilic region on the outside. Although it is not particularly limited as long as it is a lipid capable of forming a membrane, in order to simulate the reaction in a biological membrane, the same or similar to the biological membrane is preferable, and phospholipids that have been widely used in this field, for example, Diphytanoyl phosphatidylcholine (diphytanoyl phosphatidylcholine, DPhPC), dipalmitoyl phosphatidylcholine (dipalmytoyl phosphatidylcholine), palmitoyl oleoyl phosphatidylcholine (POPC), dioleoyl oil Preferred examples include choline (Dioleoyl phosphatidylcholine, DOPC) and the like. Since many of these are commercially available, commercially available products can be preferably used.
脂質二重膜の形成に用いられる溶液中のリン脂質の濃度は、脂質二重膜が形成可能な濃度であれば特に限定されないが、通常、1g/L〜50g/L程度、好ましくは5g/L〜25g/L程度である。また、リン脂質溶液の溶媒は、特に限定されないが、有機溶媒が好ましく、n-デカンのような脂肪族炭化水素溶媒が好ましい。また、リン脂質は、この溶液中でリポソームを形成してもよく、この場合には、用いられる液はリポソーム懸濁液になる。なお、チャネルタンパク質は、上記の通り、ヒドロゲルに入れてもよいが、脂質溶液に添加してもよい。 The concentration of the phospholipid in the solution used for forming the lipid bilayer membrane is not particularly limited as long as the lipid bilayer membrane can be formed, but is usually about 1 g / L to 50 g / L, preferably 5 g / L. It is about L-25g / L. The solvent of the phospholipid solution is not particularly limited, but an organic solvent is preferable, and an aliphatic hydrocarbon solvent such as n-decane is preferable. Phospholipids may also form liposomes in this solution, in which case the liquid used is a liposome suspension. In addition, although channel protein may be put into a hydrogel as above-mentioned, you may add it to a lipid solution.
次に、脂質溶液に、水系媒体をスポイトで添加する。水系媒体としては、上記の通り、水又は水を溶媒とする緩衝液が用いられる。緩衝液としては、生物適合性があることが知られている、例えばリン酸緩衝液等の周知の緩衝液を用いることができる。水系媒体を添加すると、水系媒体の周囲を脂質溶液の膜が被覆する形に自発的になる。「脂質溶液／水系媒体」は、このように水系媒体の周囲を脂質溶液の膜が被覆したものを意味する。 Next, an aqueous medium is added to the lipid solution with a dropper. As the aqueous medium, as described above, water or a buffer solution using water as a solvent is used. As the buffer solution, a known buffer solution known to be biocompatible, such as a phosphate buffer solution, can be used. When the aqueous medium is added, the aqueous medium is spontaneously formed into a form in which the membrane of the lipid solution covers the periphery. The “lipid solution / aqueous medium” means a film in which a lipid solution membrane is coated around the aqueous medium.
なお、上記のとおり、ヒドロゲルと被検試料を接触させてから、第２の領域に脂質溶液／水系媒体を入れて界面に脂質二重膜を形成してもよいが、先にこの脂質二重膜を形成した後、ヒドロゲルを被検試料と接触させてもよい。この場合も、上記と同様の条件で、被検試料中の標的物質を検出することができる。 As described above, after bringing the hydrogel and the test sample into contact, a lipid solution / aqueous medium may be put in the second region to form a lipid bilayer membrane at the interface. After forming the membrane, the hydrogel may be contacted with the test sample. Also in this case, the target substance in the test sample can be detected under the same conditions as described above.
次に、図１に示す装置を用いた標的物質の検出方法の原理を図３を参照して説明する。 Next, the principle of the target substance detection method using the apparatus shown in FIG. 1 will be described with reference to FIG.
図３の左図は、被検試料中に標的物質が含まれていない場合を模式的に示す。標的物質が存在しない場合、アプタマー２４は、直線状になり得るので、チャネルタンパク質２２のチャネルを速やかに通過することができる。図３中、下側の図は、横軸に時間、縦軸に測定された電流値をプロットした図である。アプタマー２４がチャネルタンパク質２２のチャネルを通過する際、一時的にチャネルが部分的に閉塞されるため、チャネルを流れる電流が急激に減少する。しかし、アプタマー２４は、チャネルを迅速に通過するので、電流はすぐに元に復帰する。従って、電流の低下は一時的であり、この一時的な電流の低下はスパイクシグナルとなって現れる。 The left diagram of FIG. 3 schematically shows a case where the target substance is not contained in the test sample. In the absence of the target substance, the aptamer 24 can be linear, and thus can quickly pass through the channel of the channel protein 22. In FIG. 3, the lower diagram is a diagram in which time is plotted on the horizontal axis and current values measured on the vertical axis. When the aptamer 24 passes through the channel of the channel protein 22, the channel is temporarily partially blocked, so that the current flowing through the channel rapidly decreases. However, the aptamer 24 passes quickly through the channel so that the current is quickly restored. Therefore, the current decrease is temporary, and this temporary current decrease appears as a spike signal.
一方、被検試料中に標的物質１２が含まれる場合を、図３の右図に模式的に示す。標的物質１２が含まれる場合、標的物質１２はアプタマー２４と結合する。そうすると、アプタマー２４が所定の構造をとってチャネルを通過できない大きさとなり、チャネルを閉塞し、これにより電流値が低下する。アプタマーは、チャネルを通過できないので、この閉塞は長時間持続し、このため電流値の低下も長時間持続する（図３右図の下側の図）。 On the other hand, the case where the target substance 12 is contained in the test sample is schematically shown in the right diagram of FIG. When the target substance 12 is included, the target substance 12 binds to the aptamer 24. As a result, the aptamer 24 has a predetermined structure and cannot pass through the channel, and the channel is blocked, thereby reducing the current value. Since the aptamer cannot pass through the channel, this occlusion lasts for a long time, and thus the decrease in the current value also lasts for a long time (the lower diagram in the right side of FIG. 3).
以上のとおり、被検試料中に標的物質が含まれる場合には、持続的な電流の低下が観測され、含まれない場合には、スパイクシグナルが観察される。したがって、電流測定により、被検試料中の標的物質を検出することができる。 As described above, when the target substance is included in the test sample, a continuous decrease in current is observed, and when it is not included, a spike signal is observed. Therefore, the target substance in the test sample can be detected by current measurement.
なお、図３左図に模式的に示されるように、アプタマー等の特異結合性物質は、チャネルを通過しやすいように、図３左図に示すような直線状の形状を有するものが好ましい。また、標的物質と結合した結合物が、しっかりとチャネルに突き刺さって長時間に亘ってチャネルを閉塞することが望まれるので、標的物質と結合した結合物もチャネル内に挿入される部分、すなわち、好ましくは直線状の部分を一端に有することが好ましい。アプタマーの場合、これは、上記した通り、一端に同一塩基の繰り返し領域を付加することにより達成することができるが、繰り返し領域を付加しなくても直線状となる部分が存在していれば繰り返し領域を付加する必要はない。 As schematically shown in the left diagram of FIG. 3, the specific binding substance such as an aptamer preferably has a linear shape as shown in the left diagram of FIG. 3 so as to easily pass through the channel. In addition, since it is desired that the binding substance bound to the target substance sticks firmly into the channel and closes the channel for a long time, the binding part bound to the target substance is also inserted into the channel, that is, It is preferable to have a linear portion at one end. In the case of aptamers, as described above, this can be achieved by adding a repeating region of the same base at one end, but if there is a linear part without adding a repeating region, it is repeated. There is no need to add a region.
また、下記実施例に具体的に説明するように、チャネルの閉塞率（ここで、閉塞率とは、透孔を本来流れる電流に対して、標的物質と特異結合性物質の結合物等が閉塞したことにより阻害される電流の分率）と閉塞時間は、標的物質と特異結合性物質の結合物が閉塞した場合に特徴的な閾値（一般的には高い閉塞率、長い閉塞時間）をとる。また、この閾値を満たす閉塞の頻度は、被検試料中に含まれる標的物質の濃度に依存するので、種々の既知濃度の標準試料を複数準備し、その閉塞率と閉塞時間を測定して検量線を作成することにより、標的物質の定量も可能になる。なお、標的物質を「定量」する場合、必然的に検出も行われるので、本発明の「検出方法」には標的物質を定量する場合も包含される。 Further, as will be described in detail in the following examples, the blockage rate of the channel (here, the blockage rate is the blockage of the binding substance of the target substance and the specific binding substance with respect to the current flowing through the through hole). (The fraction of the current that is inhibited by this) and the occlusion time take characteristic thresholds (generally, a high occlusion rate, a long occlusion time) when the target substance and the specific binding substance are occluded. . Since the frequency of occlusion that satisfies this threshold depends on the concentration of the target substance contained in the test sample, prepare multiple standard samples of various known concentrations, measure their occlusion rate and occlusion time, and perform calibration. By creating a line, the target substance can be quantified. In addition, since the detection is necessarily performed when the target substance is “quantified”, the “detection method” of the present invention includes the case where the target substance is quantified.
なお、上記の説明では、第１の領域及び第２の領域として、ダブルウェルチャンバーの各ウェルを用いた実施形態について説明したが、第１の領域及び第２の領域はダブルウェルチャンバーの各ウェルに限定されるものではなく、例えば、特開2014-21025号公報に示される、基板上の親水性パターン上に第１の水性溶液、脂質溶液、第２の水性溶液を順次積層していく方法により作製したものでもよく、この場合、第２の水性溶液として上記ヒドロゲルを用いることができる。すなわち、この場合、ヒドロゲルが存在する領域が第１の領域となり、第１の水性溶液と脂質溶液が存在する領域が第２の領域となる。 In the above description, the embodiment using each well of the double well chamber as the first region and the second region has been described. However, the first region and the second region are each well of the double well chamber. For example, a method of sequentially laminating a first aqueous solution, a lipid solution, and a second aqueous solution on a hydrophilic pattern on a substrate as disclosed in JP 2014-21025 A In this case, the hydrogel can be used as the second aqueous solution. That is, in this case, the region where the hydrogel is present is the first region, and the region where the first aqueous solution and the lipid solution are present is the second region.
なお、上記の説明では、特異結合性物質を用い、特異結合性物質は通過できるが、該特異結合性物質と前記標的物質との結合物は通過できないサイズのチャネルを持つチャネルタンパク質を用いる場合について説明したが、本発明はこの実施形態に限定されるものではない。例えば、非特許文献２には、リガンドと結合することによりチャネルの開閉が影響されるチャネルタンパク質を脂質二重膜に保持した装置が記載されている。本発明においてもこのようなチャネルタンパク質を脂質二重膜に保持して用いることができる。この場合、ヒドロゲルに含まれるリガンドがチャネルタンパク質と結合すると、チャネルの開閉が影響される（例えば、閉じているチャネルが開く、若しくは開く確率が高くなる、又は開いている時間が長くなる等）ので、リガンドを標的物質とすれば、特異結合性物質がなくても標的物質の検出が可能となる。このようなチャネルタンパク質の例として、非特許文献２に記載されているhBKやKcsA等、さらにコネキシン等を挙げることができる。また、標的物質とすることができるリガンドとしては、揮発性有機物質等を含む臭気物質を挙げることができるがこれらに限定されるものではない。 In the above description, a specific binding substance is used, and a specific binding substance can pass through, but a channel protein having a channel of a size that cannot pass through the binding substance between the specific binding substance and the target substance is used. Although described, the present invention is not limited to this embodiment. For example, Non-Patent Document 2 describes a device in which a channel protein whose channel opening / closing is affected by binding to a ligand is held in a lipid bilayer membrane. Also in the present invention, such a channel protein can be used while being held in a lipid bilayer membrane. In this case, when the ligand contained in the hydrogel binds to the channel protein, the opening and closing of the channel is affected (for example, the closed channel is opened, the probability of opening is increased, or the open time is increased). If the ligand is a target substance, the target substance can be detected without a specific binding substance. Examples of such channel proteins include hBK and KcsA described in Non-Patent Document 2, and connexins. Examples of the ligand that can be used as a target substance include, but are not limited to, odor substances including volatile organic substances.
実施例１
図１に模式的に示すダブルウェルチャンバーを作製した。パリレン（商品名）フィルム中の透孔の直径は、100μmであり、特許文献１記載の方法で作製した。
Example 1
A double well chamber schematically shown in FIG. 1 was produced. The diameter of the through hole in the parylene (trade name) film was 100 μm, and was prepared by the method described in Patent Document 1.
このダブルウェルチャンバーの一方のウェルに、上記のとおり、溶融したヒドロゲルを入れ、α−ヘモリシンとオメトエート結合性ＤＮＡアプタマーを添加し、ヒドロゲルの固化後、揮発性の有機リン系農薬であるオメトエートを含む空気とヒドロゲルを接触させた。次に、他方のウェルに脂質溶液、次いで緩衝液を入れ、パリレンフィルムの透孔部に脂質二重膜を自動的に形成させ、図１に示す装置を作製した。なお、図１に示すように、各ウェルには電極を配置し、電極間に電圧を印加してその間に流れる電流を測定可能とする、図１に示す回路を形成した。 In one well of this double well chamber, as described above, melted hydrogel is added, α-hemolysin and ometoate-binding DNA aptamer are added, and after hydrogel is solidified, it contains ometoate, a volatile organophosphorus pesticide. Air and hydrogel were contacted. Next, a lipid solution and then a buffer solution were placed in the other well, and a lipid bilayer membrane was automatically formed in the perforated portion of the parylene film, thereby producing the apparatus shown in FIG. As shown in FIG. 1, an electrode is arranged in each well, and a circuit shown in FIG. 1 is formed in which voltage can be applied between the electrodes and current flowing therebetween can be measured.
上記実験の詳細を以下に記載する。 Details of the experiment are described below.
試料・材料
・膜タンパク質α-ヘモリシン： 黄色ブドウ球菌由来の毒素。7量体を形成して脂質二重膜に直径1.5 nmのナノ孔を形成する。Sigma Aldrichより購入。
・DNAアプタマー： AAG CTT TTT TGA CTG ACT GCA GCG ATT CTT GAT CGC CAC GGT CTG GAA AAA GAG CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC (5’→3’)（配列番号１） [1]。Sigma Aldrichより購入。
・脂質： DPhPC（1,2-ジフィタノイル-sn-グリセロ-3-ホスフォコリン）。Avanti Polar Lipids, Inc.より購入。
・油相： n-デカン。和光純薬より購入。
・アガロース： 低融点36℃(1.5% gel, ±1.5 ℃)。Sigma Aldrichより購入。
・緩衝溶液： 1.0 M KCl、10 mM リン酸緩衝溶液、pH 7.0。試薬は和光純薬より購入。
・標的物質オメトエート： 有機リン系農薬、揮発性のある物質、本実施例では残留農薬を気体から検知した。和光純薬より購入。
Sample, material, membrane protein α-hemolysin: A toxin derived from Staphylococcus aureus. A heptamer is formed to form nanopores with a diameter of 1.5 nm in the lipid bilayer. Purchased from Sigma Aldrich.
DNA aptamer: AAG CTT TTT TGA CTG ACT GCA GCG ATT CTT GAT CGC CAC GGT CTG GAA AAA GAG CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC (5 ′ → 3 ′) (SEQ ID NO: 1) [1]. Purchased from Sigma Aldrich.
Lipid: DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine). Purchased from Avanti Polar Lipids, Inc.
-Oil phase: n-decane. Purchased from Wako Pure Chemical.
-Agarose: Low melting point 36 ° C (1.5% gel, ± 1.5 ° C). Purchased from Sigma Aldrich.
Buffer solution: 1.0 M KCl, 10 mM phosphate buffer solution, pH 7.0. Reagents are purchased from Wako Pure Chemical.
-Target substance ometoate: Organophosphorus pesticides, volatile substances, and residual pesticides in this example were detected from gas. Purchased from Wako Pure Chemical.
実験手順
1. 1wt%のアガロースを緩衝溶液で調製する。
2. アース側のチャンバーに60 度に加熱したアガロース溶液を20 μL入れる。
3. ゲル化した後に、500 μMのDNAアプタマーと300 nMヘモリシンをそれぞれ0.4μLと2 μL加え、終濃度をそれぞれ10 μMと30 nMに調製する。
4. 2.1 μLのオメトエートを 10 mLの緩衝溶液に溶かし、1 mMのオメトエート緩衝溶液を調製した後、不織紙に100 μL含ませ、デバイスと不織紙をビニール袋により密封し、1.5時間保持する。（ただし、不織布がアガロース部分などに接触しないよう留意する）
5. 電圧印加側のチャンバーに、オイルに分散させた脂質(DPhPC、20 mg/mL)を5 μL加え、さらに緩衝溶液を20 μL加える。
6. 電圧印加側チャンバーの電極に+100 mVの電圧を印加する。電流計測には、CEZ-2400（日本光電工業株式会社製）およびDigidata 1322a（Molecular Devices製）を用いた。サンプリング周波数は5 kHzとし、ベッセルフィルター1 KHz下にてデータ取得を行った。
Experimental procedure
1. Prepare 1 wt % agarose in buffer solution.
2. Add 20 μL of agarose solution heated to 60 ° C to the earth side chamber.
3. After gelation, add 500 μM DNA aptamer and 300 nM hemolysin to 0.4 μL and 2 μL, respectively, and adjust the final concentrations to 10 μM and 30 nM, respectively.
4. Dissolve 2.1 μL of ometoate in 10 mL of buffer solution to prepare 1 mM ometoate buffer solution, then add 100 μL to the nonwoven paper, seal the device and the nonwoven paper with a plastic bag, and hold for 1.5 hours To do. (However, care should be taken so that the nonwoven fabric does not come into contact with the agarose part, etc.)
5. Add 5 μL of lipid (DPhPC, 20 mg / mL) dispersed in oil and 20 μL of buffer solution to the chamber on the voltage application side.
6. Apply +100 mV to the electrode on the voltage application side chamber. For current measurement, CEZ-2400 (manufactured by Nihon Kohden Co., Ltd.) and Digidata 1322a (manufactured by Molecular Devices) were used. Sampling frequency was 5 kHz and data was acquired under a Bessel filter at 1 KHz.
計測結果
実験手順４により、標的物質であるオメトエートが不織紙から気相へと揮発し、アガロース（水溶液相）との接触によって気相から水溶液相へと分配係数に従って溶解すると考えられる。
Measurement Result According to the experimental procedure 4, it is considered that the target substance, ometoate, volatilizes from the non-woven paper to the gas phase and dissolves from the gas phase to the aqueous solution phase according to the distribution coefficient by contact with agarose (aqueous solution phase).
実験手順5および6によって、接触法[2]の原理に従って、アガロースと緩衝溶液の界面に脂質二重膜が形成される。また、ゲル中のヘモリシンが脂質二重膜に導入し、数分程度で脂質二重膜にナノ孔を形成する。ナノ孔は1.5 nmの最狭部を持ち、1 M KCl溶液中では1 nSの導電率を示す。100 mV印加下では、100 pAのステップとして観測される。 According to the experimental procedures 5 and 6, a lipid bilayer is formed at the interface between the agarose and the buffer solution according to the principle of the contact method [2]. In addition, hemolysin in the gel is introduced into the lipid bilayer, and nanopores are formed in the lipid bilayer within a few minutes. The nanopore has the narrowest part of 1.5 nm and exhibits a conductivity of 1 nS in 1 M KCl solution. Under the application of 100 mV, it is observed as a step of 100 pA.
オメトエート無しの場合（図３左図）
ヘモリシンのナノ孔はセンサ素子、DNAアプタマーは標的物質のインジケータ（指示薬）の役割を果たす。上述の通り、ナノ孔が形成されると1 nSの階段状の電流増加が観察された。強い負電荷を有するDNAアプタマーは、印加電圧により泳動力を受けてナノ孔へ到達し、ナノ孔の入口付近に滞留、あるいはナノ孔を通過する。これらの現象は、ナノ孔のイオン電流を阻害するが、閉塞率が低い（80%に満たない）、あるいは閉塞時間が短い（1秒未満）のブロッキングとして観測される。なお、図３左図の下側の図が実際の測定結果を示す。
Without ometoate (Figure 3 left)
Hemolysin nanopores serve as sensor elements and DNA aptamers serve as target substance indicators (indicators). As described above, a 1 nS stepwise increase in current was observed when nanopores were formed. A DNA aptamer having a strong negative charge receives a migration force due to an applied voltage and reaches the nanopore and stays near or passes through the nanopore. These phenomena inhibit the nanopore ion current, but are observed as blocking with a low occlusion rate (less than 80%) or a short occlusion time (less than 1 second). The lower diagram in FIG. 3 shows the actual measurement results.
オメトエート有りの場合（図３右図）
標的物質（オメトエート）が存在する場合、DNAアプタマーと標的物質が複合体を形成する。この複合体はかさ高いためにナノ孔を通過できない。イオン電流を高い閉塞率（80%以上）で長時間（1秒以上）阻害する結果が得られ、標的物質が溶液中に存在することを示すことができる。なお、図３右図の下側の図が実際の測定結果を示す。
With ometoate (right figure in Fig. 3)
When the target substance (ometoate) is present, the DNA aptamer and the target substance form a complex. This complex is bulky and cannot pass through the nanopores. A result of inhibiting the ionic current at a high occlusion rate (80% or more) for a long time (1 second or more) is obtained, and it can be shown that the target substance is present in the solution. The lower diagram in FIG. 3 shows the actual measurement results.
標的物質判定の閾値の決定
緩衝液に標的物質を混合した条件、あるいは混合しない条件において、図３と同様の電気シグナルを取得し、閉塞現象の1つ1つについて閉塞率および時間を求めて図４を作成した。先行研究により、ナノ孔をDNAが閉塞する場合、3末端のC配列は85%の電流低下率を示すと知られている [3,4]。本実験結果においても、3末端側の長いC配列がナノ孔を閉塞すると考えられるオメトエート存在下では閉塞率90%程度の閉塞現象が多く観察された。その中で、オメトエートの有無を判定するために閉塞時間を加えた閾値を設定すると、閉塞時間1.0s以上かつ閉塞率80%以上が妥当と考えられた。
Determination of threshold value for target substance determination Under the condition where the target substance is mixed in the buffer solution or not, the same electrical signal as in Fig. 3 is obtained, and the obstruction rate and time are determined for each of the obstruction phenomena. 4 was created. Previous studies have shown that when the nanopore is clogged with DNA, the 3-terminal C-sequence exhibits an 85% reduction in current [3,4]. Also in this experimental result, many occlusion phenomena with an occlusion rate of about 90% were observed in the presence of ometoate, which is thought to have a long C sequence on the 3 terminal side blocking the nanopores. Among them, when a threshold value including the occlusion time was set to determine the presence or absence of omethoate, an occlusion time of 1.0 s or more and an occlusion rate of 80% or more were considered appropriate.
文献
[1] L. Wang, X. Liu, Q. Zhang, C. Zhang, Y. Liu, K. Tu, and J. Tu, “Selection of DNA aptamers that bind to four organophosphorus pesticides”, Biotechnol Letters, vol. 34, pp. 869-874, 2012.
[2] K. Funakoshi, H. Suzuki, and S.Takeushi, “Lipid Bilayer Formation by Contacting Monolayers in a Microfluidic Device for Membrane Protein Analysis”, Anal. Chem., vol. 78, pp. 8169-8174, 2006.
[3] M. Akeson, D. Branton, J. J. Kasianowicz, E. Brandin, and D. W. Deamer., “Microsecond Time-Scale Discrimination Among Polycytidylic Acid, Polyadenylic Acid, and Polyuridylic Acid as Homopolymers or as Segments Within Single RNA Molecules”, Biophysical Journal, vol. 77, pp. 3227-3233, 1999.
[4] T. Z. Butler, J. H. Gundlach, and M.Troll, “Ionic Current Blockades from DNA and RNA Molecules in the α-Hemolysin Nanopore”, Biophysical Jounal, vol. 93, pp. 3229-3240, 2007.
Literature
[1] L. Wang, X. Liu, Q. Zhang, C. Zhang, Y. Liu, K. Tu, and J. Tu, “Selection of DNA aptamers that bind to four organophosphorus pesticides”, Biotechnol Letters, vol. 34, pp. 869-874, 2012.
[2] K. Funakoshi, H. Suzuki, and S. Takeushi, “Lipid Bilayer Formation by Contacting Monolayers in a Microfluidic Device for Membrane Protein Analysis”, Anal. Chem., Vol. 78, pp. 8169-8174, 2006.
[3] M. Akeson, D. Branton, JJ Kasianowicz, E. Brandin, and DW Deamer., “Microsecond Time-Scale Discrimination Among Polycytidylic Acid, Polyadenylic Acid, and Polyuridylic Acid as Homopolymers or as Segments Within Single RNA Molecules”, Biophysical Journal, vol. 77, pp. 3227-3233, 1999.
[4] TZ Butler, JH Gundlach, and M. Troll, “Ionic Current Blockades from DNA and RNA Molecules in the α-Hemolysin Nanopore”, Biophysical Jounal, vol. 93, pp. 3229-3240, 2007.
１０ 第１の領域
１１ 第２の領域
１２ 標的物質
１４ 脂質溶液
１６ 水系媒体
１８ 自己支持性フィルム
２０ 脂質二重膜
２１ 電極
２２ チャネルタンパク質
２４ アプタマー
DESCRIPTION OF SYMBOLS 10 1st area | region 11 2nd area | region 12 Target substance 14 Lipid solution 16 Aqueous medium 18 Self-supporting film 20 Lipid bilayer membrane 21 Electrode 22 Channel protein 24 Aptamer
Claims (19)
前記ヒドロゲルと前記脂質溶液との界面に存在する前記脂質二重膜は透孔を有し、前記電流は該透孔を介して流れ、該電流の流れ方が、前記被検試料中に前記標的物質が含まれる場合と含まれない場合とで異なる、気体状の被検試料中に含まれる標的物質の検出装置。 An apparatus for detecting a target substance contained in a gaseous test sample, the hydrogel disposed in a first region in direct contact with the test sample, and adjacent to the hydrogel via a lipid bilayer membrane A lipid bilayer-forming lipid solution / aqueous medium disposed in the second region, and means for applying a voltage between these regions and measuring a current flowing between these regions. ,
The lipid bilayer membrane present at the interface between the hydrogel and the lipid solution has a through-hole, and the current flows through the through-hole, and the current flows through the target in the test sample. An apparatus for detecting a target substance contained in a gaseous test sample, which differs depending on whether or not the substance is contained.
前記被検試料との接触の結果前記標的物質を含む、第１の領域に配置されたヒドロゲルと、該ヒドロゲルと脂質二重膜を介して隣接する、第２の領域に配置された脂質二重膜形成性の脂質溶液／水系媒体との間に電圧を印加してこれらの領域間に流れる電流を測定することを含み、
前記ヒドロゲルと前記脂質溶液との界面に存在する前記脂質二重膜は、透孔を有しており、該透孔を介して前記第１の領域と第２の領域は連通しており、前記電流は該透孔を介して流れ、該電流の流れ方が、前記被検試料中に前記標的物質が含まれる場合と含まれない場合とで異なり、測定した電流に基づき標的物質を検出する、気体状の被検試料中に含まれる標的物質の検出方法。 A method for detecting a target substance contained in a gaseous test sample,
As a result of contact with the test sample, the hydrogel disposed in the first region containing the target substance, and the lipid duplex disposed in the second region adjacent to the hydrogel via the lipid bilayer membrane Applying a voltage between the membrane-forming lipid solution / aqueous medium and measuring the current flowing between these regions,
The lipid bilayer membrane present at the interface between the hydrogel and the lipid solution has a pore, and the first region and the second region communicate with each other through the pore, An electric current flows through the through-hole, and the flow of the electric current is different depending on whether or not the target substance is included in the test sample, and the target substance is detected based on the measured current. A method for detecting a target substance contained in a gaseous test sample.
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| CN108251428A (en) * | 2018-02-05 | 2018-07-06 | 北京化工大学 | A kind of aptamers for identifying Determination of Organophosphorus Pesticide and its application |
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| JP7353597B2 (en) | 2019-10-04 | 2023-10-02 | 地方独立行政法人神奈川県立産業技術総合研究所 | Measuring instrument and method for measuring target substances using it |
| JP7511186B2 (en) * | 2019-12-10 | 2024-07-05 | 地方独立行政法人神奈川県立産業技術総合研究所 | Volatile substance concentration estimation device, volatile substance concentration sensor, volatile substance concentration estimation method and program |
| JP7526421B2 (en) * | 2020-05-26 | 2024-08-01 | 地方独立行政法人神奈川県立産業技術総合研究所 | Method for forming lipid bilayer membrane, and partition and device therefor |
| CN113109406B (en) * | 2021-04-19 | 2023-04-07 | 江苏大学 | Method for constructing electrochemiluminescence aptamer sensor for omethoate detection |
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| CN108251428A (en) * | 2018-02-05 | 2018-07-06 | 北京化工大学 | A kind of aptamers for identifying Determination of Organophosphorus Pesticide and its application |
| CN108251428B (en) * | 2018-02-05 | 2020-04-10 | 北京化工大学 | Aptamer for identifying various organophosphorus pesticides and application thereof |
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