JP2019132711A - Mass measurement kit and mass measurement method - Google Patents
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本発明は、質量測定キット及び質量測定方法に関する。 The present invention relates to a mass measurement kit and a mass measurement method.
水晶等の圧電体を用いて形成された圧電振動子や、マイクロカンチレバーをはじめとしたＭＥＭＳ共振器などの機械共振子上に何らかの物質が付着すると、その質量に応じて共振周波数が変化することが知られている。この周波数変化は極めて鋭敏であることから、ナノグラム以下の微小な質量を検出する質量センサが各種開発されている。例えば、共振子として水晶振動子を用いたＱＣＭ（Quartz Crystal Microbalance）センサが知られている。このような質量センサは、蒸着等の各種成膜装置における膜厚のモニタリングのほか、抗体など分子認識機能を有した官能膜を用いることにより、バイオセンサもしくは化学センサとして幅広い検出対象系に利用することができる。 If any substance adheres to a mechanical resonator such as a piezoelectric resonator formed using a piezoelectric material such as quartz or a MEMS resonator such as a microcantilever, the resonance frequency may change according to the mass of the material. Are known. Since this frequency change is extremely sensitive, various mass sensors that detect a minute mass of nanogram or less have been developed. For example, a QCM (Quartz Crystal Microbalance) sensor using a crystal resonator as a resonator is known. Such a mass sensor is used for a wide range of detection target systems as a biosensor or a chemical sensor by using a functional film having a molecular recognition function such as an antibody in addition to monitoring a film thickness in various film forming apparatuses such as vapor deposition. be able to.
特にバイオセンサとして利用する場合、測定対象となる生体分子等は一般に血液を始めとした液相に溶解していることから、液相測定のための発振回路や装置構成が積極的に開発されている。また、測定対象となる生体分子等は極めて低濃度であることも多いため、対象の生体分子と特異的に結合する抗体等を修飾することにより、測定対象物質に増感用の粒子を結合させることで質量を増幅する方法が知られている（例えば特許文献１参照）。 In particular, when used as a biosensor, the biomolecules to be measured are generally dissolved in the liquid phase including blood. Therefore, oscillation circuits and device configurations for liquid phase measurement have been actively developed. Yes. In addition, since the biomolecules to be measured are often in a very low concentration, sensitizing particles are bound to the substance to be measured by modifying an antibody that specifically binds to the target biomolecule. Thus, a method for amplifying mass is known (see, for example, Patent Document 1).
また、一般的な圧電振動子においては圧電基板の両面に電界発生用の電極及びその配線が配置されており、質量センサとして利用する際に実装の自由度が制限されていた。そこで、利便性向上のために無線式の圧電振動子（例えば特許文献２参照）、配線を片面のみにした圧電振動子（例えば特許文献３参照）が開発されている。 In general piezoelectric vibrators, electrodes for generating an electric field and wirings thereof are arranged on both surfaces of a piezoelectric substrate, and the degree of freedom in mounting is limited when used as a mass sensor. In order to improve convenience, a wireless piezoelectric vibrator (see, for example, Patent Document 2) and a piezoelectric vibrator having wiring on only one side (see, for example, Patent Document 3) have been developed.
高感度な質量センサの応用例として、例えば各種蛋白質マーカーやウイルスの検査などのＰＯＣＴ（Point of Care Testing）と呼ばれる検査が挙げられる。ＰＯＣＴ以外にも、食品検査、環境測定用途などの応用が期待されるが、いずれの分野においてもサンプリングされたマイクロ・ナノリットル単位の試料から対象物質を簡便な方法により迅速かつ高感度に測定することが求められる。 As an application example of a high-sensitivity mass sensor, there is an inspection called POCT (Point of Care Testing) such as inspection of various protein markers and viruses. In addition to POCT, applications such as food inspection and environmental measurement are expected. In any field, the target substance can be measured quickly and with high sensitivity using a sampled micro / nanoliter unit. Is required.
特許文献２に開示された無線式の圧電振動子によると、圧電振動子に配線を構成することが不要であるので、実装の自由度を高めることができる。しかし、このような無線式の圧電振動子では、アンテナを介して誘導電圧を発生させるために特殊な回路が必要であり、ＰＯＣＴなど簡便さが求められる用途には適していない。また、特許文献３に開示された片面励起型の圧電振動子では、表面の配線は不要であるが裏面への配線は依然として必要であり、実装の自由度に改善の余地があった。 According to the wireless piezoelectric vibrator disclosed in Patent Document 2, since it is not necessary to configure wiring in the piezoelectric vibrator, the degree of freedom in mounting can be increased. However, such a wireless piezoelectric vibrator requires a special circuit for generating an induced voltage via an antenna, and is not suitable for applications that require simplicity such as POCT. Further, in the single-side excitation type piezoelectric vibrator disclosed in Patent Document 3, wiring on the front surface is not necessary, but wiring on the back surface is still necessary, and there is room for improvement in freedom of mounting.
本発明の目的は、機械共振の周波数変動から質量を検出する際、高価で特殊な回路及び装置を使用せずに、簡便に迅速かつ高感度な測定が可能な質量測定装置及び質量測定キット並びに質量測定方法を提供することにある。 An object of the present invention is to provide a mass measuring device, a mass measuring kit, and a mass measuring device that can easily and quickly perform high-sensitivity measurement without using an expensive and special circuit and device when detecting mass from frequency fluctuations of mechanical resonance. It is to provide a mass measuring method.
圧電振動子を利用した質量センサにおいては、Sauerbreyの式により圧電振動子上に付着した質量Δｍと共振周波数の周波数変化Δｆは比例するため、特許文献１に示された手法を用いれば、測定対象の生体分子との増感用の粒子の質量差により、数式上は十万倍以上の信号増幅が可能になる。 In a mass sensor using a piezoelectric vibrator, the mass Δm adhering to the piezoelectric vibrator and the frequency change Δf of the resonance frequency are proportional to each other according to the Sauerbrey equation. Due to the mass difference between the sensitizing particles and the biomolecules, it is possible to amplify the signal by a factor of 100,000 or more.
しかし、液相での測定においては、実際の増幅率としては数倍から数十倍程度の増幅にとどまることが多い。この原因としては、液相であることによって、測定対象物質が溶媒和により既に重くなっていること、官能膜及び測定対象物質を介して結合しているため共振子表面から遠くなり振動が減衰していること、さらに測定対象物質よりも増感用の粒子のサイズが大きく立体障害が生じていること、などが挙げられる。 However, in the measurement in the liquid phase, the actual amplification factor is often only about several to several tens of times. This is because the measurement target substance is already heavy due to solvation due to the liquid phase, and the vibration is attenuated by being far from the surface of the resonator because it is bonded via the functional film and the measurement target substance. In addition, the size of the sensitizing particles is larger than that of the substance to be measured, and steric hindrance occurs.
一方で、気相における測定は研究室レベルでは行われてきたが、簡便で迅速な測定を行う実用的な方法は知られていない。気相での測定のためには、まず、液相で測定対象物質を電極に吸着させた後に乾燥させて気相とする必要があり、液相での測定よりも余分な操作が必要になる。また、乾燥操作は測定対象物質を吸着させた電極上で行う必要があるため、乾燥状態のばらつきが大きく、誤差の元になっていた。 On the other hand, measurements in the gas phase have been performed at the laboratory level, but no practical method for simple and rapid measurement is known. For measurement in the gas phase, it is necessary to first adsorb the substance to be measured in the liquid phase to the electrode and then dry it to form the gas phase, which requires extra operations than the measurement in the liquid phase. . In addition, since the drying operation needs to be performed on the electrode on which the measurement target substance is adsorbed, the variation in the dry state is large, which causes an error.
本発明は、
試料液中の測定対象の物質を気相で測定するためのキットであって、
（ａ）測定対象の物質と結合する第一の結合パートナーを表面に固定した増感用の粒子、
（ｂ） 表面に第一の浮遊電極と第二の浮遊電極が配設された圧電基板；及び
前記圧電基板の裏面と対向する面上に第一の励振電極と第二の励振電極が配設された絶縁基板；を備え、
前記第一の浮遊電極は前記第一の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置され、
前記第二の浮遊電極は前記第二の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置され、
前記第一の浮遊電極上には前記圧電基板と接する側と反対の表面に測定対象の物質と結合する第二の結合パートナーが固定化され、
前記圧電基板は前記絶縁基板から分離可能であることを特徴とする、圧電振動子；
を含んでなるキットを提供する。
The present invention
A kit for measuring a substance to be measured in a sample solution in a gas phase,
(A) sensitizing particles in which a first binding partner that binds to a substance to be measured is fixed on the surface;
(B) a piezoelectric substrate having a first floating electrode and a second floating electrode disposed on the surface; and a first excitation electrode and a second excitation electrode disposed on a surface opposite to the back surface of the piezoelectric substrate. An insulated substrate;
The first floating electrode is disposed on a straight line passing through the center of the first excitation electrode and orthogonal to the plate surface of the piezoelectric substrate,
The second floating electrode is disposed on a straight line passing through the center of the second excitation electrode and orthogonal to the plate surface of the piezoelectric substrate,
On the first floating electrode, a second binding partner that binds to a substance to be measured is immobilized on the surface opposite to the side in contact with the piezoelectric substrate,
A piezoelectric vibrator, wherein the piezoelectric substrate is separable from the insulating substrate;
A kit comprising:
本発明はまた、
（ｉ）第一の浮遊電極と第二の浮遊電極が配設された圧電基板、及び
第一の励振電極と第二の励振電極が配設された絶縁基板、を用意する工程、
（ｉｉ）前記第一の浮遊電極上に測定対象の物質と結合する第二の結合パートナーを固定する工程、
（ｉｉｉ）前記圧電基板の裏面と対向する面上に絶縁基板の第一の励振電極と第二の励振電極が配置され、
前記第一の浮遊電極は前記第一の励振電極の間を通って前記圧電基板の板面に直交する直線上に配置され、かつ
前記第二の浮遊電極は前記第二の励振電極の間を通って前記圧電基板の板面に直交する直線上に配置されるように前記圧電基板を前記絶縁基板に固定する工程、
（ｉｖ）前記第一の励振電極及び前記第二の励振電極に交流電圧を印加し、共振周波数を測定する工程、
（ｖ）前記第一の浮遊電極及び前記第二の浮遊電極に試料液を導入する工程、
（ｖｉ）前記第一の浮遊電極及び前記第二の浮遊電極に、測定対象の物質と結合する第一の結合パートナーを表面に固定した増感用の粒子を含む溶液を導入する工程、
（ｖｉｉ）前記第一の浮遊電極及び前記第二の浮遊電極から液体を除去し気相とする工程、
（ｖｉｉｉ）前記第一の励振電極及び前記第二の励振電極に交流電圧を印加し、共振周波数を測定する工程、
を含むことを特徴とする、試料液中の測定対象の物質を気相で測定するための方法、を提供する。
The present invention also provides
(I) preparing a piezoelectric substrate on which the first floating electrode and the second floating electrode are disposed, and an insulating substrate on which the first excitation electrode and the second excitation electrode are disposed;
(Ii) fixing a second binding partner that binds to the substance to be measured on the first floating electrode;
(Iii) a first excitation electrode and a second excitation electrode of the insulating substrate are disposed on a surface facing the back surface of the piezoelectric substrate;
The first floating electrode is disposed on a straight line passing between the first excitation electrodes and perpendicular to the plate surface of the piezoelectric substrate, and the second floating electrode is disposed between the second excitation electrodes. Fixing the piezoelectric substrate to the insulating substrate so that the piezoelectric substrate is disposed on a straight line that is orthogonal to the plate surface of the piezoelectric substrate.
(Iv) applying an alternating voltage to the first excitation electrode and the second excitation electrode, and measuring a resonance frequency;
(V) introducing a sample solution into the first floating electrode and the second floating electrode;
(Vi) introducing into the first floating electrode and the second floating electrode a solution containing sensitizing particles in which a first binding partner that binds to a substance to be measured is fixed on the surface;
(Vii) removing liquid from the first floating electrode and the second floating electrode to form a gas phase;
(Viii) applying an alternating voltage to the first excitation electrode and the second excitation electrode, and measuring a resonance frequency;
A method for measuring a substance to be measured in a sample solution in a gas phase.
また、本発明の実施形態の一つでは、前記第一の励振電極のスリットと前記第二の励振電極のスリットは非平行になるように絶縁基板上に配設される。 In one embodiment of the present invention, the slit of the first excitation electrode and the slit of the second excitation electrode are disposed on the insulating substrate so as to be non-parallel.
すなわち、本発明は、以下の〔１〕〜〔１６〕に関する。 That is, the present invention relates to the following [1] to [16].
〔１〕 試料液中の測定対象の物質を気相で測定するためのキットであって、
（ａ）測定対象の物質と結合する第一の結合パートナーを表面に固定した増感用の粒子、
（ｂ） 表面に第一の浮遊電極と第二の浮遊電極が配設された圧電基板；及び
前記圧電基板の裏面と対向する面上に第一の励振電極と第二の励振電極が配設された絶縁基板；を備え、
前記第一の浮遊電極は前記第一の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置され、
前記第二の浮遊電極は前記第二の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置され、
前記第一の浮遊電極上には前記圧電基板と接する側と反対の表面に測定対象の物質と結合する第二の結合パートナーが固定化され、
前記圧電基板は前記絶縁基板から分離可能であることを特徴とする、圧電振動子；
を含んでなるキット。
〔２〕 第一の浮遊電極及び第二の浮遊電極上の試料を加熱乾燥により気相とするための手段、をさらに含んでなる〔１〕に記載のキット。
〔３〕 前記増感用の粒子が金属ナノ粒子である、〔１〕又は〔２〕に記載のキット。
〔４〕 前記増感用の粒子が金ナノ粒子である、〔１〕〜〔３〕のいずれか１項に記載のキット。
〔５〕 前記増感用の粒子の平均粒子径が１０〜２００ｎｍの範囲である、〔１〕〜〔４〕のいずれか１項に記載のキット。
〔６〕 前記圧電基板が水晶振動子である、〔１〕〜〔５〕のいずれか１項に記載のキット。
〔７〕 前記第一の励振電極が、前記第二の励振電極とは異なる振動モードを励起するように絶縁基板上に配設される、〔１〕〜〔６〕のいずれか１項に記載のキット。
〔８〕 前記第一の励振電極のスリットが前記第二の励振電極のスリットに対して直交するように絶縁基板上に配設される、〔１〕〜〔７〕のいずれか１項に記載のキット。
〔９〕（ｉ）第一の浮遊電極と第二の浮遊電極が配設された圧電基板、及び
第一の励振電極と第二の励振電極が配設された絶縁基板、を用意する工程、
（ｉｉ）前記第一の浮遊電極上に測定対象の物質と結合する第二の結合パートナーを固定する工程、
（ｉｉｉ）前記圧電基板の裏面と対向する面上に絶縁基板の第一の励振電極と第二の励振電極が配置され、
前記第一の浮遊電極は前記第一の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置され、かつ
前記第二の浮遊電極は前記第二の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置されるように前記圧電基板を前記絶縁基板に固定する工程、
（ｉｖ）前記第一の励振電極及び前記第二の励振電極に交流電圧を印加し、共振周波数を測定する工程、
（ｖ）前記第一の浮遊電極及び前記第二の浮遊電極に試料液を導入する工程、
（ｖｉ）前記第一の浮遊電極及び前記第二の浮遊電極に、測定対象の物質と結合する第一の結合パートナーを表面に固定した増感用の粒子を含む溶液を導入する工程、
（ｖｉｉ）前記第一の浮遊電極及び前記第二の浮遊電極から液体を除去し気相とする工程、
（ｖｉｉｉ）前記第一の励振電極及び前記第二の励振電極に交流電圧を印加し、共振周波数を測定する工程、
を含むことを特徴とする、試料液中の測定対象の物質を気相で測定する方法。
〔１０〕 前記（ｖｉｉ）前記第一の浮遊電極及び前記第二の浮遊電極から液体を除去し気相とする工程が、加熱乾燥により液体を除去し気相とする工程である、〔９〕に記載の方法。
〔１１〕 前記増感用の粒子が金属ナノ粒子である、〔９〕又は〔１０〕に記載の方法。
〔１２〕 前記増感用の粒子が金ナノ粒子である、〔９〕〜〔１１〕のいずれか１項に記載の方法。
〔１３〕 前記増感用の粒子の平均粒子径が１０〜２００ｎｍの範囲である、〔９〕〜〔１２〕のいずれか１項に記載の方法。
〔１４〕 前記圧電基板が水晶振動子である、〔９〕〜〔１３〕のいずれか１項に記載の方法。
〔１５〕 前記絶縁基板が、第一の励振電極が第二の励振電極とは異なる振動モードを励起するように絶縁基板上に配設された絶縁基板である、〔９〕〜〔１４〕のいずれか１項に記載の方法。
〔１６〕 前記絶縁基板が、第一の励振電極のスリットが第二の励振電極のスリットに対して直交するように絶縁基板上に配設された絶縁基板である、〔９〕〜〔１５〕のいずれか１項に記載の方法。
[1] A kit for measuring a substance to be measured in a sample solution in a gas phase,
(A) sensitizing particles in which a first binding partner that binds to a substance to be measured is fixed on the surface;
(B) a piezoelectric substrate having a first floating electrode and a second floating electrode disposed on the surface; and a first excitation electrode and a second excitation electrode disposed on a surface opposite to the back surface of the piezoelectric substrate. An insulated substrate;
The first floating electrode is disposed on a straight line passing through the center of the first excitation electrode and orthogonal to the plate surface of the piezoelectric substrate,
The second floating electrode is disposed on a straight line passing through the center of the second excitation electrode and orthogonal to the plate surface of the piezoelectric substrate,
On the first floating electrode, a second binding partner that binds to a substance to be measured is immobilized on the surface opposite to the side in contact with the piezoelectric substrate,
A piezoelectric vibrator, wherein the piezoelectric substrate is separable from the insulating substrate;
A kit comprising:
[2] The kit according to [1], further comprising means for converting the sample on the first floating electrode and the second floating electrode into a gas phase by heat drying.
[3] The kit according to [1] or [2], wherein the sensitizing particles are metal nanoparticles.
[4] The kit according to any one of [1] to [3], wherein the sensitizing particles are gold nanoparticles.
[5] The kit according to any one of [1] to [4], wherein an average particle diameter of the sensitizing particles is in a range of 10 to 200 nm.
[6] The kit according to any one of [1] to [5], wherein the piezoelectric substrate is a crystal resonator.
[7] The first excitation electrode according to any one of [1] to [6], wherein the first excitation electrode is disposed on the insulating substrate so as to excite a vibration mode different from that of the second excitation electrode. Kit.
[8] The method according to any one of [1] to [7], wherein the slit of the first excitation electrode is disposed on the insulating substrate so as to be orthogonal to the slit of the second excitation electrode. Kit.
[9] (i) preparing a piezoelectric substrate on which the first floating electrode and the second floating electrode are disposed, and an insulating substrate on which the first excitation electrode and the second excitation electrode are disposed;
(Ii) fixing a second binding partner that binds to the substance to be measured on the first floating electrode;
(Iii) a first excitation electrode and a second excitation electrode of the insulating substrate are disposed on a surface facing the back surface of the piezoelectric substrate;
The first floating electrode is arranged on a straight line passing through the center of the first excitation electrode and perpendicular to the plate surface of the piezoelectric substrate, and the second floating electrode is centered on the second excitation electrode. Fixing the piezoelectric substrate to the insulating substrate so that the piezoelectric substrate is disposed on a straight line that is orthogonal to the plate surface of the piezoelectric substrate.
(Iv) applying an alternating voltage to the first excitation electrode and the second excitation electrode, and measuring a resonance frequency;
(V) introducing a sample solution into the first floating electrode and the second floating electrode;
(Vi) introducing into the first floating electrode and the second floating electrode a solution containing sensitizing particles in which a first binding partner that binds to a substance to be measured is fixed on the surface;
(Vii) removing liquid from the first floating electrode and the second floating electrode to form a gas phase;
(Viii) applying an alternating voltage to the first excitation electrode and the second excitation electrode, and measuring a resonance frequency;
A method for measuring a substance to be measured in a sample solution in a gas phase, comprising:
[10] The step (vii) of removing the liquid from the first floating electrode and the second floating electrode to form a gas phase is a step of removing the liquid by heating and drying to form a gas phase. [9] The method described in 1.
[11] The method according to [9] or [10], wherein the sensitizing particles are metal nanoparticles.
[12] The method according to any one of [9] to [11], wherein the sensitizing particles are gold nanoparticles.
[13] The method according to any one of [9] to [12], wherein an average particle diameter of the sensitizing particles is in a range of 10 to 200 nm.
[14] The method according to any one of [9] to [13], wherein the piezoelectric substrate is a crystal resonator.
[15] The insulating substrate according to [9] to [14], wherein the insulating substrate is an insulating substrate disposed on the insulating substrate such that the first excitation electrode excites a vibration mode different from that of the second excitation electrode. The method according to any one of the above.
[16] The insulating substrate is an insulating substrate disposed on the insulating substrate such that the slit of the first excitation electrode is orthogonal to the slit of the second excitation electrode. [9] to [15] The method of any one of these.
本発明の質量測定キット及び質量測定方法によれば、測定対象物質に結合した粒子によって増幅された質量を、溶液成分を脱水剤や加熱によって除去することにより、気相での高感度な測定が可能である。さらにマルチセンサで差動検出することにより、誤差の原因となる環境因子を除去し、安定して測定することができる。また、本発明に用いられる浮遊電極には配線が不要であり、浮遊電極はシンプルな形状にすることができる。したがって浮遊電極から液体を除去し気相とする工程において乾燥のばらつきを防ぐことができ、安定した測定が可能である。また、測定のたびに試料を載せた電極を使い捨てるような用途においても、浮遊電極と圧電基板を廃棄するだけで励振電極は使いまわすことができ、経済的であり環境にも優しい。すなわち、本発明により、ＰＯＣＴや食品検査、環境測定用途などに合致した迅速かつ高感度な測定が可能となる。 According to the mass measurement kit and the mass measurement method of the present invention, the mass amplified by the particles bound to the substance to be measured is removed by removing the solution component by a dehydrating agent or heating, so that highly sensitive measurement in the gas phase can be performed. Is possible. Furthermore, by performing differential detection with a multi-sensor, it is possible to remove environmental factors that cause errors and perform stable measurement. Further, the floating electrode used in the present invention does not require wiring, and the floating electrode can be formed in a simple shape. Therefore, variation in drying can be prevented in the process of removing the liquid from the floating electrode to form a gas phase, and stable measurement is possible. Further, even in an application in which the electrode on which the sample is placed is discarded every time measurement is performed, the excitation electrode can be reused simply by discarding the floating electrode and the piezoelectric substrate, which is economical and environmentally friendly. That is, according to the present invention, it is possible to perform quick and highly sensitive measurement that matches POCT, food inspection, environmental measurement applications, and the like.
本発明の「圧電振動子」によると、励振電極間に交流電圧を印加すると、浮遊電極の作用により、圧電基板の板面に平行な方向だけでなく圧電基板の板厚方向にも電界が発生する。この電界により圧電基板が発振するため、励振電極間でのインピーダンスの周波数依存性（インピーダンススペクトル）を計測することで、インピーダンスが低くなるピークを圧電基板の共振周波数として計測することができる。また、圧電基板の表面、特に浮遊電極上に物質が付着すると、圧電基板の共振周波数が変化するため、この共振周波数変化から付着物質の質量を計測することができる。 According to the “piezoelectric vibrator” of the present invention, when an AC voltage is applied between the excitation electrodes, an electric field is generated not only in the direction parallel to the surface of the piezoelectric substrate but also in the thickness direction of the piezoelectric substrate due to the action of the floating electrode. To do. Since the piezoelectric substrate oscillates due to this electric field, by measuring the frequency dependence (impedance spectrum) of the impedance between the excitation electrodes, the peak at which the impedance becomes low can be measured as the resonance frequency of the piezoelectric substrate. Further, when a substance adheres to the surface of the piezoelectric substrate, particularly on the floating electrode, the resonance frequency of the piezoelectric substrate changes, so that the mass of the attached substance can be measured from the change in the resonance frequency.
さらに本発明の圧電振動子は浮遊電極と励振電極の組を少なくとも２つ有し、第一の組を測定用、第二の組を対照とすることで余分な環境因子を除去した正確な共振周波数変化ΔＦを測定できる。すなわち、試料液を導入する前にまず共振周波数を測定し、その差分をＦｔ１とし、
Ｆｔ１＝Ｆｔ１（測定）−Ｆｔ１（対照）
試料液及び増感用の粒子を導入後に再び測定し、その差分をＦｔ２とし、
Ｆｔ２＝Ｆｔ２（測定）−Ｆｔ２（対照）
最後にＦｔ２とＦｔ１の差を計算しΔＦとすることで、
ΔＦ＝Ｆｔ２−Ｆｔ１
浮遊電極に付着した物質の質量による共振周波数変化ΔＦを正確に測定することができる。
Furthermore, the piezoelectric vibrator of the present invention has at least two pairs of floating electrodes and excitation electrodes, and the accurate resonance with the first set used for measurement and the second set used as a control to eliminate excess environmental factors. The frequency change ΔF can be measured. That is, before introducing the sample liquid, first, the resonance frequency is measured, and the difference is defined as F t1 .
F t1 = F t1 (measurement) −F t1 (control)
Measure again after introducing the sample solution and sensitizing particles, and let the difference be F t2 ,
F t2 = F t2 (measurement) −F t2 (control)
Finally, by calculating the difference between F t2 and F t1 to ΔF,
ΔF = F t2 −F t1
It is possible to accurately measure the resonance frequency change ΔF due to the mass of the substance attached to the floating electrode.
また、本発明の実施形態の一つでは、前記第一の励振電極のスリットと前記第二の励振電極のスリットは非平行になるように絶縁基板上に配設される。二つの励振電極を平行に配設した場合、どちらも同じ振動モードで振動することにより隣の素子から伝播した振動によりノイズが増える（クロストーク）おそれがある。二つの励振電極を非平行に配設し、それぞれ異なる振動モードで振動させることで振動が伝播したとしても共振周波数に与える影響は小さくなる。 In one embodiment of the present invention, the slit of the first excitation electrode and the slit of the second excitation electrode are disposed on the insulating substrate so as to be non-parallel. When the two excitation electrodes are arranged in parallel, both of them vibrate in the same vibration mode, and there is a risk that noise will increase due to vibration propagated from the adjacent element (crosstalk). Even if vibration is propagated by disposing the two excitation electrodes in parallel and oscillating in different vibration modes, the influence on the resonance frequency is reduced.
図１は、本発明の一実施形態に係る圧電基板の概略構成を示す平面図である。また、図２及び図３は、本発明の一実施形態に係る絶縁基板の概略構成を示す平面図である。図４は、図１の圧電基板を図２の絶縁基板に固定した状態での側面断面図である。図５は、さらにスペーサーを有する圧電振動子の側面断面図である。図１〜４に示されるように、本発明の一実施形態に係る圧電振動子１は、分離可能な圧電基板３と絶縁基板５を含んでなり、圧電基板２は少なくとも２つの浮遊電極２ａ、２ｂを備え、絶縁基板５は少なくとも２つの励振電極４ａ、４ｂを含む。図５に示されるように、本発明の一実施形態に係る圧電振動子１は、任意にスペーサー６を有してもよい。図１〜５は、概略図であり、厚みや幅等の関係を正確に縮尺したものではない。 FIG. 1 is a plan view showing a schematic configuration of a piezoelectric substrate according to an embodiment of the present invention. 2 and 3 are plan views showing a schematic configuration of an insulating substrate according to an embodiment of the present invention. 4 is a side sectional view of the piezoelectric substrate of FIG. 1 fixed to the insulating substrate of FIG. FIG. 5 is a side cross-sectional view of a piezoelectric vibrator having a spacer. As shown in FIGS. 1 to 4, a piezoelectric vibrator 1 according to an embodiment of the present invention includes a detachable piezoelectric substrate 3 and an insulating substrate 5, and the piezoelectric substrate 2 includes at least two floating electrodes 2 a, 2b, the insulating substrate 5 includes at least two excitation electrodes 4a and 4b. As shown in FIG. 5, the piezoelectric vibrator 1 according to one embodiment of the present invention may optionally have a spacer 6. 1 to 5 are schematic diagrams, and are not accurately scaled for relationships such as thickness and width.
本明細書においては、別段の定義がない限り、本発明に関連して使用される科学用語及び専門用語は、当業者が一般に理解する意味を有する。さらに、状況に応じて定義することが要求されない限り、単数の用語は複数を含み、複数の用語は単数を含むことが意図されている。「又は」という用語は、代替物のみを言及することが明確に示されない限り又は代替物が相互排他的でない限り、「及び／又は」を意味するために使用されるが、本明細書では、代替物のみ及び「及び／又は」の両者を意味するもとして使用される。数値範囲としてＡ〜Ｂのように数値Ａと数値Ｂとを用いて表記される場合、別段の定義がない限り、「Ａ〜Ｂ」は、Ａ以上Ｂ以下の数値範囲を意味するものとして使用される。公知の方法及び技術は、他の例示がない限り、当技術分野で周知の通常の方法によって又は一般の参考文献において記載される方法によって実施される。 In this specification, unless otherwise defined, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The term “or” is used to mean “and / or” unless expressly stated to refer only to an alternative or unless an alternative is mutually exclusive, Used only to mean both alternatives and “and / or”. When expressed using numerical values A and B such as A to B as numerical ranges, “A to B” is used to mean a numerical range from A to B unless otherwise defined. Is done. Known methods and techniques are carried out by conventional methods well known in the art or by methods described in general references, unless otherwise illustrated.
本明細書における「測定」には、測定対象物質の量を定量的又は半定量的に決定する一般的な意味の「測定」の他、測定対象物質の存在の有無を判定する「検出」の意味も含まれる。 In this specification, “measurement” includes “measurement” in a general sense for quantitatively or semi-quantitatively determining the amount of a measurement target substance, as well as “detection” for determining the presence or absence of the measurement target substance. Meaning is also included.
本発明における「結合パートナー」とは、測定対象物質を生物学的な特異性を利用して認識し結合でき、測定対象物質とともに複合体を形成することができる物質であれば、特に限定されるものではない。生物学的な特異性を利用した結合としては、例えば、抗原抗体反応、レセプター−リガンド反応、酵素−基質反応、タンパク質間相互作用（例えば、ＩｇＧとプロテインＡとの反応）、タンパク質−低分子間相互作用（例えば、アビジンとビオチンとの反応）、タンパク質−糖鎖間相互作用（例えば、レクチンと糖鎖との反応）、タンパク質−核酸間相互作用、核酸間ハイブリダイゼーション反応などを利用した結合が挙げられる。例えば、生物学的に特異的な反応として抗原抗体反応を利用する場合には、測定対象物質と結合パートナーとの組合せは、抗原（測定対象物質）と抗体（結合パートナー）との組合せ、或いは、抗体（測定対象物質）と抗原（結合パートナー）との組合せとなる。生物学的に特異的な反応として酵素−基質反応を利用する場合には、測定対象物質と結合パートナーとの組合せは、酵素（測定対象物質）と基質（結合パートナー）との組合せ、或いは、基質（測定対象物質）と酵素（結合パートナー）との組合せとなる。 The “binding partner” in the present invention is particularly limited as long as it is a substance capable of recognizing and binding the substance to be measured using biological specificity and forming a complex with the substance to be measured. It is not a thing. Examples of the binding utilizing biological specificity include, for example, antigen-antibody reaction, receptor-ligand reaction, enzyme-substrate reaction, protein-protein interaction (for example, reaction between IgG and protein A), protein-small molecule Binding using interaction (for example, reaction between avidin and biotin), protein-sugar chain interaction (for example, reaction between lectin and sugar chain), protein-nucleic acid interaction, hybridization reaction between nucleic acids, etc. Can be mentioned. For example, when an antigen-antibody reaction is used as a biologically specific reaction, a combination of a measurement target substance and a binding partner is a combination of an antigen (measurement target substance) and an antibody (binding partner), or A combination of an antibody (substance to be measured) and an antigen (binding partner). When an enzyme-substrate reaction is used as a biologically specific reaction, the combination of a substance to be measured and a binding partner is a combination of an enzyme (measuring substance) and a substrate (binding partner), or a substrate. (Measuring substance) and enzyme (binding partner).
本発明において「第一の結合パートナー」に加え「第二の結合パートナー」が用いられるとき、第二の結合パートナーは、測定対象物質を生物学的な特異性を利用して認識し結合でき、測定対象物質とともに複合体を形成することができる物質であって、第一の結合パートナーが結合する領域とは重複しない領域で測定対象物質に結合できる物質であれば特に限定されるものではない。「第一の結合パートナー」と「第二の結合パートナー」は、同じ物質であってもよく異なる物質であってもよい。通常、第二の結合パートナーが結合可能な測定対象物質の部分は第一の結合パートナーが結合可能な部分とは異なっており、第二の結合パートナーは少なくとも測定対象物質に結合する部分又は能力に関して第一の結合パートナーとは異なる物質である。しかし、測定対象物質が、第一の結合パートナーが結合可能な部分を複数有している場合には、第二の結合パートナーは第一の結合パートナーと同じ物質であってもよく、第二の結合パートナーは第一の結合パートナーが結合しない部分で測定対象物質に結合することができる。さらに、第二の結合パートナーと測定対象物質との結合は、第一の結合パートナーと測定対象物質との結合と同じ生物学的に特異的な反応を利用してもよく、異なる生物学的反応を利用してもよい。例えば、第一の結合パートナー−測定対象物質−第二の結合パートナーの組合せとして、抗原抗体反応のみを利用して、抗体（第一の結合パートナー）−抗原（測定対象物質）−抗体（第二の結合パートナー）又は抗原（第一の結合パートナー）−抗体（測定対象物質）−抗体（第二の結合パートナー）などの組合せとすることができる。或いは、抗原抗体反応と酵素−基質反応を利用して、抗体（第一の結合パートナー）−酵素（測定対象物質）−基質（第二の結合パートナー）又は酵素（第一の結合パートナー）−基質（測定対象物質）−抗体（第二の結合パートナー）などの組合せとすることもできる。 When a “second binding partner” is used in addition to the “first binding partner” in the present invention, the second binding partner can recognize and bind to the substance to be measured using biological specificity, The substance is not particularly limited as long as it is a substance that can form a complex with the measurement target substance and can bind to the measurement target substance in a region that does not overlap with the region to which the first binding partner binds. The “first binding partner” and the “second binding partner” may be the same substance or different substances. Usually, the part of the analyte to which the second binding partner can bind is different from the moiety to which the first binding partner can bind, and the second binding partner is at least in relation to the part or ability to bind to the analyte. A substance that is different from the first binding partner. However, when the substance to be measured has a plurality of moieties to which the first binding partner can bind, the second binding partner may be the same substance as the first binding partner, The binding partner can bind to the substance to be measured at a portion where the first binding partner does not bind. Furthermore, the binding between the second binding partner and the substance to be measured may utilize the same biological specific reaction as the binding between the first binding partner and the substance to be measured, and different biological reactions. May be used. For example, as a combination of a first binding partner, a measurement target substance, and a second binding partner, only an antigen-antibody reaction is used, and an antibody (first binding partner) -antigen (measurement target substance) -antibody (second Binding partner) or antigen (first binding partner) -antibody (substance to be measured) -antibody (second binding partner). Alternatively, antibody (first binding partner) -enzyme (substance to be measured) -substrate (second binding partner) or enzyme (first binding partner) -substrate using antigen-antibody reaction and enzyme-substrate reaction (Substance to be measured) —An antibody (second binding partner) or the like may be used.
本発明の結合パートナーとしては、生物学的に特異的な反応のなかでも特異性が極めて高く結合の親和性が大きい抗原抗体反応を利用して測定対象物質に結合することができる、抗体又は抗原が好ましい。さらには、天然には特異的な結合パートナーが存在しない測定対象物質に対して新たに結合パートナーを作製できる点で、抗体がより好ましい。 As a binding partner of the present invention, an antibody or an antigen that can bind to a substance to be measured using an antigen-antibody reaction having a very high specificity and a high binding affinity among biologically specific reactions. Is preferred. Furthermore, an antibody is more preferable in that a binding partner can be newly produced for a substance to be measured that does not naturally have a specific binding partner.
本発明の結合パートナーとして用いられる「抗体」は、測定対象物質に対して十分な特異性と親和力を示すことができれば、必ずしも免疫グロブリン分子全体の構造が維持されていなくてもよく、抗体の抗原結合性断片であってもよい。抗体の抗原結合能は、抗体の可変部に支配されており、抗体の定常部は必ずしも存在しなくてもよい。従って、本発明の「抗体」としては、５種類の免疫グロブリン分子（ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ）の他、これらの分子の可変部からなる断片である、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、ＦａｂからＶＬを取り除いたＦｄ、一本鎖Ｆｖフラグメント（ｓｃＦｖ）及びその二量体であるｄｉａｂｏｄｙ、又はｓｃＦｖからＶＬを取り除いた単一ドメイン抗体（ｓｄＡｂ）などを用いることができるが、これらに限定されない。 The “antibody” used as the binding partner of the present invention may not necessarily maintain the structure of the entire immunoglobulin molecule as long as it can exhibit sufficient specificity and affinity for the substance to be measured. It may be a binding fragment. The antigen-binding ability of an antibody is governed by the variable part of the antibody, and the constant part of the antibody does not necessarily have to exist. Therefore, the “antibody” of the present invention includes five types of immunoglobulin molecules (IgG, IgM, IgA, IgD, IgE), as well as fragments consisting of the variable regions of these molecules, Fab, Fab ′, F ( ab ′) 2 , Fd in which VL is removed from Fab, single chain Fv fragment (scFv) and its dimer diabody, or single domain antibody (sdAb) in which VL is removed from scFv However, it is not limited to these.
本発明の抗体は、商業的に入手することもできるし、公知の標準的な方法によって作製することもできる。測定対象物質に対する抗体を作製する場合には、測定対象物質でウサギ、マウス、ラット、モルモット、ロバ、ヤギ、ヒツジ、ニワトリなどの実験動物を免疫し、測定対象物質に特異的に結合する抗体を動物体内で生成させ、抗体を含む抗血清又はポリクローナル抗体を調製するか、又は、抗体産生に関わる細胞をミエローマ細胞と融合させたのちクローン化してモノクローナル抗体を調製することができる。或いは、遺伝子工学的な手法により、化学的に合成した抗体遺伝子を大腸菌などに発現させて、動物体内では生成されない構造をもつ人工抗体をin vitroで作製することもできる。
本発明の抗体として抗原結合性断片を用いる場合には、公知の方法により、前記のように作製された抗体を酵素消化することにより得ることができる。パパインによる分解でＦａｂが得られ、ペプシンによる処理でＦ（ａｂ’）２が得られ、Ｆ（ａｂ’）２を還元処理することによりＦａｂ’が得られる。或いは、遺伝子操作により、抗体の重鎖可変部（ＶＨ）と軽鎖可変部（ＶＬ）を可動性に富むリンカーペプチドで連結することによりｓｃＦｖを作製することができる。
The antibody of the present invention can be obtained commercially or can be prepared by a known standard method. When preparing an antibody against a substance to be measured, immunize experimental animals such as rabbits, mice, rats, guinea pigs, donkeys, goats, sheep, and chickens with the substance to be measured, and use antibodies that specifically bind to the substance to be measured. An antiserum or polyclonal antibody containing an antibody can be prepared in an animal body, or a cell involved in antibody production can be fused with a myeloma cell and then cloned to prepare a monoclonal antibody. Alternatively, a chemically synthesized antibody gene can be expressed in Escherichia coli or the like by genetic engineering techniques, and an artificial antibody having a structure that cannot be produced in an animal body can be produced in vitro.
When an antigen-binding fragment is used as the antibody of the present invention, it can be obtained by enzymatic digestion of the antibody prepared as described above by a known method. Fab is obtained by degradation with papain, F (ab ′) 2 is obtained by treatment with pepsin, and Fab ′ is obtained by reducing F (ab ′) 2 . Alternatively, scFv can be prepared by linking the heavy chain variable region (V H ) and light chain variable region (V L ) of an antibody with a linker peptide that is highly mobile.
本発明により測定することのできる「測定対象の物質」としては、生物学的な特異性を利用してそれに結合できる結合パートナーが存在すれば如何なる物質であってもよく、例えば、タンパク質（抗原、抗体、レセプター、酵素、レクチン等）、ペプチド、糖鎖（単糖、オリゴ糖、多糖等の糖鎖）、脂質、核酸、低分子化合物、ホルモン（ステロイドホルモン、アミンホルモン、ペプチドホルモン等）、腫瘍マーカー、アレルギー物質、農薬、環境ホルモン、乱用薬物、ウイルス、又は細胞（細菌、血球等）等が挙げられるが、これらに限定されない。 The “substance to be measured” that can be measured according to the present invention may be any substance as long as there is a binding partner that can bind to it using biological specificity, for example, a protein (antigen, Antibodies, receptors, enzymes, lectins, etc.), peptides, sugar chains (sugar chains such as monosaccharides, oligosaccharides, polysaccharides), lipids, nucleic acids, low molecular compounds, hormones (steroid hormones, amine hormones, peptide hormones, etc.), tumors Examples include, but are not limited to, markers, allergic substances, agricultural chemicals, environmental hormones, drugs of abuse, viruses, cells (bacteria, blood cells, etc.) and the like.
前記の測定対象物質を含有し、本発明による測定に供される試料液としては、血液（全血、血漿、血清）、リンパ液、唾液、尿、大便、汗、粘液、涙、随液、鼻汁、頸部又は膣の分泌液、精液、胸膜液、羊水、腹水、中耳液、関節液、胃吸引液、組織・細胞等の抽出液や破砕液等の生体液の他、食品、土壌、植物の抽出液や破砕液等の溶液や、河水、温泉水、飲料水、汚染水等を含む、ほとんど全ての液体試料が挙げられる。 Sample liquids containing the above-mentioned substances to be measured and used for the measurement according to the present invention include blood (whole blood, plasma, serum), lymph, saliva, urine, stool, sweat, mucus, tears, free liquid, nasal discharge In addition to cervical or vaginal secretions, semen, pleural fluid, amniotic fluid, ascites, middle ear fluid, joint fluid, gastric suction fluid, biological fluids such as tissue and cell extracts and crushed fluids, food, soil, Almost all liquid samples are included, including solutions such as plant extracts and crushed liquids, river water, hot spring water, drinking water, contaminated water, and the like.
本発明において、「気相」で測定するとは、浮遊電極が気体に接する状態で測定を行うことを言う。浮遊電極が接する気体は、測定の妨げとなるものでなければ任意の気体であってよいが、空気、乾燥空気、乾燥窒素、アルゴン又はこれらの混合気体であることが好ましく、簡便かつ迅速に測定を行う観点から、空気又は乾燥空気が好ましい。 In the present invention, the measurement in the “gas phase” means that the measurement is performed in a state where the floating electrode is in contact with the gas. The gas in contact with the floating electrode may be any gas as long as it does not interfere with the measurement, but is preferably air, dry air, dry nitrogen, argon, or a mixed gas thereof, and is easily and quickly measured. From the viewpoint of performing air, air or dry air is preferable.
本発明において、浮遊電極上の試料から溶液を除去し気相とする手段は、溶液を除去するための公知の任意の手段であってよいが、簡便かつ迅速に測定を行う観点から、脱水剤の使用、風乾、加熱乾燥又はこれらの手段を組み合わせて用いることが好ましい。 In the present invention, the means for removing the solution from the sample on the floating electrode to form a gas phase may be any known means for removing the solution, but from the viewpoint of simple and rapid measurement, the dehydrating agent Use, air drying, heat drying or a combination of these means.
本発明の「脱水剤」は、乾燥窒素などの気体では乾燥効率が悪く、また固体の乾燥剤では固形物が残留する恐れがあるため、感応膜表面及び吸着層への濡れ性が高く、かつ水と任意の割合で混合可能な揮発性液体であることが好ましい。例えば、エタノールやメタノールなどのアルコール類、アセトンなどのケトン類が好ましい。また、これらの混合物、及び適切な濃度の水との混合物であっても良い。また、脱水剤には、共振子表面の吸着層を強化する目的でホルムアルデヒドやグルタルアルデヒドをはじめとした架橋剤等の任意の揮発性成分を添加しても良い。脱水剤を圧電基板に供給する手段は特に問わないが、例えば、電極表面に滴下する方法、セルに満たされた脱水剤に浸漬する方法、マイクロ流路を用いる方法などが挙げられる。また、元の試料中に存在していた未反応物の、脱水後の残留及び吸着を防ぐために、まず水系の洗浄液もしくは緩衝液を供給して洗浄もしくは希釈を実施し、続いて脱水剤を供給することが望ましい。さらにこのとき、段階的に脱水剤濃度を上げてもよい。 The “dehydrating agent” of the present invention has poor drying efficiency with a gas such as dry nitrogen, and solids may remain with a solid drying agent, so that the wettability to the sensitive membrane surface and the adsorption layer is high, and It is preferably a volatile liquid that can be mixed with water in any proportion. For example, alcohols such as ethanol and methanol, and ketones such as acetone are preferable. Moreover, a mixture with these mixtures and water of a suitable density | concentration may be sufficient. Moreover, you may add arbitrary volatile components, such as crosslinking agents including formaldehyde and glutaraldehyde, to the dehydrating agent in order to strengthen the adsorption layer on the surface of the resonator. The means for supplying the dehydrating agent to the piezoelectric substrate is not particularly limited, and examples thereof include a method of dropping on the electrode surface, a method of immersing in a dehydrating agent filled in a cell, and a method of using a microchannel. In addition, in order to prevent unreacted substances present in the original sample from remaining and adsorbed after dehydration, first, wash or dilute by supplying an aqueous cleaning solution or buffer, followed by supplying a dehydrating agent. It is desirable to do. At this time, the concentration of the dehydrating agent may be increased stepwise.
本発明の「加熱乾燥」の加熱の手段としては、電熱線による抵抗加熱、電磁場による誘導加熱もしくは誘電加熱、ペルチェ素子等によるヒートポンプ加熱、レーザーやハロゲンランプ等による光加熱などが利用できる。これらの発熱体及び熱源は共振子基板上に直接作り込んでも良いし、共振子付近に配置しても良い。加熱を高速に精度良く行うためには、測温抵抗体、サーミスタ、熱電対などの温度センサを共振子基板上もしくは近傍に配置して熱源の出力を制御することが望ましく、複数の温度センサを共振子近傍に配置し、ブリッジ回路を構成する等により共振子中心部の温度を擬似的に測定することはさらに望ましい。また、溶液を完全に除去するため、設定温度は除去する溶液の沸点よりも高いことが望ましいが、参照子表面の含水率を一定に制御できるのであれば、沸点以下の温度に設定しても良い。
本発明において、特に、増感用の粒子を導入後に浮遊電極上の試料から溶液を除去し気相とする手段は、加熱乾燥又は加熱乾燥と他の手段との併用が好ましい。増感用の粒子は加熱乾燥の過程で浮遊電極と低温焼結を起こし安定な質量測定に寄与すると考えらえる。加熱乾燥の温度は、好ましくは５０〜１５０℃、より好ましくは１００℃である。加熱乾燥の時間は、０．５〜１０分間、より好ましくは１〜５分間である。
As the heating means of the “heat drying” of the present invention, resistance heating with a heating wire, induction heating or dielectric heating with an electromagnetic field, heat pump heating with a Peltier element, light heating with a laser, a halogen lamp, or the like can be used. These heating elements and heat sources may be formed directly on the resonator substrate, or may be arranged near the resonator. In order to perform heating quickly and accurately, it is desirable to control the output of the heat source by arranging temperature sensors such as resistance temperature detectors, thermistors, and thermocouples on or near the resonator substrate. It is further desirable to artificially measure the temperature at the center of the resonator by arranging it near the resonator and forming a bridge circuit. In order to completely remove the solution, it is desirable that the set temperature is higher than the boiling point of the solution to be removed. However, if the moisture content on the surface of the reference element can be controlled to be constant, it may be set to a temperature below the boiling point. good.
In the present invention, in particular, the means for removing the solution from the sample on the floating electrode after introducing the sensitizing particles to form a gas phase is preferably heat drying or heat drying in combination with other means. The sensitizing particles are considered to contribute to stable mass measurement by causing low-temperature sintering with the floating electrode during the heat-drying process. The temperature of heat drying is preferably 50 to 150 ° C, more preferably 100 ° C. The heat drying time is 0.5 to 10 minutes, more preferably 1 to 5 minutes.
本発明の「増感用の粒子」とは、その表面に第一の結合パートナーを固定することができ、測定対象の物質と結合したときに増感効果を得ることができる粒子であれば何であってもよい。測定対象物質との質量差が増幅率となるため、密度が高い金属ナノ粒子を含んでなる金属コロイドが好ましく、なかでも表面修飾が容易で腐食のない金ナノ粒子を含んでなる金コロイドがさらに好ましい。
増感用の粒子は、小さいと軽すぎるために増幅率が低く、大きいと重すぎるために感応膜及び測定対象物質との結合力が不足して吸着できなくなることから、その平均粒子径は、好ましくは約１０〜２００ｎｍ、より好ましくは約２０〜１５０ｎｍの範囲である。
なお、本明細書において、動的光散乱法により求められる値を、平均粒子径とする。
The “sensitizing particle” of the present invention is any particle that can fix the first binding partner on its surface and can obtain a sensitizing effect when bound to the substance to be measured. There may be. Since the mass difference from the substance to be measured becomes an amplification factor, metal colloids containing high-density metal nanoparticles are preferred, and among them, gold colloids containing gold nanoparticles that are easy to surface-modify and have no corrosion are further included. preferable.
Since the particles for sensitization are too light if they are small, the amplification factor is low, and if they are too large, they are too heavy and cannot be adsorbed due to insufficient binding force between the sensitive membrane and the substance to be measured. Preferably it is about 10-200 nm, More preferably, it is the range of about 20-150 nm.
In the present specification, the value obtained by the dynamic light scattering method is defined as the average particle diameter.
本発明の増感用の粒子は、金属ナノ粒子、特に金ナノ粒子であって、その平均粒子径が、約１０〜２００ｎｍの範囲である金属ナノ粒子が好ましく挙げられる。金属ナノ粒子は、浮遊電極上に導入した後、加熱乾燥の過程で浮遊電極と低温焼結を起こし安定な質量測定に寄与すると考えらえる。金属ナノ粒子は、小さいと常温でも焼結・凝集してしまい取り扱いづらく、大きいと低温焼結を起こしづらくなるため、前述の平均粒子径の範囲であることが好ましい。 The particles for sensitization of the present invention are preferably metal nanoparticles, particularly gold nanoparticles, and the average particle diameter thereof is in the range of about 10 to 200 nm. The metal nanoparticles are considered to contribute to stable mass measurement by introducing low-temperature sintering with the floating electrode in the process of heat drying after being introduced onto the floating electrode. If the metal nanoparticles are small, they are sintered and agglomerated even at room temperature and are difficult to handle. If they are large, it is difficult to cause low-temperature sintering.
本発明の「浮遊電極」は、任意の導電性材料を成膜して形成される。浮遊電極を形成する導電性材料としては、例えば、金、白金、チタン、クロム、アルミニウム、ニッケル、ニッケル系合金、銀等の金属；シリコン；カーボン；カーボンナノチューブ；ポリピロール、ポリアニリン等の合成有機高分子；ＤＮＡ等の生体由来の有機高分子等が挙げられ、好ましくは導電率が高く、かつ増感用の金属ナノ粒子の低温焼結時に強固な結合が形成される金、白金、銀、クロム等が用いられる。 The “floating electrode” of the present invention is formed by depositing an arbitrary conductive material. Examples of the conductive material for forming the floating electrode include gold, platinum, titanium, chromium, aluminum, nickel, nickel-based alloys, silver and other metals; silicon; carbon; carbon nanotubes; synthetic organic polymers such as polypyrrole and polyaniline. An organic polymer derived from a living body such as DNA, etc., preferably gold, platinum, silver, chromium, etc. that have high conductivity and that form a strong bond during low-temperature sintering of metal nanoparticles for sensitization Is used.
本発明の浮遊電極は、例えば、略円形状、略矩形状等、任意の適切な形状とすることができる。浮遊電極の圧電基板に沿う方向における幅は、好ましくは約１〜１２ｍｍであり、さらに好ましくは約２〜５ｍｍである。また、浮遊電極の厚みは、好ましく約０．０１〜１μｍであり、さらに好ましくは約０．１〜０．５μｍである。 The floating electrode of the present invention can have any suitable shape such as a substantially circular shape or a substantially rectangular shape. The width of the floating electrode in the direction along the piezoelectric substrate is preferably about 1 to 12 mm, more preferably about 2 to 5 mm. The thickness of the floating electrode is preferably about 0.01 to 1 μm, more preferably about 0.1 to 0.5 μm.
第１の浮遊電極と第２の浮遊電極の間の距離（電極間隔）は、好ましくは約１．０〜５．０ｍｍであり、さらに好ましくは約１．５ｍｍ〜５．０ｍｍである。電極間隔が短すぎると振動の干渉が発生するため望ましくない。 The distance (electrode spacing) between the first floating electrode and the second floating electrode is preferably about 1.0 to 5.0 mm, more preferably about 1.5 mm to 5.0 mm. If the electrode interval is too short, vibration interference occurs, which is not desirable.
本発明の「圧電基板」は、表面に吸着した質量の変化を共振周波数の変化として検出できる機械共振子を含んでなる基板であればよく、特定の基板に限定されるものではない。機械共振子としてはＰＺＴ（チタン酸ジルコン酸鉛）やチタン酸バリウムなどの圧電振動子などが挙げられるが、周波数安定性の高さから、水晶振動子が好ましい。本発明の圧電基板が水晶振動子である場合、その切出し方は図１の上下方向をＸ軸として、ＡＴカット、ＳＣカットなど任意のカットであってよいが、励振電極の方向によって２つ以上の振動モードで振動させることができるカットが好ましく、ＡＴカットであることが好ましい。圧電基板の形状は、任意の適切な形状とすることができる。圧電基板の厚みは、好ましくは約１〜１０００μｍ、さらに好ましくは約１０〜５００μｍであり、１００μｍが特に好ましい。 The “piezoelectric substrate” of the present invention may be any substrate including a mechanical resonator that can detect a change in mass adsorbed on the surface as a change in resonance frequency, and is not limited to a specific substrate. Examples of the mechanical resonator include piezoelectric vibrators such as PZT (lead zirconate titanate) and barium titanate. A crystal vibrator is preferable because of high frequency stability. When the piezoelectric substrate of the present invention is a crystal resonator, the cut-out method may be any cut such as AT cut and SC cut with the vertical direction in FIG. 1 as the X axis, but two or more depending on the direction of the excitation electrode A cut that can be vibrated in the vibration mode is preferred, and an AT cut is preferred. The shape of the piezoelectric substrate can be any suitable shape. The thickness of the piezoelectric substrate is preferably about 1 to 1000 μm, more preferably about 10 to 500 μm, and particularly preferably 100 μm.
本発明の浮遊電極と圧電基板を含む部分は、励振電極や絶縁基板を含む部分とは独立しており、分離可能である。
浮遊電極と励振電極の位置関係がずれることは、共振周波数のずれを引き起こし、測定ノイズの原因となる。そのため、試料なしでの共振周波数を測定してから一連の測定が終了するまでの間は、圧電基板が絶縁基板に固定されていることが好ましい。一連の測定が終了した後には圧電基板を絶縁基板から分離することができる。本発明の一実施形態では圧電基板及びそれに配設された浮遊電極は一測定毎に使い捨てられる。
The portion including the floating electrode and the piezoelectric substrate of the present invention is independent of the portion including the excitation electrode and the insulating substrate and can be separated.
A deviation in the positional relationship between the floating electrode and the excitation electrode causes a deviation in the resonance frequency and causes measurement noise. Therefore, it is preferable that the piezoelectric substrate is fixed to the insulating substrate from when the resonance frequency without the sample is measured until the series of measurements is completed. After a series of measurements are completed, the piezoelectric substrate can be separated from the insulating substrate. In one embodiment of the present invention, the piezoelectric substrate and the floating electrode disposed thereon are disposable for each measurement.
本発明の「励振電極」は、絶縁基板の表面であって、圧電基板と対向する面上に配設される。励振電極は略円形状、略矩形状等の任意の適切な形状に設計され得る。励振電極はスリットによって二つの部分に分かれており、それぞれ配線を介して外部の共振周波数を測定する手段と接続され、それぞれの間で交流電圧を印加する出力端子として機能する。振動エネルギーの閉じ込め効果／及びコンダクタンスを高くするため、スリットの位置は励振電極の中心を通ることが望ましい。本明細書において、「励振電極の中心」とは、励振電極の外接円の中心をいう。 The “excitation electrode” of the present invention is disposed on the surface of the insulating substrate that faces the piezoelectric substrate. The excitation electrode can be designed in any appropriate shape such as a substantially circular shape or a substantially rectangular shape. The excitation electrode is divided into two parts by a slit, and is connected to a means for measuring an external resonance frequency via a wiring, and functions as an output terminal for applying an AC voltage therebetween. In order to increase the confinement effect / and conductance of vibration energy, it is desirable that the slit position passes through the center of the excitation electrode. In this specification, “the center of the excitation electrode” refers to the center of the circumscribed circle of the excitation electrode.
前記「外部の共振周波数を測定する手段」は、浮遊電極の質量変化に伴う共振周波数の変化を測定できる手段であれば何であってもよいが、好ましくは発振回路と周波数カウンタによる測定手段である。発振回路としては、公知の任意の発振回路を用いることができる。周波数カウンタとしては公知の任意の周波数カウンタを用いることができる。 The “means for measuring the external resonance frequency” may be any means as long as it can measure the change in the resonance frequency accompanying the mass change of the floating electrode, but is preferably a measurement means using an oscillation circuit and a frequency counter. . Any known oscillation circuit can be used as the oscillation circuit. Any known frequency counter can be used as the frequency counter.
励振電極は、任意の導電性材料をパターニングして形成される。励振電極を形成する導電性材料としては、浮遊電極と同様の導電性材料が挙げられ、好ましくは金、白金、銀、クロム等が用いられる。励振電極の厚みは、好ましくは約０．００１〜１μｍ、さらに好ましくは約０．０１〜１μｍである。
励振電極のスリットの幅は、好ましくは約１００〜６００μｍであり、特に好ましくは約１００μｍである。スリットは、圧電基板と絶縁基板を固定したときに、圧電基板の特定の振動モードを励起することができる方向に形成されることが好ましい。複数の励振電極のスリットはそれぞれ平行であってもよいが、絶縁基板が複数の振動モードを有する場合には、励振電極のスリットはそれぞれ異なる振動モードを励起することができるように非平行であることが好ましい。本発明の一実施形態では、第１の励振電極のスリットは第２の励振電極のスリットと直交する向きに形成される。
The excitation electrode is formed by patterning an arbitrary conductive material. Examples of the conductive material for forming the excitation electrode include the same conductive material as that of the floating electrode, and gold, platinum, silver, chromium, or the like is preferably used. The thickness of the excitation electrode is preferably about 0.001 to 1 μm, more preferably about 0.01 to 1 μm.
The width of the slit of the excitation electrode is preferably about 100 to 600 μm, particularly preferably about 100 μm. The slit is preferably formed in a direction in which a specific vibration mode of the piezoelectric substrate can be excited when the piezoelectric substrate and the insulating substrate are fixed. The slits of the plurality of excitation electrodes may be parallel to each other, but when the insulating substrate has a plurality of vibration modes, the slits of the excitation electrodes are non-parallel so that different vibration modes can be excited. It is preferable. In one embodiment of the present invention, the slit of the first excitation electrode is formed in a direction orthogonal to the slit of the second excitation electrode.
絶縁基板は、ガラス、アルミナ等の絶縁体で形成される。絶縁基板の厚みは、好ましくは０．１ｍｍ以上である。 The insulating substrate is formed of an insulator such as glass or alumina. The thickness of the insulating substrate is preferably 0.1 mm or more.
本発明の圧電振動子は、スペーサーを有していてもよい。スペーサーは、圧電振動子の使用状態において、圧電基板を載置するための部材である。圧電振動子がスペーサーを有さない場合、圧電振動子の使用状態において、圧電基板は励振電極の上に直接載置される。圧電振動子がスペーサーを有する場合、圧電振動子の使用状態における、圧電基板と絶縁基板との間隔は、スペーサーによって規定される。換言すると、圧電基板及び絶縁基板は、スペーサーによって所定の間隔で配置される。このとき、圧電基板と絶縁基板とは、互いに略平行に配置されることが好ましい。スペーサーは、例えばレジスト、フィルム、スライドガラス、カバーガラス又はプラスチック等によって形成される。スペーサーは、間隔を規定し得る限り、形状や配置は限定されない。また、スペーサー以外の、圧電基板と絶縁基板との間隔を規定し得る他の構成で代用してもよい。 The piezoelectric vibrator of the present invention may have a spacer. The spacer is a member for mounting the piezoelectric substrate when the piezoelectric vibrator is in use. When the piezoelectric vibrator does not have a spacer, the piezoelectric substrate is directly placed on the excitation electrode in the usage state of the piezoelectric vibrator. When the piezoelectric vibrator has a spacer, the distance between the piezoelectric substrate and the insulating substrate in the usage state of the piezoelectric vibrator is defined by the spacer. In other words, the piezoelectric substrate and the insulating substrate are arranged at a predetermined interval by the spacer. At this time, the piezoelectric substrate and the insulating substrate are preferably disposed substantially parallel to each other. The spacer is formed of, for example, a resist, a film, a slide glass, a cover glass, or plastic. The shape and arrangement of the spacer are not limited as long as the interval can be defined. Further, other configurations other than the spacer that can define the interval between the piezoelectric substrate and the insulating substrate may be substituted.
本発明の圧電振動子は、スペーサーと圧電基板の間に、又はスペーサーの代わりに間隔を規定しうる、隔壁を有していてもよい。隔壁は例えば、ガラス又はプラスチック等で形成することができる。隔壁を備えることにより測定対象などを励振電極や絶縁基板に接触させることなく測定を行うことができる。 The piezoelectric vibrator of the present invention may have a partition wall that can define an interval between the spacer and the piezoelectric substrate or instead of the spacer. The partition wall can be formed of, for example, glass or plastic. By providing the partition wall, measurement can be performed without bringing the object to be measured into contact with the excitation electrode or the insulating substrate.
以下、実施例により本発明をより具体的に説明するが、本発明はこれら実施例により何ら限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
作製例１ 圧電振動子の作製
（１）圧電基板の作製
圧電基板として、長軸２０ｍｍ×短軸１０ｍｍ×厚さ１００μｍのＡＴカット（短軸方向をＸ軸とする）の水晶基板を用意した。圧電基板上に、下地として金を１５０ｎｍ、その上にクロム５０ｎｍをスパッタリングし、合計厚さ２００ｎｍの浮遊電極を成膜した。浮遊電極は、直径３．５ｍｍの略円形状とし、電極間隔が３．５ｍｍとなるように二か所に成膜した。すなわち、二つの浮遊電極の中心間の距離は７．０ｍｍである。圧電基板の概略構成を図１に示す。
（２）平行型の絶縁基板の作製
アルミナ製の絶縁基板上に、金／クロムをスパッタリングすることにより励振電極を成膜した。励振電極は、１００μｍ幅のスリットを有する直径２．５ｍｍの略円形とし、電極間隔が４．５ｍｍとなるように２か所に成膜した。すなわち、二つの励振電極の中心間の距離は７．０ｍｍである。また、励起電極は、圧電基板を絶縁基板に固定したときに、圧電基板のＸ軸（短軸方向）と励起電極のスリットとが平行になるように成膜した。励振電極から配線を伸ばし発振回路に接続した。絶縁基板の概略構成を図２に示す。
（３）非平行の絶縁基板の作製
二つの励振電極のうちの一方の励振電極はスリットが圧電基板のＸ軸（短軸方向）と平行になるように、他方の励振電極はスリットが圧電基板のＸ軸と直交するように、励振電極を成膜することを除いては、（２）と同様にして絶縁基板を作成した。絶縁基板の概略構成を図３に示す。
Production Example 1 Production of Piezoelectric Vibrator (1) Production of Piezoelectric Substrate As a piezoelectric substrate, an AT-cut quartz substrate having a major axis of 20 mm, a minor axis of 10 mm, and a thickness of 100 μm (the minor axis direction is taken as the X axis) was prepared. A floating electrode having a total thickness of 200 nm was formed on a piezoelectric substrate by sputtering gold of 150 nm as a base and chromium of 50 nm thereon. The floating electrode was formed into a substantially circular shape with a diameter of 3.5 mm, and was formed at two locations so that the electrode spacing was 3.5 mm. That is, the distance between the centers of the two floating electrodes is 7.0 mm. A schematic configuration of the piezoelectric substrate is shown in FIG.
(2) Production of parallel-type insulating substrate An excitation electrode was formed on an alumina insulating substrate by sputtering gold / chromium. The excitation electrode was formed into a substantially circular shape having a diameter of 2.5 mm having a slit having a width of 100 μm, and was formed at two locations so that the electrode interval was 4.5 mm. That is, the distance between the centers of the two excitation electrodes is 7.0 mm. The excitation electrode was formed so that the X-axis (short axis direction) of the piezoelectric substrate and the slit of the excitation electrode were parallel when the piezoelectric substrate was fixed to the insulating substrate. The wiring was extended from the excitation electrode and connected to the oscillation circuit. A schematic configuration of the insulating substrate is shown in FIG.
(3) Production of non-parallel insulating substrate One of the two excitation electrodes has a slit parallel to the X axis (short axis direction) of the piezoelectric substrate, and the other excitation electrode has a piezoelectric substrate. An insulating substrate was prepared in the same manner as (2) except that the excitation electrode was formed so as to be orthogonal to the X axis. A schematic configuration of the insulating substrate is shown in FIG.
実施例１ 電極の位置ずれのノイズ測定
（１）平行型
作製例１（２）で作製した平行型の絶縁基板に、作製例１（１）で作製した圧電基板を固定した。試料を導入しない状態で、気相で二つの電極の共振周波数を測定し、周波数差Ｆ（Ｆ＝Ｆａ−Ｆｂ，Ｈｚ）を記録した。その後、絶縁基板から圧電基板を分離し、再度固定してからＦを記録する操作を繰り返した。結果を図５に示す。１８回のデータから、Ｆの標準偏差は１０７７（Ｈｚ）、最大値と最小値の差は３８６２（Ｈｚ）であった。
（２）非平行型
平行型の絶縁基板の代わりに作製例１（３）で作製した非平行型の絶縁基板を用いることを除いては、（１）と同様にＦを測定した。結果を図６に示す。１８回のデータから、Ｆの標準偏差は８７５（Ｈｚ）、最大値と最小値の差は２７３６（Ｈｚ）であった。
平行型と非平行型の比較は、意外にも、二つの電極が同じ条件になる平行型よりも、電極がそれぞれ異なる振動モードで共振する非平行型の方がＦのばらつきが小さい結果となった。この結果は非平行型の方が、圧電基板と絶縁基板との位置ずれに起因する測定ノイズを抑えられることを意味する。
Example 1 Noise Measurement of Electrode Position Shift (1) Parallel Type The piezoelectric substrate manufactured in Preparation Example 1 (1) was fixed to the parallel type insulating substrate manufactured in Preparation Example 1 (2). The resonance frequency of the two electrodes was measured in the gas phase without introducing the sample, and the frequency difference F (F = Fa−Fb, Hz) was recorded. Thereafter, the operation of separating the piezoelectric substrate from the insulating substrate, fixing it again and recording F was repeated. The results are shown in FIG. From the 18 data, the standard deviation of F was 1077 (Hz), and the difference between the maximum value and the minimum value was 3862 (Hz).
(2) Non-parallel type F was measured in the same manner as (1) except that the non-parallel type insulating substrate manufactured in Preparation Example 1 (3) was used instead of the parallel type insulating substrate. The results are shown in FIG. From the 18 data, the standard deviation of F was 875 (Hz), and the difference between the maximum and minimum values was 2736 (Hz).
The comparison between the parallel type and the non-parallel type surprisingly results in a smaller variation in F in the non-parallel type in which the electrodes resonate in different vibration modes than in the parallel type in which the two electrodes have the same conditions. It was. This result means that the non-parallel type can suppress the measurement noise caused by the positional deviation between the piezoelectric substrate and the insulating substrate.
実施例２ 気相での質量増感の予備試験
（１）ｃＴｎＩ検出用の圧電基板の調整
作製例１（１）と同様の方法で作製した圧電基板を、硫酸と過酸化水素水溶液（５０％）の２：１混合溶液に１０分間浸漬した。基板を純水で洗浄しエアガンで乾燥した。密閉容器中で圧電基板にＯＤＳ（オクタデシルトリメトキシシラン）を滴下し、１００℃で１２時間加熱し、圧電基板上にＳＡＭ（自己組織化単分子膜）を形成させ浮遊電極以外の水晶基板表面を疎水性とした。抗ｃＴｎＩモノクローナル抗体の２０μｇ／ｍＬ水溶液を、浮遊電極上に５μＬずつ滴下し、２時間静置した後、純水で洗浄しエアガンで乾燥した。ブロッキング溶液として浮遊電極上に、１重量％ＢＳＡ（ウシ血清アルブミン）のＰＢＳ（リン酸緩衝生理食塩水）溶液を１０μＬずつ滴下した後、純水で洗浄しエアガンで乾燥した。
（２）事前共振周波数の測定
（１）で調整した圧電基板を、作製例１（３）で作製した非平行型絶縁基板に固定し、絶縁基板を温度調節機に接続されたホットプレート上に設置した。大気中、室温で二つの電極の共振周波数を測定し、周波数差Ｆｔ１（Ｆｔ１＝Ｆｔ１（ａ）−Ｆｔ１（ｂ），Ｈｚ）を記録した。
（３）金コロイドの導入
抗マウスＩｇＧポリクローナル抗体を固定した金ナノ粒子（平均粒子径８０ｎｍ）を、１重量％ＢＳＡのＰＢＳ溶液に分散させ、ＯＤ５５０（光学濃度）＝０〜１６．０とした金コロイド溶液を調整した。前記金コロイド溶液１０μＬを、一方の浮遊電極２ａにのみ滴下し、１０分間静置した。純水で洗浄後、１００℃で３分間加熱して乾燥させた。
（４）吸着後共振周波数の測定
大気中、室温で二つの電極の共振周波数を測定し、周波数差Ｆｔ２（Ｆｔ２＝Ｆｔ２（ａ）−Ｆｔ２（ｂ），Ｈｚ）を記録した。金コロイド濃度ＯＤ５５０ 対 周波数変化量ΔＦ（ΔＦ＝Ｆｔ２−Ｆｔ１、Ｈｚ）を図７に示す。
図７に示される通り、金コロイドの濃度に応じて周波数変化が増大していることがわかる。金コロイドをほぼ定量的に検出できていることから、本来の測定対象の測定においても金コロイドによる質量増感が有用であることが予想される。
Example 2 Preliminary test of mass sensitization in gas phase (1) Preparation of piezoelectric substrate for cTnI detection A piezoelectric substrate prepared by the same method as in Preparation Example 1 (1) was prepared by using sulfuric acid and hydrogen peroxide aqueous solution (50% ) For 2 minutes. The substrate was washed with pure water and dried with an air gun. ODS (octadecyltrimethoxysilane) is dropped onto the piezoelectric substrate in a sealed container and heated at 100 ° C. for 12 hours to form a SAM (self-assembled monolayer) on the piezoelectric substrate, and the surface of the quartz substrate other than the floating electrode is formed. Hydrophobic. A 20 μg / mL aqueous solution of an anti-cTnI monoclonal antibody was dropped on a floating electrode by 5 μL each, allowed to stand for 2 hours, washed with pure water, and dried with an air gun. As a blocking solution, 10 μL each of 1 wt% BSA (bovine serum albumin) in PBS (phosphate buffered saline) was dropped on the floating electrode, washed with pure water, and dried with an air gun.
(2) Measurement of pre-resonance frequency The piezoelectric substrate adjusted in (1) is fixed to the non-parallel type insulating substrate manufactured in Manufacturing Example 1 (3), and the insulating substrate is placed on a hot plate connected to a temperature controller. installed. The resonance frequency of the two electrodes was measured in the atmosphere at room temperature, and the frequency difference F t1 (F t1 = F t1 (a) −F t1 (b), Hz) was recorded.
(3) Introduction of gold colloid Gold nanoparticles (average particle diameter of 80 nm) immobilized with anti-mouse IgG polyclonal antibody were dispersed in 1 wt% BSA in PBS to obtain OD550 (optical density) = 0 to 16.0. A colloidal gold solution was prepared. 10 μL of the colloidal gold solution was dropped only on one floating electrode 2a and allowed to stand for 10 minutes. After washing with pure water, it was dried by heating at 100 ° C. for 3 minutes.
(4) Measurement of resonance frequency after adsorption The resonance frequency of the two electrodes was measured in the atmosphere at room temperature, and the frequency difference F t2 (F t2 = F t2 (a) −F t2 (b), Hz) was recorded. FIG. 7 shows the colloidal gold concentration OD550 versus the frequency variation ΔF (ΔF = F t2 −F t1 , Hz).
As shown in FIG. 7, it can be seen that the frequency change increases with the concentration of the gold colloid. Since gold colloid can be detected almost quantitatively, it is expected that mass sensitization with gold colloid is also useful in the measurement of the original measurement object.
実施例３ ｃＴｎＩの検出試験
（１）ｃＴｎＩ検出用の圧電基板の調整
実施例２（１）と同様にして調整した。
（２）事前共振周波数の測定
（１）で調整した圧電基板を、作製例１（３）で作製した非平行型の絶縁基板に固定し、絶縁基板を温度調節機に接続されたホットプレート上に設置した。大気中、室温で二つの電極の共振周波数を測定し、周波数差Ｆｔ１（Ｆｔ１＝Ｆｔ１（ａ）−Ｆｔ１（ｂ），Ｈｚ）を記録した。
（３）試料液の導入
測定対象であるｃＴｎＩを、重量％ＢＳＡのＰＢＳ溶液に希釈し、０〜１０μｇ／ｍＬとした溶液を試料液とした。試料液１０μｍＬを一方の浮遊電極２ａにのみ滴下し、３０分間静置した。純水で洗浄後、１００℃で３分間加熱乾燥した。
（４）吸着後共振周波数の測定
大気中、室温で二つの電極の共振周波数を測定し、周波数差Ｆｔ２（Ｆｔ２＝Ｆｔ２（ａ）−Ｆｔ２（ｂ），Ｈｚ）を記録した。周波数変化量ΔＦ２（ΔＦ２＝Ｆｔ２−Ｆｔ１、Ｈｚ）を図８にwithout AuNPとして図示した。
（５）増感用の粒子の導入
抗ｃＴｎＩモノクローナル抗体を固定した金ナノ粒子（平均粒子径８０ｎｍ）を、１重量％ＢＳＡのＰＢＳ溶液に分散させ、ＯＤ５５０＝５．０とした金コロイド溶液を調整した。金コロイド溶液を調整した。前記金コロイド溶液１０μＬを、一方の浮遊電極２ａにのみ滴下し、１０分間静置した。純水で洗浄後、１００℃で３分間加熱して乾燥させた。
（６）増感後共振周波数の測定
大気中、室温で二つの電極の共振周波数を測定し、周波数差Ｆｔ３（Ｆｔ３＝Ｆｔ３（ａ）−Ｆｔ３（ｂ），Ｈｚ）を記録した。周波数変化量ΔＦ３（ΔＦ３＝Ｆｔ３−Ｆｔ１、Ｈｚ）を図８にwith AuNPとして図示した。
図８は、without AuNPのデータに示される、増感用の粒子なしでは検出できない１μｇ／ｍＬのような極めて微量な測定対象であっても、増感用の粒子を加えることによって検出可能になることを示している。
Example 3 cTnI Detection Test (1) Adjustment of cTnI Detection Piezoelectric Substrate Adjustment was performed in the same manner as in Example 2 (1).
(2) Measurement of pre-resonance frequency The piezoelectric substrate adjusted in (1) is fixed to the non-parallel type insulating substrate manufactured in Manufacturing Example 1 (3), and the insulating substrate is mounted on a hot plate connected to a temperature controller. Installed. The resonance frequency of the two electrodes was measured in the atmosphere at room temperature, and the frequency difference F t1 (F t1 = F t1 (a) −F t1 (b), Hz) was recorded.
(3) Introduction of sample solution The cTnI to be measured was diluted in a PBS solution of wt% BSA to obtain a solution of 0 to 10 μg / mL. 10 μmL of the sample solution was dropped only on one floating electrode 2a and allowed to stand for 30 minutes. After washing with pure water, it was dried by heating at 100 ° C. for 3 minutes.
(4) Measurement of resonance frequency after adsorption The resonance frequency of the two electrodes was measured in the atmosphere at room temperature, and the frequency difference F t2 (F t2 = F t2 (a) −F t2 (b), Hz) was recorded. The frequency change amount ΔF 2 (ΔF 2 = F t2 −F t1 , Hz) is illustrated as without AuNP in FIG.
(5) Introduction of particles for sensitization A gold colloid solution in which gold nanoparticles (average particle diameter of 80 nm) to which an anti-cTnI monoclonal antibody is immobilized is dispersed in a PBS solution of 1% by weight BSA to make OD550 = 5.0 is obtained. It was adjusted. A colloidal gold solution was prepared. 10 μL of the colloidal gold solution was dropped only on one floating electrode 2a and allowed to stand for 10 minutes. After washing with pure water, it was dried by heating at 100 ° C. for 3 minutes.
(6) Measurement of resonance frequency after sensitization The resonance frequency of the two electrodes was measured in the atmosphere at room temperature, and the frequency difference F t3 (F t3 = F t3 (a) −F t3 (b), Hz) was recorded. . The frequency change amount ΔF 3 (ΔF 3 = F t3 −F t1 , Hz) is shown as “with AuNP” in FIG.
FIG. 8 shows that even a very small amount of measurement target such as 1 μg / mL that cannot be detected without sensitizing particles shown in the data of without AuNP can be detected by adding sensitizing particles. It is shown that.
実施例４ 使用済み圧電基板の電子顕微鏡写真撮影
実施例２で金コロイドを導入して測定した圧電基板を、硫酸と過酸化水素水溶液（５０％）の２：１混合溶液に１０分間浸漬した。基板を純水で洗浄しエアガンで乾燥した。その後、ＳＥＭ像を撮影した。結果を図１０に示す。
図１０は、硫酸過水で有機物を完全に除去した後にも、金ナノ粒子が浮遊電極の金及びクロム表面上に残留していることを示す。実施例２（３）の工程のうち、金コロイド導入後の１００℃３分での加熱工程により、電極表面と金ナノ粒子の間に抗体−抗原の結合よりも強固な結合が生成したと考えられる。この強固な結合は気相での安定な質量測定に有用である。
Example 4 Electron micrograph of used piezoelectric substrate The piezoelectric substrate measured by introducing gold colloid in Example 2 was immersed in a 2: 1 mixed solution of sulfuric acid and aqueous hydrogen peroxide (50%) for 10 minutes. The substrate was washed with pure water and dried with an air gun. Thereafter, an SEM image was taken. The results are shown in FIG.
FIG. 10 shows that the gold nanoparticles remain on the gold and chromium surfaces of the floating electrode even after the organics are completely removed with sulfuric acid / hydrogen peroxide. In the step of Example 2 (3), it is considered that the heating step at 100 ° C. for 3 minutes after introducing the gold colloid produced a stronger bond than the antibody-antigen bond between the electrode surface and the gold nanoparticle. It is done. This strong bond is useful for stable mass measurement in the gas phase.
本発明の質量測定キット及び質量測定方法によれば、極微量の対象物質を特異的に、簡便かつ迅速な方法で高感度に測定することができる。すなわち、本発明により、ＰＯＣＴや食品検査、環境測定用途などに合致した迅速かつ高感度な測定が可能であり、本発明は産業上の利用可能性を有している。 According to the mass measurement kit and the mass measurement method of the present invention, a very small amount of a target substance can be specifically measured with high sensitivity by a simple and rapid method. That is, according to the present invention, it is possible to perform quick and highly sensitive measurement that matches POCT, food inspection, environmental measurement applications, etc., and the present invention has industrial applicability.
１ 圧電振動子
２ａ， ２ｂ 浮遊電極
３ 圧電基板
４ａ， ４ｂ 励振電極
５ 絶縁基板
６ スペーサー
DESCRIPTION OF SYMBOLS 1 Piezoelectric vibrator 2a, 2b Floating electrode 3 Piezoelectric substrate 4a, 4b Excitation electrode 5 Insulating substrate 6 Spacer
Claims (16)
（ａ）測定対象の物質と結合する第一の結合パートナーを表面に固定した増感用の粒子、
（ｂ） 表面に第一の浮遊電極と第二の浮遊電極が配設された圧電基板；及び
前記圧電基板の裏面と対向する面上に第一の励振電極と第二の励振電極が配設された絶縁基板；を備え、
前記第一の浮遊電極は前記第一の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置され、
前記第二の浮遊電極は前記第二の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置され、
前記第一の浮遊電極上には前記圧電基板と接する側と反対の表面に測定対象の物質と結合する第二の結合パートナーが固定化され、
前記圧電基板は前記絶縁基板から分離可能であることを特徴とする、圧電振動子；
を含んでなるキット。 A kit for measuring a substance to be measured in a sample solution in a gas phase,
(A) sensitizing particles in which a first binding partner that binds to a substance to be measured is fixed on the surface;
(B) a piezoelectric substrate having a first floating electrode and a second floating electrode disposed on the surface; and a first excitation electrode and a second excitation electrode disposed on a surface opposite to the back surface of the piezoelectric substrate. An insulated substrate;
The first floating electrode is disposed on a straight line passing through the center of the first excitation electrode and orthogonal to the plate surface of the piezoelectric substrate,
The second floating electrode is disposed on a straight line passing through the center of the second excitation electrode and orthogonal to the plate surface of the piezoelectric substrate,
On the first floating electrode, a second binding partner that binds to a substance to be measured is immobilized on the surface opposite to the side in contact with the piezoelectric substrate,
A piezoelectric vibrator, wherein the piezoelectric substrate is separable from the insulating substrate;
A kit comprising:
第一の励振電極と第二の励振電極が配設された絶縁基板、を用意する工程、
（ｉｉ）前記第一の浮遊電極上に測定対象の物質と結合する第二の結合パートナーを固定する工程、
（ｉｉｉ）前記圧電基板の裏面と対向する面上に絶縁基板の第一の励振電極と第二の励振電極が配置され、
前記第一の浮遊電極は前記第一の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置され、かつ
前記第二の浮遊電極は前記第二の励振電極の中心を通って前記圧電基板の板面に直交する直線上に配置されるように前記圧電基板を前記絶縁基板に固定する工程、
（ｉｖ）前記第一の励振電極及び前記第二の励振電極に交流電圧を印加し、共振周波数を測定する工程、
（ｖ）前記第一の浮遊電極及び前記第二の浮遊電極に試料液を導入する工程、
（ｖｉ）前記第一の浮遊電極及び前記第二の浮遊電極に、測定対象の物質と結合する第一の結合パートナーを表面に固定した増感用の粒子を含む溶液を導入する工程、
（ｖｉｉ）前記第一の浮遊電極及び前記第二の浮遊電極から液体を除去し気相とする工程、
（ｖｉｉｉ）前記第一の励振電極及び前記第二の励振電極に交流電圧を印加し、共振周波数を測定する工程、
を含むことを特徴とする、試料液中の測定対象の物質を気相で測定する方法。 (I) preparing a piezoelectric substrate on which the first floating electrode and the second floating electrode are disposed, and an insulating substrate on which the first excitation electrode and the second excitation electrode are disposed;
(Ii) fixing a second binding partner that binds to the substance to be measured on the first floating electrode;
(Iii) a first excitation electrode and a second excitation electrode of the insulating substrate are disposed on a surface facing the back surface of the piezoelectric substrate;
The first floating electrode is arranged on a straight line passing through the center of the first excitation electrode and perpendicular to the plate surface of the piezoelectric substrate, and the second floating electrode is centered on the second excitation electrode. Fixing the piezoelectric substrate to the insulating substrate so that the piezoelectric substrate is disposed on a straight line that is orthogonal to the plate surface of the piezoelectric substrate.
(Iv) applying an alternating voltage to the first excitation electrode and the second excitation electrode, and measuring a resonance frequency;
(V) introducing a sample solution into the first floating electrode and the second floating electrode;
(Vi) introducing into the first floating electrode and the second floating electrode a solution containing sensitizing particles in which a first binding partner that binds to a substance to be measured is fixed on the surface;
(Vii) removing liquid from the first floating electrode and the second floating electrode to form a gas phase;
(Viii) applying an alternating voltage to the first excitation electrode and the second excitation electrode, and measuring a resonance frequency;
A method for measuring a substance to be measured in a sample solution in a gas phase, comprising:
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