JP2013002816A - Measuring method using biosensor with temperature compensation - Google Patents
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本発明は、測定溶液中の生体分子の定量に際して、測定データの信頼性を向上させるための、温度補正を伴うバイオセンサーによる測定方法に関する。 The present invention relates to a measurement method using a biosensor with temperature correction in order to improve the reliability of measurement data when quantifying biomolecules in a measurement solution.
従来、固体支持体表面にリガンド分子を固定化し、それに相互作用する物質の吸着過程を解析することによって、物質の濃度や相互作用パラメータを算出する技術には、表面プラズモン共鳴（ＳＰＲ）を利用する方法、水晶発振子マイクロバランス（ＱＣＭ）を利用する方法、反射光の干渉を利用する方法等がある。
支持体表面に固定化した物質Ｌに対して、可逆的に相互作用する物質Ｓの結合及び解離は下記の関係で示すことができる。尚、式中「→」は、結合速度定数ｋｏｎの結合反応を示し、「←」は、解離速度定数ｋｏｆｆの解離反応を示すものとする。
Ｌ＋Ｓ⇔ＬＳ
この時、ＳのＬに対する結合初期速度（lnitial blndlng rate）は下記式（１）で表すことができる。またｋｏｆｆが十分小さいとき及び［ＬＳ］が十分小さい時に、式（１）は式（２）に近似することができる。この時、あらかじめｋｏｎ、ｋｏｆｆが既知であれば、ある一定量［Ｌ］のＬが固定化された表面に対するＳの結合初期速度を測定することによって、溶液中のＳの濃度［Ｓ］を定量することができる。
結合初期速度＝ｋｏｎ［Ｓ］［Ｌ］−ｋｏｆｆ［ＬＳ］・・・（１）
≒ｋｏｎ［Ｓ］［Ｌ］・・・（２）
尚、本明細書中において、［］により囲まれたものは、囲まれた物質の濃度を示すものとする。
Conventionally, surface plasmon resonance (SPR) is used as a technique for calculating the concentration and interaction parameters of a substance by immobilizing a ligand molecule on the surface of a solid support and analyzing the adsorption process of the substance that interacts with the ligand molecule. A method using a crystal oscillator microbalance (QCM), a method using interference of reflected light, and the like.
The binding and dissociation of the substance S that interacts reversibly with the substance L immobilized on the surface of the support can be expressed by the following relationship. In the formula, “→” indicates a binding reaction with a binding rate constant k on , and “←” indicates a dissociation reaction with a dissociation rate constant k off .
L + S⇔LS
At this time, the initial bonding rate (lnitial blndlng rate) of S to L can be expressed by the following equation (1). Also, when k off is sufficiently small and [LS] is sufficiently small, equation (1) can be approximated to equation (2). At this time, if k on and k off are known in advance, the concentration of S in the solution [S] is measured by measuring the initial binding velocity of S to the surface on which a certain amount [L] of L is immobilized. Can be quantified.
Initial binding speed = k on [S] [L] −k off [LS] (1)
≒ k on [S] [L] (2)
In addition, in this specification, what is enclosed by [] shall show the density | concentration of the enclosed substance.
この方法を利用してサンプル中の標的分子の濃度を算出する方法の例としては非特許文献１がある。ここでは圧電素子表面に固定化した抗ミオグロビン抗体に対するミオグロビンの結合の初期速度を算出し、この初期速度が溶液中ミオグロビン濃度と相関することを利用したサンプル中のミオグロビン濃度測定法を紹介している。
ここで言うｋｏｎは測定環境の温度によって変化することが知られている。ある一定温度の元であらかじめ測定されたｋｏｎと上記の式（２）を用いて濃度未知のＳの濃度［Ｓ］を求める時、あらかじめｋｏｎを測定した際の測定温度と濃度未知のＳの濃度を測定する時の測定温度が異なるとその分誤差が生じる。
濃度未知のＳの濃度を測定する時の測定温度を、あらかじめｋｏｎを測定した際の測定温度に一致させるためには、センサー周辺に恒温層を必要とするが、これによって装置のコストが高くなるという問題があった。また温度が一定になるまで時間がかかるなどの問題があった。
Non-Patent Document 1 is an example of a method for calculating the concentration of a target molecule in a sample using this method. Here, we calculate the initial rate of binding of myoglobin to the anti-myoglobin antibody immobilized on the surface of the piezoelectric element, and introduce a method for measuring myoglobin concentration in samples using the fact that this initial rate correlates with the concentration of myoglobin in solution. .
Here, k on is known to change depending on the temperature of the measurement environment. When obtaining the concentration [S] of unknown concentration S using k on measured in advance at a certain temperature and the above equation (2), the measured temperature and the unknown concentration S when measuring k on in advance. If the measurement temperature at the time of measuring the concentration of is different, an error will occur accordingly.
The measured temperature when measuring the concentration of an unknown concentration of S, in order to match the measured temperature when measured in advance k on is around the sensor requires a constant temperature layer, whereby high cost of the apparatus There was a problem of becoming. There is also a problem that it takes time until the temperature becomes constant.
本発明においては、固体表面に固定化されたリガンドに対する試料中の結合分子の吸着量の測定により試料中の吸着分子の濃度を測定する方法において、測定環境の温度変化による影響を補正する温度補正を伴うバイオセンサーによる測定方法を提供することを目的とする。 In the present invention, in the method of measuring the concentration of adsorbed molecules in a sample by measuring the amount of adsorbed molecules in the sample with respect to the ligand immobilized on the solid surface, the temperature correction for correcting the influence of temperature change in the measurement environment It aims at providing the measuring method by the biosensor accompanied by.
本発明の温度補正を伴うバイオセンサーによる測定方法は、請求項１に記載の通り、測定槽内において固体支持体表面に固定されたリガンドに相互作用可能な試料中の標的物質の吸着量を測定することによって標的物質の濃度を測定する方法であって、測定中に測定槽近傍又は測定槽内の溶液の環境温度を計測し、前記環境温度に基づいて測定結果を補正することを特徴とする。
請求項２記載の本発明は、請求項１記載の発明において、前記標的物質の前記固定化リガンドへの吸着初期速度から前記標的物質の濃度を定量することを特徴とする。
請求項３記載の本発明は、請求項１又は２記載の発明において、標的物質とリガンドの対ごとの相互作用に関与するパラメータの温度依存性のデータで測定結果を補正することを特徴とする。
請求項４記載の本発明は、請求項１乃至３の何れか１項に記載の発明において、前記相互作用に関与するパラメータがアレニウスの式の頻度因子Ａと活性化自由エネルギーＥ、又はそれらと他パラメータの組み合わせでなるパラメータであることを特徴とする。
請求項５記載の本発明は、請求項１乃至４の何れか１項に記載の発明において、前記試料は、全血、血漿及び血清の何れかであることを特徴とする。
請求項６記載の本発明は、請求項１乃至５の何れか１項に記載の発明において、前記標的物質は、モノクローナル抗体、キメラモノクローナル抗体、ヒト化モノクローナル抗体、ヒトモノクローナル抗体及びマウスモノクローナル抗体、並びに、抗体の抗原結合部位を含む融合タンパク質及びレセプターの抗原結合部位を含む融合タンパク質の中の何れかであることを特徴とする。
The measurement method using a biosensor with temperature correction according to the present invention measures the amount of adsorption of a target substance in a sample capable of interacting with a ligand immobilized on the surface of a solid support in a measurement tank. A method for measuring the concentration of a target substance by measuring the ambient temperature of a solution in the vicinity of or in a measurement tank during measurement, and correcting the measurement result based on the ambient temperature .
The present invention according to claim 2 is characterized in that, in the invention according to claim 1, the concentration of the target substance is quantified from the initial adsorption rate of the target substance to the immobilized ligand.
The present invention according to claim 3 is characterized in that, in the invention according to claim 1 or 2, the measurement result is corrected with temperature-dependent data of a parameter involved in the interaction of each pair of the target substance and the ligand. .
According to a fourth aspect of the present invention, in the invention according to any one of the first to third aspects, the parameter involved in the interaction is a frequency factor A and an activation free energy E in the Arrhenius equation, or The parameter is a combination of other parameters.
The invention according to claim 5 is the invention according to any one of claims 1 to 4, characterized in that the sample is any one of whole blood, plasma and serum.
The present invention according to claim 6 is the invention according to any one of claims 1 to 5, wherein the target substance is a monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, or a mouse monoclonal antibody, And any one of a fusion protein containing an antigen-binding site of an antibody and a fusion protein containing an antigen-binding site of a receptor.
環境温度を測定して標的吸着分子の吸着量からサンプル溶液中の標的分子の濃度を算出する時の温度変化を原因とする誤差を補正することによって、従来より正確な測定が可能となる。
また測定溶液周辺に恒温層を配置する必要がなくなり、装置構成が大幅に簡略される。
By measuring the environmental temperature and correcting the error caused by the temperature change when calculating the concentration of the target molecule in the sample solution from the amount of the target adsorbed molecule, more accurate measurement than before can be achieved.
Moreover, it is not necessary to arrange a thermostatic layer around the measurement solution, and the apparatus configuration is greatly simplified.
一例として結合分子のリガンドへの結合の結合初速度から濃度を換算する方法を考える。結合初期速度と結合分子の濃度の関係は上記式（２）である。ここでｋｏｎは溶液温度に依存するパラメータであり、この温度依存性は下記（３）のアレニウスの式で表すことができる。
ｋｏｎ＝Ａｅ−Ｅ／ＲＴ・・・（３）
ここでＡは定数（頻度因子）、Ｅは活性化エネルギー、Ｒは気体定数、Ｔは絶対温度（Ｋ）である。
式（３）を式（２）に代入して式（４）を得る。
結合初期速度＝Ａｅ−Ｅ／ＲＴ［Ｓ］［Ｌ］・・・（４）
式（４）の両辺の自然対数をとると下記式（５）になる。
Ln結合初期速度＝−Ｅ／ＲＴ＋LnＡ・［Ｓ］［Ｌ］・・・（５）
この式から、ある濃度［Ｓ］でのＬに対する結合初速度を種々の温度で算出し、Ln結合初期速度を縦軸にして、１／Ｔを横軸にするとプロットは直線を描き、傾きから−Ｅ／Ｒ、切片からLnＡ・［Ｌ］［Ｓ］が算出できる。ここで［Ｓ］は既知、［Ｌ］はセンサー表面に固定化されたリガンドの固定化量なので既知とすると、ＥとＡを算出することができる。このようにして算出したＡとＥを式（４）に代入すると結合初速度の温度に対する関係式が得られる。これを変形して式（６）を得る。
［Ｓ］＝結合初期速度／（Ａｅ−Ｅ／ＲＴ［Ｌ］）・・・（６）
式（６）ではＡ，Ｅ，［Ｌ］が既知であり、結合初速度と環境温度Ｔを測定することで試料中の分子濃度［Ｓ］を測定することが可能となる。
環境温度の測定はなるべく測定槽の近傍が望ましく、白金温度センサー等の温度センサーを用いて行う。測定溶液内に温度センサーを入れることで行ってもよい。
また、この方法は、濃度測定を行う標的物質毎にあらかじめ上記の方法によりＡ，Ｅを算出しておいて式（６）を求めておく必要がある。
As an example, consider a method of converting the concentration from the initial binding rate of binding of a binding molecule to a ligand. The relationship between the initial binding velocity and the concentration of the binding molecule is the above formula (2). Here, k on is a parameter depending on the solution temperature, and this temperature dependency can be expressed by the following Arrhenius equation (3).
k on = Ae− E / RT (3)
Here, A is a constant (frequency factor), E is activation energy, R is a gas constant, and T is an absolute temperature (K).
Substituting equation (3) into equation (2) yields equation (4).
Initial binding speed = Ae− E / RT [S] [L] (4)
Taking the natural logarithm of both sides of Equation (4) gives Equation (5) below.
Ln coupling initial velocity = −E / RT + LnA · [S] [L] (5)
From this equation, the initial binding velocity for L at a certain concentration [S] is calculated at various temperatures, the initial velocity of Ln binding is taken as the vertical axis, and 1 / T is taken as the horizontal axis, the plot draws a straight line from the slope -LnA · [L] [S] can be calculated from E / R and intercept. Here, if [S] is known and [L] is the amount of ligand immobilized on the sensor surface, E and A can be calculated. By substituting A and E calculated in this way into equation (4), a relational expression for the temperature of the initial bond speed is obtained. This is transformed to obtain equation (6).
[S] = bonding initial speed / (Ae− E / RT [L]) (6)
In Formula (6), A, E, and [L] are known, and the molecular concentration [S] in the sample can be measured by measuring the initial binding velocity and the environmental temperature T.
The ambient temperature is preferably measured in the vicinity of the measuring tank as much as possible, and is performed using a temperature sensor such as a platinum temperature sensor. You may carry out by putting a temperature sensor in a measurement solution.
In this method, A and E must be calculated in advance by the above method for each target substance whose concentration is to be measured to obtain Equation (6).
実際には測定装置、センサーの製造時にこれらの測定を前もって行い、式（６）を得ておいて、装置やソフトウエアに組み込んでおき、試験者が結合初期速度を測定すると、その結合初期速度とその時の環境温度の値を装置側に取り込み、自動的に温度補正された分子濃度が算出されるようにすることが望ましい。 Actually, these measurements are performed in advance when manufacturing the measuring device and sensor, and Equation (6) is obtained and incorporated in the device or software. When the tester measures the initial binding speed, the initial binding speed is obtained. It is desirable that the value of the ambient temperature at that time is taken into the apparatus side so that the temperature-corrected molecular concentration is automatically calculated.
尚、相互作用に関与するパラメータとしては、アレニウスの式の頻度因子Ａと活性化自由エネルギーＥ、又はそれらと他パラメータの組み合わせでなるパラメータ、例えば、式（５）における−Ｅ／Ｒを使用することができる。 As a parameter involved in the interaction, a frequency factor A and activation free energy E in the Arrhenius equation, or a parameter composed of a combination of these and other parameters, for example, -E / R in equation (5) is used. be able to.
これらの温度依存性のキャリブレーション方式は水晶振動子マイクロバランス法に限るものではなく、標的分子の吸着量を測定する方式のバイオセンサー、例えば、表面プラズモン共鳴素子等の全て適用できる。 These temperature-dependent calibration methods are not limited to the quartz crystal microbalance method, and all biosensors that measure the amount of adsorption of target molecules, such as surface plasmon resonance elements, can be applied.
また、本発明の測定対象となる試料について特に制限はなく、例えば、全血、血漿、血清等を測定対象とすることができる。
また、標的物質についても、例えば、モノクローナル抗体、キメラモノクローナル抗体、ヒト化モノクローナル抗体、ヒトモノクローナル抗体及びマウスモノクローナル抗体、並びに、抗体の抗原結合部位を含む融合タンパク質及びレセプターの抗原結合部位を含む融合タンパク質等を使用することができる。
Moreover, there is no restriction | limiting in particular about the sample used as the measuring object of this invention, For example, whole blood, plasma, serum, etc. can be made into a measuring object.
As for the target substance, for example, a monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody and a mouse monoclonal antibody, and a fusion protein containing an antigen binding site of an antibody and a fusion protein containing an antigen binding site of a receptor Etc. can be used.
固体支持体として水晶振動子を構成する金表面を用い、バイオセンサーシステムとして水晶振動子マイクロバランス法を採用した。水晶振動子を発振回路、周波数カウンターに接続し水晶振動子の周波数を一定時間毎に測定するようにした。
その水晶振動子の表面に標的物質と相互作用可能なリガンドタンパク質Ｌを固定化し緩衝溶液中に浸漬する。濃度を測定したい標的物質Ｓを含むサンプル溶液を緩衝溶液に一定量添加（終濃度６.６ｎＭ）すると標的物質が水晶振動子表面のリガンドに吸着し、水晶振動子の振動数が減少する。振動数減少の時間変化を計測し、結合初期速度を算出する。このときの反応模式図を図１に示す。
The gold surface constituting the crystal unit was used as the solid support, and the crystal unit microbalance method was used as the biosensor system. The crystal unit was connected to an oscillation circuit and a frequency counter, and the frequency of the crystal unit was measured at regular intervals.
Ligand protein L capable of interacting with the target substance is immobilized on the surface of the crystal unit and immersed in a buffer solution. When a certain amount of sample solution containing the target substance S whose concentration is to be measured is added to the buffer solution (final concentration: 6.6 nM), the target substance is adsorbed to the ligand on the surface of the crystal unit, and the frequency of the crystal unit is reduced. Measure the change in frequency with time and calculate the initial bond speed. The reaction schematic diagram at this time is shown in FIG.
この時、水晶振動子が内部に配置された測定容器の近傍に温度センサーを配置し、環境温度（雰囲気温度）を計測した。本実施例では、環境温度を１５℃、２５℃、３７℃と変化させたときの結合初期速度を算出した。
算出した結合初期速度を、上記式（５）に代入し、Ln初期の傾きを縦軸、温度の逆数である１／Ｔを横軸にプロットすると図２のようになる。この直線の傾きから−Ｅ／Ｒ、切片からLnＡ・［Ｓ］［Ｌ］が算出できる。ここでＲは定数、［Ｓ］はこの実験で用いた標的物質の終濃度で、６.６ｎＭ、［Ｌ］はリガンドの固定化量で既知であるため、ＥとＡを算出することができる。
At this time, a temperature sensor was disposed in the vicinity of the measurement container in which the crystal resonator was disposed, and the environmental temperature (atmosphere temperature) was measured. In this example, the initial bonding speed when the environmental temperature was changed to 15 ° C., 25 ° C., and 37 ° C. was calculated.
When the calculated initial bond speed is substituted into the above equation (5), the initial slope of Ln is plotted on the vertical axis, and the inverse of temperature 1 / T is plotted on the horizontal axis, as shown in FIG. -E / R can be calculated from the slope of this straight line, and LnA · [S] [L] can be calculated from the intercept. Here, R is a constant, [S] is the final concentration of the target substance used in this experiment, 6.6 nM, and [L] is known as the amount of ligand immobilized, so E and A can be calculated. .
ここで算出されたＡとＥを下記式（６）に代入することで、標的物質Ｓと結合初期速度の温度依存性の関係式が得られる。
［Ｓ］＝結合初期速度／（Ａｅ−Ｅ／ＲＴ［Ｌ］）・・・（６）
By substituting A and E calculated here into the following formula (6), a relational expression of the temperature dependence of the target substance S and the initial binding velocity is obtained.
[S] = bonding initial speed / (Ae− E / RT [L]) (6)
実際に測定を行うときは、結合初期速度と温度Ｔを測定し、式（６）に代入することで標的物質Ｓの濃度［Ｓ］を得る。
ここでは初期速度の温度依存性の一例を示したが、固定化するリガンド、測定したい標的物質を変更する毎にこれらの測定を行い、ＡとＥを算出し式（６）を得ておく必要がある。ただしＡとＥの値は相互作用する分子同士が同じであれば常に一定であるので、センサーの製造時にあらかじめ算出しておいた関係式を測定システムに組み込んでおけば、試験者は測定された初期速度と環境温度から特に追加の操作なしで正しい濃度の値を得ることができる。
When actually performing the measurement, the initial binding velocity and the temperature T are measured and substituted into the equation (6) to obtain the concentration [S] of the target substance S.
Here, an example of the temperature dependence of the initial velocity is shown, but it is necessary to perform these measurements each time the ligand to be immobilized and the target substance to be measured are changed, and calculate A and E to obtain equation (6). There is. However, since the values of A and E are always constant as long as the interacting molecules are the same, if the relational expression calculated in advance at the time of manufacturing the sensor is incorporated in the measurement system, the tester can measure it. The correct concentration value can be obtained from the initial speed and ambient temperature without any additional operation.
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| WO2009041554A1 (en) * | 2007-09-28 | 2009-04-02 | Hitachi Chemical Company, Ltd. | Sensor, sensor system, portable sensor system, method for analyzing metal ions, substrate for mounting, method for analyzing plating inhibitory chemical species, method for analyzing produced compound, and method for analyzing monovalent copper chemical species |
| WO2010082497A1 (en) * | 2009-01-15 | 2010-07-22 | 株式会社アルバック | Method for measuring systemic concentration of protein-containing medicament |
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| US20040046963A1 (en) * | 2002-09-07 | 2004-03-11 | Lackritz Hilary S. | Bioanalysis systems including optical integrated circuit |
| JP2005241537A (en) * | 2004-02-27 | 2005-09-08 | Matsushita Electric Ind Co Ltd | Detected substance measuring instrument |
| JP2008517298A (en) * | 2004-10-19 | 2008-05-22 | ベクトン・ディキンソン・アンド・カンパニー | Method for correcting light emission value and method for determining corrected concentration of analyte |
| WO2009041554A1 (en) * | 2007-09-28 | 2009-04-02 | Hitachi Chemical Company, Ltd. | Sensor, sensor system, portable sensor system, method for analyzing metal ions, substrate for mounting, method for analyzing plating inhibitory chemical species, method for analyzing produced compound, and method for analyzing monovalent copper chemical species |
| WO2010082497A1 (en) * | 2009-01-15 | 2010-07-22 | 株式会社アルバック | Method for measuring systemic concentration of protein-containing medicament |
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