JP4078615B2 - Method for measuring surface plasmon resonance - Google Patents
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Description
本発明は、泡が混入しても、容易に泡が溶解除去される表面プラズモン共鳴の測定方法に関する。 The present invention relates to a method for measuring surface plasmon resonance in which bubbles are easily dissolved and removed even if bubbles are mixed.
光学的なセンサー技術として表面プラズモン共鳴（ＳＰＲ）が知られている。ＳＰＲは金属薄膜に光を照射して反射光をモニターし、金属薄膜上の屈折率の変化を検出する方法である。ＳＰＲはバイオ分野、環境分野、工業分野へ応用されており、表面に固定化した生体分子の相互作用解析、抗原抗体反応モニター、糖度モニターなどに用いられている。 Surface plasmon resonance (SPR) is known as an optical sensor technology. SPR is a method of detecting a change in refractive index on a metal thin film by irradiating the metal thin film with light and monitoring reflected light. SPR is applied to the bio field, the environment field, and the industrial field, and is used for interaction analysis of biomolecules immobilized on the surface, antigen-antibody reaction monitor, sugar content monitor and the like.
最近はバイオ分野の生体分子相互作用解析に注目が集まっており、ＳＰＲによるラベルフリーかつリアルタイム解析からＫｉｎｅｔｉｃｓデータを得る試みがなされている。平衡状態を評価するだけでなく、結合、解離、反応などの速度を解析したＫｉｎｅｔｉｃｓデータは生体分子の機能を明らかにする上で非常に有用な情報となる。 Recently, attention has been focused on biomolecular interaction analysis in the bio field, and attempts have been made to obtain Kinetics data from label-free and real-time analysis by SPR. Kinetics data that analyzes not only the equilibrium state but also the kinetics of binding, dissociation, reaction, etc. is very useful information for clarifying the functions of biomolecules.
通常のＳＰＲは基本的に一点のみの測定であるため、スループット性に欠ける。そこで同時に複数点の解析が可能なＳＰＲイメージング技術が注目されている。ＳＰＲイメージングにおいては、複数の生体分子を金蒸着表面に固定化したアレイを作製し、生体分子が固定化された部位の反射光強度をリアルタイムでモニターすることで、リアルタイムにＫｉｎｅｔｉｃｓデータを得ることができる（非特許文献１）。 Since normal SPR is basically a measurement of only one point, it lacks throughput. Therefore, SPR imaging technology capable of analyzing a plurality of points simultaneously has attracted attention. In SPR imaging, it is possible to obtain Kinetics data in real time by preparing an array in which a plurality of biomolecules are immobilized on a gold deposition surface and monitoring the reflected light intensity of the site where the biomolecules are immobilized in real time. Yes (Non-Patent Document 1).
従来のＳＰＲでは測定点は狭い流路内で行う方式であり、泡が混入しても狭い流路内を泡が通過するために、泡が滞留する恐れが少なかった。最近ではランニングバッファと測定対象物質（アナライト）を含む溶液の間に恣意的に泡を混入させて、アナライトを含む溶液が希釈されず、完全に分け隔てられる技術が開発されている。 In the conventional SPR, the measurement point is a method performed in a narrow channel, and even if bubbles are mixed, the bubbles pass through the narrow channel, so that there is little possibility that the bubbles will stay. Recently, a technique has been developed in which bubbles are arbitrarily mixed between a running buffer and a solution containing a substance to be measured (analyte) so that the solution containing the analyte is not diluted and is completely separated.
しかし、ＳＰＲイメージング法においてはチップ上の広い面積に対して液を流すため、泡が一旦混入すると、泡の部分は流れにくく、周囲の領域に流れる傾向となる。その結果、泡は全く除去されず、残ったままとなる問題点があった。特にチップをセットし、液を流し始めたときに泡がチップ上に残る場合が多く、容易には泡は除去されなかった。泡が存在すると、泡の存在する場所は全く測定できなく状態となるため、好ましくない。従って、測定前に泡を完全に除去する必要がある。 However, in the SPR imaging method, since the liquid is flowed over a large area on the chip, once the bubbles are mixed, the bubbles are difficult to flow and tend to flow to the surrounding area. As a result, there was a problem that bubbles were not removed at all and remained. In particular, when the chip was set and the liquid started to flow, bubbles often remained on the chip, and the bubbles were not easily removed. If bubbles are present, the location where the bubbles are present cannot be measured at all, which is not preferable. Therefore, it is necessary to completely remove the bubbles before the measurement.
泡の混入を予防する手段として、ランニングバッファやアナライトを含む溶液を事前に脱気しておく手段が公知である。しかし、この手段は予防する手段としては効果があるかもしれないが、泡が混入してしまった場合、除去する手段としてはそれほど効果的ではない。 As a means for preventing the mixing of bubbles, a means for previously degassing a solution containing a running buffer or an analyte is known. However, this means may be effective as a preventive means, but is not very effective as a means for removing bubbles when they are mixed.
センサー面への試料液供給通路を設け、前記試料液供給通路内に、供給されてきた試料液中の気泡をトラップする凹所を設けたＳＰＲ装置が開発・開示されている（特許文献１）。試料中に泡が混入していた場合、センサー面に泡が入るのを防ぐ優れた技術である。しかし、一旦入った泡を取り除く方法は示されていない。 An SPR device in which a sample liquid supply passage to the sensor surface is provided and a recess for trapping bubbles in the supplied sample liquid is provided in the sample liquid supply passage has been developed and disclosed (Patent Document 1). . This is an excellent technique for preventing bubbles from entering the sensor surface when bubbles are mixed in the sample. However, it does not show how to remove the foam once it has entered.
また、従来のＳＰＲ装置においてはフローセルの入口側の内部容量を微小化し、アナライトのインジェクションからセンサー表面までの到達までのレスポンスが短くなるようには工夫されたものがあるものの、フローセルの出口部分はそのまま開放されて廃液とする場合が多く、出口側に工夫を加えたものはほとんどみられなかった。
本発明の課題は、フローセル内に泡が混入したとしても、容易に泡が除去可能な表面プラズモン共鳴の測定方法に関する。 The subject of this invention is related with the measuring method of the surface plasmon resonance which can remove a bubble easily even if a bubble mixes in a flow cell.
本発明者らは鋭意検討した結果、以下に示す手段により、上記課題を解決できることを見出した。
１．チップ表面に液を流すことのできるフローセルを有する表面プラズモン共鳴装置を用いる表面プラズモン共鳴の測定方法であり、フローセルの出口に接続された流路の断面積の最小部分が０．０４０ｍｍ２以下であり、ならびにフローセルの入口に接続された流路の断面積の最小部分をフローセルの出口に接続された流路の断面積の最小部分より大きくする、もしくはフローセルの入口に接続された流路の長さをフローセルの出口に接続された流路の長さより短くすることを特徴とする表面プラズモン共鳴の測定方法。
２．前記フローセルに流速５０μｌ／ｍｉｎ以上で液を送液する１の表面プラズモン共鳴の測定方法。
３．1または２のフローセルの出口に接続された流路が、断面積０．０４０ｍｍ ２ 以下の流路部分が３０ｃｍ以上存在しているフローセルの出口側のチューブである表面プラズモン共鳴の測定方法。
４．チップ表面に液を流すことのできるフローセルを有する表面プラズモン共鳴装置を用いる表面プラズモン共鳴の測定方法であり、フローセルの流路でチップ表面のセンサー部と液が接触するための領域における圧力が１．０×１０４Ｐａ以上となる条件で液を流すことを特徴とする１〜３のいずれかの表面プラズモン共鳴の測定方法。
As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
1. This is a surface plasmon resonance measurement method using a surface plasmon resonance apparatus having a flow cell capable of flowing a liquid on the chip surface, and the minimum portion of the cross-sectional area of the flow path connected to the outlet of the flow cell is 0.040 mm 2 or less. Ri, and larger than the minimum portion of the cross-sectional area of the flow path of the minimal portion which is connected to the outlet of the flow cell of the cross-sectional area of the connected flow path to the inlet of the flow cell or flow cell inlet mouth connected to flow paths of A method of measuring surface plasmon resonance, characterized in that the length is shorter than the length of a flow path connected to the outlet of the flow cell .
2. The surface plasmon resonance measuring method of 1 which sends a liquid to the said flow cell by the flow rate of 50 microliters / min or more .
3. A method for measuring surface plasmon resonance, wherein the flow path connected to the outlet of the flow cell 1 or 2 is a tube on the outlet side of the flow cell in which a flow path portion having a cross-sectional area of 0.040 mm 2 or less is 30 cm or more.
4). This is a surface plasmon resonance measurement method using a surface plasmon resonance apparatus having a flow cell capable of flowing a liquid on the chip surface, and the pressure in the region where the liquid is in contact with the sensor part on the chip surface in the flow channel of the flow cell is 1. The method for measuring surface plasmon resonance according to any one of 1 to 3 , wherein the liquid is allowed to flow under conditions of 0 × 10 4 Pa or more .
本発明の表面プラズモン共鳴測定方法は泡が混入しても容易に溶解除去することができる測定方法であり、操作性が大幅に改善される。 The surface plasmon resonance measurement method of the present invention is a measurement method that can be easily dissolved and removed even if bubbles are mixed, and the operability is greatly improved.
以下に本発明を詳細に説明する。本発明で使用されるＳＰＲ装置はフローセルを有している。フローセルの材質、形状は特に限定されるものではなく、材質としてはポリジメチルシロキサン（ＰＤＭＳ）などのシリコン系ポリマー、ポリエーテルエーテルケトン（ＰＥＥＫ）などの抗薬品性を有するポリマー、チタンなどの抗腐食性の金属などの材質が挙げられ、形状としては一体型のマイクロ流路セル、固体の板とチップの間にガスケットを挟み込んで流路部分を確保したセル、Ｏリングで液漏れを防いだセルなどが挙げられる。加工方法としては、削り出し、焼成、エッチング、射出など、それぞれの素材に適合したものであれば、特に限定されるものではない。 The present invention is described in detail below. The SPR device used in the present invention has a flow cell. The material and shape of the flow cell are not particularly limited. Examples of the material include silicon-based polymers such as polydimethylsiloxane (PDMS), polymers having anti-chemical properties such as polyetheretherketone (PEEK), and anti-corrosion such as titanium. The shape of the material is an integrated micro-channel cell, a cell with a gasket sandwiched between a solid plate and a chip, and a cell that prevents liquid leakage with an O-ring. Etc. The processing method is not particularly limited as long as it is suitable for each material, such as cutting, baking, etching, and injection.
また、チップは基板の表面に測定対象物質と何らか結合や相互作用を示す物質が設置されたセンサー部を有し、センサー部はアレイ状に配置されていることができる。 In addition, the chip has a sensor part in which a substance that exhibits some binding or interaction with the measurement target substance is disposed on the surface of the substrate.
本発明に使用されるフローセルはチップ表面のセンサー部に液を供給して流すことができ、液の入口と出口を有する。ここで、フローセルの流路でチップ表面のセンサー部と液が接触するための領域を接液部と呼ぶ。また、単にフローセル内部という場合はこの接液部を意味する。フローセルの入口側は特に限定されるものではないが、少なくとも５０μｌ／ｍｉｎ以上の流速で水を供給できる能力を有するポンプが接続されていることが好ましい。ポンプの種類も特に限定されるものではなく、シリンジポンプ、プランジャーポンプ、ギアポンプなどが挙げられる。また、加圧タンクを用いることもできる。 The flow cell used in the present invention can supply and flow a liquid to the sensor part on the chip surface, and has an inlet and an outlet for the liquid. Here, an area where the liquid contacts the sensor part on the chip surface in the flow path of the flow cell is called a liquid contact part. In addition, simply referring to the inside of the flow cell means this liquid contact portion. The inlet side of the flow cell is not particularly limited, but a pump having a capability of supplying water at a flow rate of at least 50 μl / min is preferably connected. The type of pump is not particularly limited, and examples thereof include a syringe pump, a plunger pump, and a gear pump. A pressurized tank can also be used.
本発明において使用されるフローセルの出口に接続された流路は細くなるように設計されており、流路中の液の進行方向に対して垂直方向の切断面における断面積は０．０４ｍｍ2以下が好ましく、さらに好ましくは０．０３ｍｍ2以下である。
The flow path connected to the outlet of the flow cell used in the present invention is designed to be thin, and the cross-sectional area in the cut surface perpendicular to the liquid traveling direction in the flow path is 0.04 mm 2 or less. Is more preferable, and 0.03 mm 2 or less is more preferable.
ここで、フローセルの出口に接続された流路とは、フローセルの出口側のチューブを表し、以下、フローセルの出口に接続された流路を出口側流路と記することがある。
Here, the flow path connected to the outlet of the flow cell represents a tube on the outlet side of the flow cell , and hereinafter, the flow path connected to the outlet of the flow cell may be referred to as an outlet-side flow path.
Ｈａｇｅｎ−Ｐｏｉｓｅｕｉｌｌｅの式（式１）より、流路の相当半径の４乗に、圧力損失は反比例する。 From the Hagen-Poiseillele equation (Equation 1), the pressure loss is inversely proportional to the fourth power of the equivalent radius of the flow path.
式１において、符号は以下の通りである。
ｐ0−ｐL：静圧差 [Ｐａ]
μ：流体の粘度 [Ｐａ・ｓ]
Ｌ：流路の長さ [ｍ]
＜ｕ＞：平均断面速度 [ｍ／ｓ]
Ｒ：流路の相当半径 [ｍ]
Ｖ：流速 [ｍ3／ｓ]
In Equation 1, the symbols are as follows.
p 0 −p L : Static pressure difference [Pa]
μ: Fluid viscosity [Pa · s]
L: Length of flow path [m]
<U>: Average cross-sectional velocity [m / s]
R: Equivalent radius of channel [m]
V: Flow velocity [m 3 / s]
流路を細くすることで、フローセル内部に圧力を与えることが可能である。圧力を加わると、混入した泡が液中に溶解しやすくなり、容易に泡が除去されるため好ましい。流路の断面形状は耐圧上、円形であることが好ましい。また素材としてはＳＵＳなどの金属、ＰＥＥＫ、テフゼルなどの耐圧性、耐薬品性に優れたポリマーが好ましい。ＰＥＥＫ製のチューブが耐圧性、耐薬品性の面で特に好ましい。 By narrowing the flow path, it is possible to apply pressure to the inside of the flow cell. It is preferable to apply pressure because the mixed bubbles easily dissolve in the liquid and the bubbles are easily removed. The cross-sectional shape of the channel is preferably circular in terms of pressure resistance. The material is preferably a metal such as SUS or a polymer excellent in pressure resistance and chemical resistance such as PEEK or Tefzel. A PEEK tube is particularly preferable in terms of pressure resistance and chemical resistance.
フローセルに送液する液の流速は５０μｌ／ｍｉｎ以上であることが好ましい。当然、液漏れはないため、フローセル出口の流速も、入口と同じ流速である。これも前記理由と同様にフローセル内部に圧力を与え、泡を溶解除去しやすくするためである。流速を上げることで断面平均速度＜ｕ＞が増大し、その結果圧力損失が増加する。さらに好ましい流速は１００μｌ／ｍｉｎ以上であり、場合によっては、流速を上げたり下げたりする方法、瞬間的に圧力波を与え、一気に泡を押し流す方法なども含む。 The flow rate of the liquid sent to the flow cell is preferably 50 μl / min or more. Of course, since there is no liquid leakage, the flow velocity at the outlet of the flow cell is the same as that at the inlet. This is also because the pressure is applied to the inside of the flow cell to facilitate dissolution and removal of the bubbles as in the above reason. Increasing the flow rate increases the cross-sectional average speed <u>, resulting in an increase in pressure loss. Furthermore, a preferable flow rate is 100 μl / min or more. In some cases, a method of raising or lowering the flow rate, a method of momentarily applying a pressure wave, and pushing away bubbles at once are included.
出口側流路の長さは３０ｃｍ以上、さらには４０ｃｍ以上であることが好ましい。これも長ければ長いほど圧力損失が生じるためである。出口側流路の液出口末端は基本的に開放系であり、排出され液は廃液として廃棄される。 The length of the outlet side channel is preferably 30 cm or more, and more preferably 40 cm or more. This is because the longer this is, the more pressure loss occurs. The liquid outlet end of the outlet side channel is basically an open system, and the discharged liquid is discarded as waste liquid.
出口側流路の断面積が、液出口末端までの間で変化している場合、断面積が０．０４ｍｍ2以下である部分が少しでも含まれている場合は本発明に含まれる。この場合、断面積０．０４０ｍｍ2以下の流路部分が３０ｃｍ以上存在していることが好ましい。 In the case where the cross-sectional area of the outlet-side channel changes between the liquid outlet and the end of the liquid outlet, the present invention includes a case where even a portion having a cross-sectional area of 0.04 mm 2 or less is included. In this case, it is preferable that a flow path portion having a cross-sectional area of 0.040 mm 2 or less is 30 cm or more.
また、フローセルの入口に接続された流路（以下入口側流路と総称することがある）は、ポンプに負担をかけず、フローセルに効率よく圧をかけるために、同一の液を同一の流速で流した際に、入口側流路で生じる圧力損失が出口側流路で生じる圧力損失より小さいことが好ましい。
上記の具体的な方法としては、入口側流路の断面積の最少部分を出口側流路の断面積の最少部分より大くする、入口側流路の長さを出口側流路の長さより短くする、等の方法が挙げられる。
Further, an inlet connected to flow paths of the flow cell (Ru Kotogaa be collectively referred to as the inlet side flow path below) without burdening the pump, in order to apply efficient pressure flow cell, the same liquid to the same When flowing at a flow rate, it is preferable that the pressure loss generated in the inlet side flow path is smaller than the pressure loss generated in the outlet side flow path.
As a specific method of the above, the minimum part of the cross-sectional area of the inlet-side channel is made larger than the minimum part of the cross-sectional area of the outlet-side channel, and the length of the inlet-side channel is made longer than the length of the outlet-side channel. The method of shortening etc. is mentioned.
本発明のＳＰＲ測定方法においては、接液部の圧力は１．０×１０4Ｐａ以上であることが好ましい。圧力が高いことで混入した泡が溶解して除去されるためである。前述の方法を適宜採用することにより、接液部の圧力を１．０×１０4Ｐａ以上とすることがる。 In the SPR measurement method of the present invention, the pressure in the wetted part is preferably 1.0 × 10 4 Pa or more. This is because the mixed bubbles are dissolved and removed due to the high pressure. By appropriately adopting the above-described method, the pressure of the liquid contact part may be 1.0 × 10 4 Pa or more.
以下に実施例を示して本発明を具体的に説明するが、本発明は実施例に限定されるものではない。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.
［実施例］ [Example]
図１に示すフローセル（材質：ＰＥＥＫ）を備えたＳＰＲイメージング装置（ＭｕｌｔｉＳＰＲｉｎｔｅｒ（Ｒ）：東洋紡績製）にＭｕｌｔｉＳＰＲｉｎｔｅｒ（Ｒ） ＣＯＯＨチップ（東洋紡績製）をセットした。このフローセルに、断面直径０．１７ｍｍで長さ６０ｃｍのＰＥＥＫ製チューブを接続し、２５０μｌ／ｍｉｎの流速で脱気処理を施した水を流した。脱気処理は超音波を当て、アスピレータで１０分間減圧させた。チューブの断面積は０．０２３ｍｍ2と計算され、０．０４０ｍｍ2以下である。ＳＰＲ装置の温度は３０℃に保って実験を行った。
水を通液直後は図２のように泡が混入したものの、その泡は１０分以内に消失した。空気が液中に溶解したためである。このフローセル内の圧力を以下のパラメータをＨａｇｅｎ−Ｐｏｉｓｅｕｉｌｌｅの式に代入し算出した。
流体の粘度：μ＝０．７９７×１０-3 [Ｐａ・ｓ]
流路の長さ：Ｌ＝０．６ [ｍ]
流路の相当半径：Ｒ＝８．５×１０-5 [ｍ]
流速：Ｖ＝４．２×１０-9 [ｍ3／ｓ]
計算の結果、静圧差は９．７×１０4Ｐａであり、１．０×１０4Ｐａ以上であった。泡の除去が完了したのちは、流速を１００μｌ／ｍｉｎに落として、実験を行った。流速１００μｌ／ｍｉｎにおけるフローセル内圧力は３．９×１０4Ｐａと計算され、測定中においても１．０×１０4Ｐａ以上の高い圧力を保っている。この場合、測定中に泡が混入しても、泡が消失する可能性が高く、泡によって測定が中断される危険性が少ない。
A MultiSPRinter (R) COOH chip (manufactured by Toyobo) was set in an SPR imaging apparatus (MultiSPRinter (R): manufactured by Toyobo) equipped with a flow cell (material: PEEK) shown in FIG. A PEEK tube having a cross-sectional diameter of 0.17 mm and a length of 60 cm was connected to the flow cell, and degassed water was flowed at a flow rate of 250 μl / min. In the deaeration process, ultrasonic waves were applied and the pressure was reduced by an aspirator for 10 minutes. The cross-sectional area of the tube was calculated to 0.023 mm 2, it is 0.040 mm 2 or less. The experiment was conducted while maintaining the temperature of the SPR device at 30 ° C.
Immediately after passing water, although the foam was mixed as shown in FIG. 2, the foam disappeared within 10 minutes. This is because air was dissolved in the liquid. The pressure in the flow cell was calculated by substituting the following parameters into the Hagen-Poiseille equation.
Fluid viscosity: μ = 0.797 × 10 −3 [Pa · s]
Length of flow path: L = 0.6 [m]
Equivalent radius of flow path: R = 8.5 × 10 −5 [m]
Flow velocity: V = 4.2 × 10 −9 [m 3 / s]
As a result of the calculation, the static pressure difference was 9.7 × 10 4 Pa, which was 1.0 × 10 4 Pa or more. After the removal of the bubbles was completed, the experiment was performed with the flow rate lowered to 100 μl / min. The pressure in the flow cell at a flow rate of 100 μl / min is calculated to be 3.9 × 10 4 Pa, and a high pressure of 1.0 × 10 4 Pa or higher is maintained even during measurement. In this case, even if bubbles are mixed during the measurement, there is a high possibility that the bubbles disappear, and there is little risk that the measurement is interrupted by the bubbles.
［比較例］
図１に示すフローセル（材質：ＰＥＥＫ）を備えたＳＰＲイメージング装置（ＭｕｌｔｉＳＰＲｉｎｔｅｒ（Ｒ）：東洋紡績製）にＭｕｌｔｉＳＰＲｉｎｔｅｒ（Ｒ） ＣＯＯＨチップ（東洋紡績製）をセットした。このフローセルに、断面直径０．２５ｍｍで長さ２０ｃｍのＰＥＥＫ製チューブを接続し、２５０μｌ／ｍｉｎの流速で水を流した。チューブの断面積は０．０４９ｍｍ2と計算され、０．０４ｍｍ2以上であり本発明を満たさない。
実施例と同様に水を通液直後に泡が混入し、水を流したが、泡は３０分経過した段階で、やや小さくなったものの消失しなかった。このフローセル内の圧力をＨａｇｅｎ−Ｐｏｉｓｅｕｉｌｌｅの式から計算したところ、６．９×１０3Ｐａであり、１．０×１０4Ｐａ以下であった。圧力が不十分であり、混入した泡は容易には溶解消失しない。
[Comparative example]
A MultiSPRinter (R) COOH chip (manufactured by Toyobo) was set in an SPR imaging apparatus (MultiSPRinter (R): manufactured by Toyobo) equipped with a flow cell (material: PEEK) shown in FIG. A PEEK tube having a cross-sectional diameter of 0.25 mm and a length of 20 cm was connected to the flow cell, and water was allowed to flow at a flow rate of 250 μl / min. The cross-sectional area of the tube was calculated to 0.049 mm 2, it does not satisfy the there present invention with 0.04 mm 2 or more.
As in the example, bubbles were mixed immediately after passing water, and water was allowed to flow, but the bubbles did not disappear after 30 minutes, although they were slightly smaller. When the pressure in the flow cell was calculated from the Hagen-Poiseille equation, it was 6.9 × 10 3 Pa and 1.0 × 10 4 Pa or less. The pressure is insufficient and the mixed bubbles do not dissolve and disappear easily.
本発明の表面プラズモン共鳴測定方法は泡が混入しても容易に溶解除去することができる測定方法であり、操作性が大幅に改善され、産業界に貢献すること大である。 The surface plasmon resonance measurement method of the present invention is a measurement method that can be easily dissolved and removed even if bubbles are mixed in, and greatly improves operability and contributes to the industry.
Claims (4)
This is a surface plasmon resonance measurement method using a surface plasmon resonance apparatus having a flow cell capable of flowing a liquid on the chip surface, and the pressure in the region where the liquid is in contact with the sensor part on the chip surface in the flow channel of the flow cell is 1. The method for measuring surface plasmon resonance according to any one of claims 1 to 3 , wherein the liquid is allowed to flow under a condition of 0 x 10 4 Pa or more .
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