JP7333928B2 - Measurement method for electrochemical unlabeled nucleic acid detection - Google Patents
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Description
本発明は、電気的化学的に核酸を検出する方法に関する。 The present invention relates to methods for electrochemically detecting nucleic acids.
核酸と呼ばれるＤＮＡやＲＮＡは、生体の遺伝情報を含むことから遺伝子診断や細菌叢の同定などに利用されている。また最近では、血清や尿、唾液に存在するＲＮＡ（マイクロＲＮＡ）が、がん診断の指標として利用できる可能性が示され、ヘルスケア用途での在宅で利用できるセンサとして核酸検出技術の実用化が望まれている。 DNA and RNA, which are called nucleic acids, contain the genetic information of living organisms, and are therefore used for genetic diagnosis, identification of bacterial flora, and the like. Recently, it has been shown that RNA (microRNA) present in serum, urine, and saliva can be used as an index for cancer diagnosis, and the practical application of nucleic acid detection technology as a sensor that can be used at home for healthcare purposes. is desired.
これまで核酸を検出する技術としては検出対象となるターゲット核酸と相互作用する捕捉核酸を溶液中で混合、またはターゲット核酸を捕捉核酸を固定した基板上で作用させ、相互作用した二本鎖核酸に特異的に作用する蛍光標識物質または相互作用に伴って蛍光が変化する予め核酸に標識された蛍光物質を光学装置を用いて検出する方法が用いられてきた。検出感度が優れている一方で大型・高価で精密な光学装置を必要とする点で、在宅利用を想定したセンサとしては不向きであった。 Until now, as a technique for detecting nucleic acids, the capture nucleic acid that interacts with the target nucleic acid to be detected is mixed in a solution, or the target nucleic acid is allowed to act on a substrate on which the capture nucleic acid is immobilized, and the interacting double-stranded nucleic acid A method has been used in which an optical device is used to detect a fluorescent labeling substance that acts specifically or a fluorescent substance that is previously labeled on a nucleic acid and whose fluorescence changes upon interaction. Although it has excellent detection sensitivity, it is not suitable as a sensor for home use because it requires a large, expensive, and precise optical device.
また、ターゲット核酸と捕捉核酸が相互作用した二本鎖核酸に特異的に作用する電子メディエーター標識物質を電気化学装置で検出する原理のセンサ開発も報告されているが（特許文献１、２を参照のこと）、電気化学式は装置の小型化に有利である一方、核酸特異的試薬や標識が依然として必要という問題点がある。二本鎖核酸に特異的に作用する蛍光および電子メディエーター試薬や標識物質は発がん性を有する潜在的な危険性を否定できず、取り扱いや廃棄も含めて在宅での利用では安全性の確保が問題となる。 In addition, the development of a sensor based on the principle of detecting an electron mediator-labeled substance that specifically acts on a double-stranded nucleic acid in which a target nucleic acid and a capture nucleic acid interact with an electrochemical device has been reported (see Patent Documents 1 and 2). ), while the electrochemical method is advantageous for miniaturization of the device, it still requires nucleic acid-specific reagents and labels. Fluorescent and electronic mediator reagents and labeling substances that act specifically on double-stranded nucleic acids cannot be denied the potential danger of carcinogenicity. becomes.
非標識で核酸を検出する技術としてインピーダンスを測定（特許文献３、４）やＦＥＴの伝達特性変化を測定（非特許文献１）する方法がある。しかしインピーダンス測定やＦＥＴの伝達特性変化の測定には電流と電圧を同時に制御かつ測定が高精度に可能な大型な装置が必要であり、在宅での利用を想定した小型の装置化は困難である。 Techniques for detecting nucleic acids without labeling include a method for measuring impedance (Patent Documents 3 and 4) and a method for measuring changes in transfer characteristics of FET (Non-Patent Document 1). However, impedance measurement and measurement of changes in transfer characteristics of FET require a large-scale device capable of simultaneously controlling and measuring current and voltage at the same time. .
これまで在宅で利用できる核酸センサが実現しないのは、上記の問題点があったためである。 The reason why a nucleic acid sensor that can be used at home has not been realized so far is because of the above problems.
核酸はリン酸基に負電荷イオンを持っており、その負電荷を電気化学的に測定することができれば標識物質を用いることなく核酸の検出が可能である。しかしターゲット核酸をセンサ基板上の捕捉核酸と相互作用させて二本鎖核酸として検出する場合には、核酸のリン酸基の負電荷イオンによる核酸同士の静電反発を抑えて結合させるために一般的には生理食塩水と同程度の１００から２００ｍＭの濃度の塩化ナトリウム（ＮａＣｌ）の存在下で実施する必要がある。 Nucleic acids have negatively charged ions in their phosphate groups, and if the negative charges can be electrochemically measured, nucleic acids can be detected without using a labeling substance. However, when the target nucleic acid is allowed to interact with the captured nucleic acid on the sensor substrate and is detected as a double-stranded nucleic acid, the electrostatic repulsion between the nucleic acids caused by the negatively charged ions of the phosphate groups of the nucleic acids is suppressed and binding is generally performed. Practically, it should be performed in the presence of sodium chloride (NaCl) at a concentration of 100 to 200 mM, which is similar to physiological saline.
電気化学においてイオン溶液中で電極の溶液界面での電荷密度変化を測定する場合には溶液内のイオンが動いて電場を遮蔽するデバイ遮蔽が生じ、遮蔽が有効となる電極からの距離であるデバイ長は溶液中のイオン強度に依存する。デバイ長は数式１で表される。δがデバイ長、εは比誘電率、ε０は真空の誘電率、ｋはボルツマン定数、Ｔは絶対温度、ｑは電荷、Ｉはイオン強度である。
センサ基板からの距離について、バイオセンサで一般的に生体分子を基板に固定化する方法であるアビジン－ビオチン結合法で、センサ基板１の表面にアビジンタンパク質２を介して捕捉核酸として１０塩基ビオチン化核酸３を固定しターゲット核酸として１０塩基核酸４を作用させた場合のそれぞれの分子の大きさとデバイ長δとのおおよその関係を図１に示す。 Regarding the distance from the sensor substrate, biotinylation of 10 bases as a capture nucleic acid on the surface of the sensor substrate 1 via avidin protein 2 is performed by the avidin-biotin binding method, which is a method of generally immobilizing biomolecules on a substrate in biosensors. FIG. 1 shows the approximate relationship between the size of each molecule and the Debye length δ when the nucleic acid 3 is immobilized and the 10-base nucleic acid 4 is allowed to act as the target nucleic acid.
アビジンタンパク質はおよそ６ｎｍ×３ｎｍの楕円状球体であり二本鎖核酸の１０塩基当たりの長さは３．４ｎｍである。測定にはセンサ基板から６．４ｎｍあるいは９．４ｎｍの範囲内での検出が必要である。それに対して１００から２００ｍＭの濃度の塩化ナトリウム（ＮａＣｌ）溶液中でのデバイ長δは数式１より０．７ｎｍから１ｎｍと算出される。二本の核酸の相互作用はデバイ遮蔽の外側で起こっていて二本鎖形成に伴う核酸のリン酸基の負電荷イオンの増加を電荷密度の増加として検出することはできない。また捕捉核酸として１０塩基核酸５をセンサ基板上に直接固定できたとしても３分の２以上はデバイ遮蔽の外に出るため正しく測定することができない。 The avidin protein is an ellipsoidal sphere of approximately 6 nm×3 nm, and the length per 10 bases of the double-stranded nucleic acid is 3.4 nm. Measurement requires detection within 6.4 nm or 9.4 nm from the sensor substrate. On the other hand, the Debye length δ in a sodium chloride (NaCl) solution with a concentration of 100 to 200 mM is calculated from Equation 1 to be 0.7 nm to 1 nm. Since the interaction of two nucleic acids occurs outside the Debye shield, the increase in negatively charged ions on the phosphate groups of the nucleic acids accompanying double strand formation cannot be detected as an increase in charge density. Moreover, even if the 10-base nucleic acid 5 can be directly immobilized on the sensor substrate as a capture nucleic acid, two-thirds or more of it is outside the Debye shielding, so that it cannot be measured correctly.
上記のように、核酸を検出する方法として二本鎖核酸形成を検出する場合、核酸同士の負電荷による静電反発を緩和するため高濃度の塩化ナトリウム（ＮａＣｌ）溶液中で相互作用させる必要があるが、電気化学的には電荷を検出できる電極からの距離、デバイ長が分子サイズ以下に短くなってしまい、核酸本来のリン酸基の負電荷に基づく電荷密度の変化として核酸を検出することができない二律背反の問題が生じる。 As described above, when detecting double-stranded nucleic acid formation as a method of detecting nucleic acids, it is necessary to interact in a high-concentration sodium chloride (NaCl) solution in order to reduce electrostatic repulsion due to negative charges between nucleic acids. However, electrochemically, the distance from the electrode where the charge can be detected, the Debye length, is shorter than the molecular size, and the nucleic acid cannot be detected as a change in charge density based on the negative charge of the phosphate group inherent in the nucleic acid. There is a problem of antinomy that cannot be done.
本発明の目的は、核酸同士の静電反発を抑えて二本鎖核酸を形成させつつ核酸が検出可能なデバイ長を確保できる方法を提供することで、電気化学式において核酸特異的標識試薬を用いることなく汎用的な電位差測定で核酸を検出可能にすることである。 An object of the present invention is to provide a method that can secure a Debye length that allows nucleic acids to be detected while suppressing electrostatic repulsion between nucleic acids to form a double-stranded nucleic acid, using a nucleic acid-specific labeling reagent in an electrochemical method. It is to enable detection of nucleic acids by general-purpose potentiometric measurement without using
本発明における核酸の二本鎖形成を検出する測定方法は、核酸が本来持つリン酸基の負電荷イオンを使って電荷密度の変化として核酸を標識試薬を用いることなく測定する方法であって、核酸同士の静電反発を抑えるために塩化ナトリウムの代わりに生体由来の有機多価カチオン分子、例えば、スペルミジンを用いたこと、電極からの距離を短くするために捕捉核酸を電極から約１ｎｍの距離で直接固定したことである。 The measurement method for detecting double-strand formation of a nucleic acid in the present invention is a method for measuring a nucleic acid as a change in charge density using negatively charged ions of a phosphate group inherent in the nucleic acid without using a labeling reagent, In order to suppress the electrostatic repulsion between nucleic acids, a bio-derived organic polyvalent cation molecule such as spermidine was used instead of sodium chloride. It is fixed directly by
本発明は、生体由来の有機多価カチオン分子を用いることで１ｍＭ程度の低濃度の陽電荷イオンであっても多点相互作用により核酸のリン酸アニオンと作用し核酸のリン酸アニオンの静電反発を抑制し、かつ、溶液中のイオン強度を低下させてデバイ長を分子サイズと同程度にまで確保したことと合わせて捕捉核酸を電極から約１ｎＭの距離で直接固定したことで、ターゲット核酸の結合を標識試薬を用いずに核酸本来の負電荷イオンに基づく電極上の電荷密度変化として汎用的な電位差計で経時的に測定できるようになった。 In the present invention, by using organic polyvalent cation molecules derived from living organisms, even positively charged ions at a low concentration of about 1 mM act with the phosphate anions of nucleic acids through multipoint interactions, and the static electricity of the phosphate anions of nucleic acids is achieved. The target nucleic acid was directly fixed at a distance of about 1 nM from the electrode by suppressing the repulsion and reducing the ionic strength in the solution to secure the Debye length to the same extent as the molecular size. binding can be measured over time by a general-purpose potentiometer as a change in charge density on the electrode based on the negatively charged ions inherent in nucleic acids without using a labeling reagent.
また用いる試薬の安全性が高いため、研究・開発の現場だけでなく、病院や一般家庭での日常診断用の核酸センサ用途として用いることができる。 In addition, since the reagent used is highly safe, it can be used not only in research and development sites but also as a nucleic acid sensor for daily diagnosis in hospitals and general households.
以下、本発明の実施の形態について説明する。
図２は、電極６上に捕捉核酸として任意の配列をもつ１０塩基ＤＮＡ７を固定した核酸センサの表面設計を示す模式図である。電極の表面素材には金を使用する。ＤＮＡ７は５’末端にアミノ基をもつものを用いてＤＮＡ７をアミド結合を介して炭素鎖長２の末端にカルボキシル基を持つアルキルチオール８で金電極６の表面に固定する。金とアルキルチオールは金－チオール吸着で固定される。アルキルチオールと捕捉ＤＮＡのアミド結合は水溶性の縮合剤を用いたアミンカップリング反応により生成させた。ＤＮＡ７が固定されていない金電極の隙間は炭素鎖長２の末端に水酸基を持つアルキルチオール９を固定する。電極表面からＤＮＡの末端までの距離はおよそ４．４ｎｍほどである。
BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below.
FIG. 2 is a schematic diagram showing the surface design of a nucleic acid sensor in which a 10-base DNA 7 having an arbitrary sequence is immobilized on an electrode 6 as a capture nucleic acid. Gold is used as the surface material of the electrodes. A DNA 7 having an amino group at the 5' end is used, and the DNA 7 is immobilized on the surface of the gold electrode 6 via an amide bond with an alkylthiol 8 having a carbon chain length of 2 and having a carboxyl group at the end. Gold and alkylthiols are immobilized by gold-thiol adsorption. An amide bond between the alkylthiol and the capture DNA was formed by an amine coupling reaction using a water-soluble condensing agent. Alkyl thiol 9 having a hydroxyl group at the end of carbon chain length 2 is fixed in the gap of the gold electrode where DNA 7 is not fixed. The distance from the electrode surface to the end of the DNA is approximately 4.4 nm.
図３に電気化学測定を行うための実験装置の模式図を示す。汎用的な電位差計１０に捕捉ＤＮＡを固定したセンサ電極１１およびガラス参照電極１２を接続する。水溶液を満たしたビーカー１３にセンサ電極１１およびガラス参照電極１２を浸し水溶液は撹拌子１４で撹拌する。水溶液としてターゲットＤＮＡの検出実験では塩化ナトリウムの代わりに有機多価カチオン分子としてスペルミジン２００μＭを含む溶液中において実施する。 FIG. 3 shows a schematic diagram of an experimental apparatus for performing electrochemical measurements. A sensor electrode 11 with captured DNA immobilized thereon and a glass reference electrode 12 are connected to a general-purpose potentiometer 10 . The sensor electrode 11 and the glass reference electrode 12 are immersed in a beaker 13 filled with an aqueous solution, and the aqueous solution is stirred with a stirrer 14 . The target DNA detection experiment in an aqueous solution is carried out in a solution containing 200 μM spermidine as an organic polyvalent cation molecule instead of sodium chloride.
捕捉ＤＮＡと配列が相補的なターゲットＤＮＡまたは１塩基のみ相補的でないミスマッチＤＮＡとの相互作用による電荷密度の変化をそれぞれ参照電極との電位差変化として電位差計で測定する。それぞれの測定結果の比較において核酸中の１塩基のミスマッチに起因する結合挙動の差異から配列の違いを検出する。 The change in charge density due to the interaction between the captured DNA and the target DNA that is complementary in sequence or the mismatched DNA that is not complementary in only one base is measured by a potentiometer as a change in potential difference with respect to the reference electrode. A difference in sequence is detected from a difference in binding behavior caused by a single base mismatch in the nucleic acid when comparing the measurement results.
［実施例１］
以下、本発明の測定法を用いた非標識核酸検出の方法の実施例を示す。
核酸には以下の配列の１０塩基のＤＮＡ（ユーロフィンジェノミクス社製）を使用した。
捕捉ＤＮＡ ＮＨ２－５’－ＡＧＣＴＴＧＧＧＡＡ－３’
ターゲットＤＮＡ ３’－ＴＣＧＡＡＣＣＣＴＴ－５’
ミスマッチＤＮＡ ３’－ＴＣＧＡＣＣＣＣＴＴ－５’
[Example 1]
Examples of methods for detecting unlabeled nucleic acids using the assay method of the present invention are shown below.
A 10-base DNA (manufactured by Eurofins Genomics) having the following sequence was used as the nucleic acid.
capture DNA NH 2 -5′-AGCTTGGGAA-3′
target DNA 3'-TCGAACCCTT-5'
Mismatched DNA 3'-TCGA C CCCTT-5'
金電極の表面の洗浄のためにＰｉｒａｎｈａ溶液（硫酸：過酸化水素水＝３：１）を滴下して５分間放置した後に超純水で洗い流した。さらにこの操作を２回繰り返した。４ｍＭの３，３’－Ｄｉｔｈｉｏｄｉｐｒｏｐｉｏｎｉｃ ａｃｉｄと４０ｍＭのＢｉｓ（２－ｈｙｄｒｏｘｙｅｔｈｙｌ）ｄｉｓｕｌｆｉｄｅを含む混合水溶液を調製し、混合水溶液を洗浄した金電極に滴下して３０分間室温で放置した。その後、金電極を超純水で洗浄した。０．５２Ｍの（１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩水溶液と０．８７ＭのＮ－ヒドロキシスクシンイミド水溶液を等量混合し、混合溶液を金表面に滴下し３０分間室温で放置した。反応後、超純水で洗浄した。１μＭの捕捉ＤＮＡを含む１０ｍＭのＨＥＰＥＳ－ＮａＯＨ（ｐＨ８．０）緩衝液を滴下し６０分間のアミンカップリング反応を行った。その反応溶液に反応後５ｍＭのエタノールアミンを含む１０ｍＭのＨＥＰＥＳ－ＮａＯＨ（ｐＨ８．０）緩衝液を等量添加し１０分間放置した。超純水でセンサ表面を洗浄後、参照電極（ＢＡＳ社製、ＲＥ－１Ｂ）とともに電位差計（Ａｇｉｌｅｎｔ社製３４４０５Ａ）にセットした。ビーカーには２００μＭのスペルミジンを含む水溶液を５ｍＬ入れた。水溶液の温度を２０℃に調節して、終濃度（ｆ．ｃ．）が５０ｎＭになるようにターゲットＤＮＡを測定ビーカーへ添加した。測定後、金電極を超純水で洗浄し、再びビーカー１３に２００μＭのスペルミジンを含む水溶液に入れ換えて、ミスマッチＤＮＡを添加した。 For cleaning the surface of the gold electrode, a Piranha solution (sulfuric acid:hydrogen peroxide solution=3:1) was dropped and left for 5 minutes, followed by rinsing with ultrapure water. Furthermore, this operation was repeated twice. A mixed aqueous solution containing 4 mM 3,3'-Dithiodipropionic acid and 40 mM Bis(2-hydroxyethyl) disulfide was prepared, and the mixed aqueous solution was dropped onto the washed gold electrode and left at room temperature for 30 minutes. After that, the gold electrode was washed with ultrapure water. Equal amounts of 0.52 M (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution and 0.87 M N-hydroxysuccinimide aqueous solution were mixed, and the mixed solution was dropped onto the gold surface and left at room temperature for 30 minutes. After the reaction, the reaction solution was washed with ultrapure water, and a 10 mM HEPES-NaOH (pH 8.0) buffer solution containing 1 μM capture DNA was added dropwise to carry out an amine coupling reaction for 60 minutes. Then, an equal volume of 10 mM HEPES-NaOH (pH 8.0) buffer containing 5 mM ethanolamine was added and allowed to stand for 10 minutes.After washing the sensor surface with ultrapure water, a reference electrode (manufactured by BAS, RE-1B) was added. 5 mL of an aqueous solution containing 200 μM spermidine was placed in a beaker, and the temperature of the aqueous solution was adjusted to 20° C. to a final concentration (f.c.) of 50 nM. After the measurement, the gold electrode was washed with ultrapure water, the beaker 13 was again replaced with an aqueous solution containing 200 μM spermidine, and mismatched DNA was added.
測定結果を図４に示す。
ターゲットＤＮＡを添加した場合には結合に伴う負電荷密度の増加による電極電位の減少１５が観察された。一方、ミスマッチＤＮＡの添加では電極電位が変化しない様子１６が観察された。従って検出対象である核酸を標識物質を用いることなく汎用的な電位差測定装置で経時的に捕捉ＤＮＡに結合する様子を測定でき、一塩基のみ配列が異なる核酸を識別できる核酸センサとなることが示された。
以上、説明した電気化学的に非標識で核酸を検出することのできる核酸センサは、潜在的に発がん性を有する危険な標識物質を用いることなく汎用的な安価でシンプルな電位差系で核酸の一塩基ミスマッチの違いを検出できることから、安全、小型で安価な核酸センサが実現でき、専用の施設での利用に限らず在宅でのヘルスケア用途向けの核酸センサの手法として用いることができる。
The measurement results are shown in FIG.
When target DNA was added, a decrease in electrode potential 15 due to an increase in negative charge density accompanying binding was observed. On the other hand, it was observed that the addition of mismatched DNA did not change the electrode potential 16 . Therefore, it is possible to measure how the nucleic acid to be detected binds to the captured DNA over time with a general-purpose potentiometric device without using a labeling substance. was done.
As described above, the nucleic acid sensor that can electrochemically detect nucleic acids without labeling is a versatile, inexpensive, and simple potentiometric system that does not use potentially carcinogenic and dangerous labeling substances. Since the difference in base mismatches can be detected, a safe, compact, and inexpensive nucleic acid sensor can be realized, and it can be used as a nucleic acid sensor method not only for use in dedicated facilities but also for home healthcare applications.
１ センサ基板
２ アビジンタンパク質
３ 捕捉用１０塩基ビオチン化核酸
４ ターゲット１０塩基核酸
５ 捕捉用１０塩基核酸
６ 金電極
７ １０塩基核酸
８ アミンカップリングでＤＮＡと結合した炭素鎖長２のアルキルチオール
９ 末端が水酸基の炭素鎖長２のアルキルチオール
１０ 電位差計
１１ センサ電極
１２ ガラス参照電極
１３ ビーカー
１４ 撹拌子
１５ 捕捉用ＤＮＡへのターゲットＤＮＡ添加による電位差応答
１６ 捕捉用ＤＮＡへの一塩基ミスマッチＤＮＡ添加による電位差応答
1 sensor substrate 2 avidin protein 3 10-base biotinylated nucleic acid for capture 4 target 10-base nucleic acid 5 10-base nucleic acid for capture 6 gold electrode 7 10-base nucleic acid 8 alkylthiol with a carbon chain length of 2 bound to DNA by amine coupling 9 end 10 Potentiometer 11 Sensor electrode 12 Glass reference electrode 13 Beaker 14 Stirrer 15 Potential difference response due to addition of target DNA to capture DNA 16 Potential difference due to addition of single base mismatch DNA to capture DNA response
Claims (3)
核酸のリン酸アニオンの静電反発を抑えるため、検出対象となる核酸よりも大きさの小さい有機多価カチオン分子を使用して、前記核酸と塩基配列既知の核酸とで二本鎖核酸を形成し、
前記塩基配列既知の核酸をセンサ表面に末端で固定するとともに、電位差計を用いる核酸検出の測定方法。 A nucleic acid detection method that does not use a labeling substance,
In order to suppress the electrostatic repulsion of the phosphate anion of the nucleic acid , an organic polyvalent cation molecule smaller in size than the nucleic acid to be detected is used to form a double-stranded nucleic acid with the nucleic acid and a nucleic acid with a known base sequence. death,
A measurement method for nucleic acid detection using a potentiometer while immobilizing the nucleic acid having a known base sequence on the sensor surface at the terminal thereof .
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| JP2002223755A (en) | 2000-11-30 | 2002-08-13 | Toshiba Corp | Method and apparatus for detecting nucleic acid and container for detecting nucleic acid |
| JP2003090815A (en) | 2001-09-18 | 2003-03-28 | Japan Science & Technology Corp | Method for electrochemically detecting gene, and nucleic acid tip |
| JP2004097173A (en) | 2002-07-17 | 2004-04-02 | Toyo Kohan Co Ltd | Solid support having electrostatic layer and its use |
| JP2007512810A (en) | 2003-11-10 | 2007-05-24 | ジーンオーム サイエンシーズ、インク． | Nucleic acid detection method with increased sensitivity |
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| JP2003090815A (en) | 2001-09-18 | 2003-03-28 | Japan Science & Technology Corp | Method for electrochemically detecting gene, and nucleic acid tip |
| JP2004097173A (en) | 2002-07-17 | 2004-04-02 | Toyo Kohan Co Ltd | Solid support having electrostatic layer and its use |
| JP2007512810A (en) | 2003-11-10 | 2007-05-24 | ジーンオーム サイエンシーズ、インク． | Nucleic acid detection method with increased sensitivity |
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