JP2006322878A - Method and apparatus for analyzing ligands in sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly accurate ligand analysis method which enables reduction of the influence of nonspecific adsorbates. <P>SOLUTION: In the sample analysis method, separating and removing nonspecific adsorbates, which have been produced due to the binding reaction between a sample containing ligands and a receptor which is specific to the ligands, applying external oscillations and detecting the changes generated by the separation and removal of the nonspecific adsorbates as a stable width of surface plasmon resonance angles enables verifying the completion of the separation and the removal of the nonspecific adsorbates. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for analyzing a ligand in a sample and an apparatus for analyzing a ligand in a sample.

  The present invention relates to a method for analyzing a ligand in a sample and an apparatus for analyzing a ligand in a sample. More specifically, the present invention uses the principle of surface plasmon resonance and applies at least one of electrical vibration, magnetic vibration, and mechanical vibration. The present invention relates to a technique for vibrating the receptor, separating and removing non-specifically adsorbed substances, and confirming the achievement of the separation and removal.

  The present invention relates to a surface plasmon resonance apparatus, and can be preferably used to improve measurement accuracy and reproducibility by separating and removing non-specifically adsorbed substances, and further relates to a method and apparatus for separating and purifying biomolecules. By applying this, it can be used for an efficient biomolecule separation and purification apparatus.

  In biochemical reactions, the reaction of binding of a receptor and a ligand is important. For example, in an antigen-antibody reaction, an antibody binds to an antigen as a receptor and an antigen as a ligand. For example, in an enzyme reaction, an enzyme is bound as a receptor and an enzyme substrate is bound as a ligand. By utilizing such a reaction, it is possible to detect the ligand contained in the sample.

For example, when a receptor is bonded to the surface of a metal thin film and a ligand is bonded to the receptor, the dielectric constant near the metal thin film changes. A method of detecting the change by using the surface plasmon phenomenon and analyzing the binding amount between the ligand and the receptor is known (see Non-Patent Document 1). In this method, first, the metal thin film as described above is irradiated with light from the back of the surface to which the receptor is bonded at an angle satisfying the total reflection condition. Only when the wave number of the surface plasmon excited by the incident light and the wave number of the evanescent wave derived from the excitation light coincide with each other, a part of the light amount of the incident light is used for exciting the surface plasmon. The amount of reflected light decreases. Changes in the dielectric constant in the vicinity of the metal thin film can be measured by, for example, measuring the reflected light by changing the angle of the incident light and obtaining the angle of the incident light that maximizes absorption, or maintaining the angle of the incident light constant and absorbing it. There is known a method for obtaining the angle of reflected light that maximizes the angle. Also. A technique (for example, refer to Patent Document 1) for controlling the separation and movement of a material on a metal thin film by applying an electric field that is an electric vibration to the back surface of the metal thin film on which the receptor is fixed. There have been proposed techniques for measuring a large amount of data at once (for example, see Patent Document 2) and techniques for measuring the follow-up frequency of receptor molecules by applying external vibration to the receptor region.
Anal. Chem. , 1988, 70, 2019-2024) JP 2003-65947 A JP 2003-75336 A

  However, the conventional method for analyzing and evaluating the binding reaction between the ligand and the receptor is that the ligand and the non-measurement substance are adsorbed on a site other than the specific binding site of the receptor, adsorbed on the surface of the metal thin film, and the ligand molecule. Nonspecific adsorption, such as binding between the two, occurs, and due to the influence, the binding reaction between the ligand and the receptor cannot be accurately analyzed and evaluated, and it is difficult to obtain reproducibility and measurement accuracy.

  Therefore, the present invention solves the above-described conventional problems, and by applying a constant frequency external vibration to the receptor region, non-specifically adsorbed substances that may occur in the reaction between the ligand and the receptor are separated and removed, and Confirms the separation and removal of non-specifically adsorbed substances, thereby reducing the influence of non-specifically adsorbed substances with high measurement accuracy and reproducibility, and analyzing the ligand in the sample and analyzing the ligand in the sample The purpose is to provide a device.

  In order to achieve the above object, the analysis method of the present invention comprises a sample containing a ligand and a receptor capable of specifically binding to the ligand immobilized on one side thereof, and an optical prism disposed on the back side thereof. A metal thin film capable of causing surface plasmon resonance, an irradiation means for irradiating measurement light, a light receiving means for receiving reflected light of the measurement light, an analysis means for analyzing a ligand bound to the receptor, and the receptor fixed An application means for applying external vibration to the surface is prepared, the sample and the metal thin film are brought into contact with each other, the ligand in the sample is bound to the receptor, and the receptor of the metal thin film is formed by the irradiation means. The surface opposite to the surface on which is fixed is irradiated with measurement light, and the light receiving means receives the reflected light of the measurement light reflected on the surface of the metal thin film. The analysis means detects a change in surface plasmon resonance angle caused by a change in dielectric constant in the vicinity of the metal thin film from the reflected light, and the external vibration applying means fixes the receptor of the metal thin film. In a method of analyzing the ligand in the sample bound to the receptor from the frequency characteristics by applying external vibration to the surface, applying a constant frequency external vibration to the metal thin film The immobilized receptor is vibrated, and the non-specifically adsorbed substance is separated and removed by Coulomb force in addition to the vibration force in the case of the vibration force of the ligand and the electric external vibration.

  Further, the surface plasmon resonance angle is changed by the vibration of the receptor, and a width is generated in the surface plasmon resonance angle. The width of the surface plasmon resonance angle is gradually stabilized by separation and removal of the non-specifically adsorbed substance. The point in time when the stable width is detected is that the nonspecifically adsorbed substance is separated and removed, and it is confirmed that the nonspecifically adsorbed substance is separated and removed.

  As described above, in the analysis method and apparatus of the present invention, it is possible to selectively separate and remove only non-specifically adsorbed substances having a weaker binding force than specific adsorbed substances by applying constant frequency external vibration. It becomes.

  In addition, according to the immunoassay method and measurement apparatus of the present invention, it is possible to perform measurement with reduced influence of a non-specifically adsorbed substance by confirming a stable surface plasmon resonance angle width.

  Furthermore, by measuring the width of the surface plasmon resonance angle immediately after vibration and the width of the stable surface plasmon resonance angle in a sample in which a plurality of substances are mixed, the ratio of the specific adsorbed substance and the nonspecific adsorbed substance can be detected. it can.

  In the analysis method and apparatus of the present invention, it is preferable that at least one of the receptor and the ligand is charged. This is because the dielectric constant on the surface of the metal thin film is likely to change when electrical vibration is applied as external vibration. Examples of combinations of receptor and ligand include antigen and antibody, antibody and antigen, hormone and hormone receptor, hormone receptor and hormone, polynucleotide and polynucleotide receptor, polynucleotide receptor and polynucleotide, enzyme inhibitor, and the like. Examples include enzymes, enzymes and enzyme inhibitors, enzyme substrates and enzymes, enzymes and enzyme substrates, and the like.

  The mechanism of the change in dielectric constant near the metal thin film will be described below. When external vibration is applied to the receptor and the ligand, the metal follows the metal thin film, and the receptor and the ligand are concentrated on the surface of the thin film. Then, the molecular density of the evanescent region in the metal thin film changes. This change in molecular density changes the dielectric constant of the evanescent region, that is, the dielectric constant in the vicinity of the metal thin film.

  In the analysis method of the present invention, the applying means is preferably means for applying at least one of electric vibration, magnetic vibration and mechanical vibration, and more preferably means for applying at least electric vibration. preferable.

  In addition, a means for receiving the reflected light of the measurement light reflected on the surface of the metal thin film on which the receptor is fixed is prepared, and the separation of the nonspecifically adsorbed substance is performed at the time when the vibration width of the surface plasmon resonance angle is stabilized. The mechanism for achieving the removal will be described below. By applying an AC electric field, which is a constant frequency electrical external vibration, to the receptor and the ligand, the ligand and the receptor vibrate at a constant frequency. As a result, the dielectric constant of the evanescent region in the metal thin film undergoes a constant change, and the surface plasmon resonance angle included in the measurement light reflected on the surface of the metal thin film on which the receptor is fixed has a certain width. Changes called vibration. When non-specific adsorption occurs, the receptor and the ligand vibrate by applying a constant frequency electrical external vibration. A non-specifically adsorbing substance having a weaker binding than the specific binding between the receptor and the ligand is generated by applying a vibration force and an alternating electric field generated by the receptor and the ligand by the electrical external vibration. Since the change in the dielectric constant of the evanescent region is stable, the surface plasmon resonance angle included in the measurement light reflected on the surface of the metal thin film on which the receptor is fixed has a stable width. Detected as Further, when the non-specifically adsorbed substance is not removed and separated, it can be separated and removed by changing the predetermined voltage stepwise.

  FIG. 1 shows an optimal form of an immunoassay apparatus according to the first embodiment of the present invention.

  In FIG. 1, 1 is a metal thin film / upper electrode, 2 is a lower electrode, 3 is a receptor fixed to the metal thin film / upper electrode, 4 is a ligand specifically adsorbed to the receptor, 5 is a substance non-specifically adsorbed to the receptor, 6 Is an AC source, 7 is a frequency divider that converts the frequency of the AC source 6 to 1 / n, 8 and 18 are band-pass active filters that pass only a specific frequency in proportion to the external clock frequency, 9 is an AC electric field, 10 Is a light source, 11 is a prism, 12 is measurement light, 13 is a light receiving device, 14 is reflected light, 15 is a reflected light dark part in which a part of the light amount is reduced by plasmon excitation, 16 is a capacitor, 17 is an amplifier, and 19 is a surface. A detector 20 that detects the vibration width of the plasmon resonance angle, and 20 is an A / D converter that converts an analog signal obtained by the detector 19 into a digital signal.

  Non-specific to ligand 4 and receptor 3 specifically adsorbed to receptor 3 by introducing a sample into a metal thin film / upper electrode 1 and lower electrode 2 and receptor 3 fixed to metal thin film / upper electrode 1 The adsorbed substance 5 is bonded. Here, a unit comprising an AC source 6 and a frequency divider 7 is connected to the metal thin film / upper electrode 1 and the lower electrode 2. An AC electric field 9 which is a constant frequency electrical external vibration having a frequency of nf is applied by the AC source 6 to vibrate the receptor 3 by changing the direction of the molecule. Due to the vibration of the receptor 3, the ligand 4 and the nonspecifically adsorbed substance 5 also vibrate. In general, the bond between the receptor and the metal thin film has a stronger binding force than the bond between the receptor and the ligand, and the specific bond between the receptor and the ligand has a stronger binding force than the non-specific bond with the receptor. For this reason, a Coulomb force is generated between the electrodes by the AC electric field 9 which is an electrical external vibration. Then, the nonspecific adsorption material 5 having the weakest binding force due to the vibration force and Coulomb force of the receptor is released into the fluid, and the nonspecific adsorption material 5 is separated and removed.

  The light source 10 is composed of a ROHM AlGaAs double heterojunction visible light semiconductor laser, a Matsushita Nitto collimator lens, and a Sigma Kogyo polarizing beam splitter. Only the polarized light is applied to the metal thin film / upper electrode 1 made of JEOL Laser gold through the JEOL Laser prism 11 while changing the incident angle θ. As a method of changing the incident angle, the light source 10 may be driven and the measurement light 12 may be scanned on the metal thin film / upper electrode 1, or the light source 10 may be fixed and a reflecting mirror such as a polygon mirror scanner may be driven. 12 may be scanned.

  The measurement light 12 applied to the metal thin film / upper electrode 1 is totally reflected by the metal thin film / upper electrode 1 to generate reflected light 14. When the metal thin film / upper electrode 1 is irradiated with the measurement light 12 at a specific incident angle θ, an evanescent wave is generated, and a part of the light amount is used for excitation of the plasmon wave called surface plasmon resonance. A reduced reflected light dark portion 15 is generated. Using a light receiving device 13 that converts the intensity of the reflected light 14 into a voltage using a National Semiconductor operational amplifier and resistance element in a CCD area image sensor with 1024 × 252 effective pixels manufactured by Hamamatsu Photonics, Inc., the intensity of the reflected light 14 Is detected.

  Here, it is the electronic polarization component of the receptor that dominates the dielectric constant for the measurement light in the evanescent wave region. Therefore, if the receptor 3 in the evanescent region on the surface of the metal thin film / upper electrode 1 is changed in direction by the AC electric field 9. Since the density center of the electrons also changes, the dielectric constant changes, and thus the specific angle of the reflected light dark portion 15 where plasmon resonance occurs also vibrates in the range surrounding the light receiving device 13.

  Therefore, it is possible to separate and remove non-specifically adsorbed substances that have been an adverse effect of detecting the interaction between receptors and ligands by applying electrical external vibration, reducing the effects of non-specifically adsorbed substances. It is possible to analyze and evaluate the binding between the receptor and the ligand. Further, the reflected light 14 including the reflected light dark portion 15 is detected by the photodetector 13, and the AC component is amplified through the amplifier 17. The active filter 18 removes components other than the external vibration signal component, and the detector 19 compares the phase of the external vibration and the phase of the signal component of the external vibration included in the reflected light. The obtained analog signal is converted into a digital signal by the A / D converter 20.

Although the embodiment shown in FIG. 1 has the metal thin film / electrode as the upper part and the electrode as the lower part, it goes without saying that it is effective even if the upper and lower sides are reversed or left and right.
Although the embodiment of FIG. 1 is a nonspecific adsorption substance bound to a receptor, it includes all nonspecific adsorption substances that inhibit measurement, such as adsorption to a metal thin film surface and nonspecific adsorption to a ligand bound to a receptor. Needless to say.

  2 (a) to 2 (b), in one example of the apparatus of the present invention, a surface plasmon resonance curve measured without installing a receptor in the form of a metal thin film and a surface plasmon resonance curve measured by installing water as a receptor. An example is shown. In each figure, the horizontal axis represents the incident angle of the measurement light 12, and the vertical axis represents the intensity of the reflected light 5. FIG. 2A shows a surface plasmon resonance curve when no receptor is used, and FIG. 2B shows a surface plasmon resonance curve when water is used as the receptor. In this measurement, no external vibration is applied. As shown in FIGS. 2A and 2B, the angle at which surface plasmon resonance occurs varies depending on the presence or absence of a receptor. That is, it is shown that the change of the surface plasmon resonance angle accompanying the change of the dielectric constant near the metal thin film can be measured by an example of the apparatus of the present invention.

  FIG. 3 is a schematic diagram showing a change with time of separation and removal of a non-specifically adsorbed substance by an alternating electric field which is an electrical external vibration.

  In FIG. 3, 21 and 29 are metal thin film and electrodes, 22 and 30 are AC electric fields, 23 and 31 are receptors bound to the metal thin films and electrodes 21 and 29, 24 and 32 are ligands bound to the receptors 23 and 31, 25 And 33 are non-specifically adsorbed substances bound to the receptors 23 and 31, 26 and 34 are measurement light, 27 and 35 are reflected light, 28 and 36 are reflected light dark portions where a part of the light amount is reduced by plasmon excitation, 37 is The width of the reflected light dark part where a part of the light quantity due to unstable plasmon excitation is reduced by the non-specific adsorption separation and removal stage, and 38 is a part of the light quantity due to plasmon excitation stabilized by the non-specific adsorption substance separation and removal The width of the reflected light dark part.

  In FIG. 3, the receptor 23 in the vicinity of the metal thin film / upper electrode 21 vibrates by applying an alternating electric field 22 which is an electrical external vibration. In the stage where the non-specifically adsorbed substance 25 bound to the receptor 23 is separated and removed, the vibration width 37 of the reflection dark line portion 28 where plasmon resonance occurs causes unstable vibration. Subsequently, by applying an alternating electric field 30 that is an electrical external vibration, the nonspecifically adsorbed substance 33 bound to the receptor 31 is separated and removed. As the non-specific adsorption substance 33 is separated and removed, the vibration of the reflection dark line portion 36 in which plasmon resonance occurs gradually becomes a certain stable width 38. By detecting the maximum value or the average value of the stable vibration width 38, separation and removal of the nonspecifically adsorbed substance can be confirmed. The principle of separation and removal of the nonspecifically adsorbed substance is based on the force that causes the receptor 31 to vibrate and the Coulomb force when an AC electric field is applied.

  In the sample used in the present invention, bovine serum albumin (manufactured by Sigma-Aldrich, used as a ligand) was added to 1% phosphate buffer prepared by adding sodium hydroxide to phosphoric acid (manufactured by Sigma-Aldrich) and adjusted to pH 7.0. Was prepared to 10 μg / ml. The receptor is a bovine serum albumin antibody (manufactured by Sigma-Aldrich).

  In FIGS. 1 and 3, an AC electric field that is an electric external vibration is selected as an external vibration having a constant frequency. However, an AC electric field that is an electric external vibration can be applied, although a magnetic vibration or a mechanical external vibration is possible. It is more effective.

  From the above, non-specifically adsorbed substances are removed by applying external vibration, and the stable width of the surface plasmon resonance angle is detected, thereby achieving separation and removal of substances non-specifically adsorbed on the receptor molecules. It can be confirmed.

The present inventor speculates that the above-mentioned result was caused by the following mechanism. FIG. 4 is a procedure for confirming separation and removal of nonspecific adsorption of a ligand by an electric field. A receptor prepared by adding sodium hydroxide to phosphoric acid (manufactured by Sigma-Aldrich) and adding a ligand to a 1% phosphate buffer adjusted to pH 7.0, and a receptor immobilized on the metal thin film and upper electrode, Reacted. As ligands, bovine serum albumin (manufactured by Sigma-Aldrich, molecular weight 66 kDa, dissociation constant (Kd) 6.6 × 10 ⁻¹² M) and insulin (manufactured by Sigma-Aldrich, molecular weight 6 kDa, dissociation constant (Kd) 2 × 10 ⁻¹² ). M) are prepared, and bovine serum albumin antibody (manufactured by Sigma-Aldrich, used as a receptor) for bovine serum albumin-containing samples and insulin antibody (manufactured by Sigma-Aldrich, used as a receptor) for samples containing insulin. And reacted with each other. FIG. 4 compares the influence of the binding state of bovine serum albumin and bovine serum albumin antibody with insulin and insulin antibody by applying an electric field in the direction from metal thin film and upper electrode 1 to lower electrode 2 and sweeping the voltage. It is a thing. (A) is insulin and (b) is bovine serum albumin. (A) is a separation and removal step of non-specifically adsorbed substances, (B) is a steady state of binding between a ligand and a receptor, (C) is dissociation of the ligand, and (D) is a receptor from which the ligand is separated. It is done. FIG. 4 shows that the dissociation state of the ligand and the receptor is different by pulling the potential gradient by each substance. The dissociation constant of insulin and insulin antibody is 2 × 10 ⁻¹² M, the dissociation constant of bovine serum albumin and bovine serum albumin antibody is 6 × 10 ⁻¹² M, and the dissociation between bovine serum albumin and bovine serum albumin antibody The constant is large. That is, it is shown from the known data that the binding between insulin and the insulin antibody is stronger than that of the bovine serum albumin and the bovine serum albumin antibody, but this is also shown in FIG. Yes. That is, in general, the bond between the receptor and the metal thin film has a stronger binding force than the specific bond between the receptor and the ligand, and the bond between the receptor and the ligand has a stronger binding force than the bond between the receptor and the non-specific adsorbent. Because it is strong, non-specific adsorption is performed by separating and removing non-specific adsorbed substances and detecting the stable width of the surface plasmon resonance angle without dissociating the bond between the receptor and the metal thin film by applying external vibration. It is considered that the analysis of the ligand in the sample in which the influence of the nonspecific adsorption substance is reduced can be realized by confirming the achievement of the separation and removal of the substance.

  The method for analyzing a ligand in a sample and an apparatus for analyzing a ligand in a sample according to the present invention separate and remove nonspecifically adsorbed substances by external vibration, and achieve separation and removal of nonspecifically adsorbed substances. By checking, it is possible to perform measurement with reduced influence of non-specifically adsorbed substances, which has been an adverse effect of conventional measurement.

The schematic diagram which shows the apparatus which analyzes the ligand in the sample which reduced the influence of the non-specific adsorption substance which showed an example of this invention The figure which shows an example of the surface plasmon resonance curve measured without installing a receptor on a metal thin film in an example of the apparatus of this invention, and the surface plasmon resonance curve measured by installing water as a receptor, (a) No receptor Surface plasmon resonance curve diagram in the case of (b), (b) Surface plasmon resonance curve diagram when water is used as the receptor The schematic diagram which shows the time-dependent change of separation and removal of a nonspecific adsorption substance by an alternating electric field in an example of the apparatus of the present invention. Schematic diagram comparing the effects of substances bound to receptors fixed to a metal thin film by sweeping voltage in an example of the device of the present invention.

Explanation of symbols

1, 2, 29 Metal thin film and upper electrode 2 Lower electrode 3, 23, 31 Receptor fixed on metal thin film and upper electrode 4, 24, 32 Ligand adsorbed specifically to receptor 5, 25, 33 Non-specific adsorbed to receptor Substance 6 AC source 7 Frequency divider for dividing frequency of AC source 8, 18 Active filter that passes only specific frequency 9, 22, 30 AC electric field 10 Light source 11 Prism 12, 26, 34 Measuring light 13 Light receiving device 14, 27, 35 Reflected light 15, 28, 36 Reflected light dark portion in which a part of the amount of light is reduced by plasmon excitation 16 Capacitor 17 Amplifier 19 Detector 20 for detecting the vibration width of the surface plasmon resonance angle The analog signal obtained by the detector 19 is digitally converted A / D converter to convert to signal 37 Unstable plasmo by non-specific adsorption separation and removal stage The width of the reflected light dark portion in which a part of the light amount is reduced due to plasmon excitation becomes stable by the width 38 nonspecific adsorption material separation and removal of the reflected light dark portion partially decreased the amount of light by the excitation

Claims (11)

A sample containing a ligand, a receptor capable of specifically binding to the ligand, fixed on one side thereof, an optical prism disposed on the back side thereof, a metal thin film capable of causing surface plasmon resonance, and irradiation with measurement light An irradiating means, a light receiving means for receiving reflected light of the measurement light, and an analyzing means for analyzing a ligand bound to the receptor are prepared, and the sample and the metal thin film are brought into contact with each other to thereby form the ligand in the sample. Was bonded to the receptor, and the irradiation means irradiated measurement light onto the surface of the metal thin film opposite to the surface on which the receptor was fixed, and was reflected on the surface of the metal thin film by the light receiving means. A change in surface plasmon resonance angle caused by a change in dielectric constant in the vicinity of the metal thin film is received from the reflected light by the analyzing means by receiving the reflected light of the measurement light. A method for analyzing the ligand in a sample to be detected, comprising: applying means for applying an external vibration to a region where the receptor is fixed; and applying the external vibration to thereby detect the ligand and the receptor. It is detected that the separation and removal of only the non-specific adsorption substance is achieved without separating the specific adsorption as a change in the vibration width of the surface plasmon resonance angle which is a signal component detected from the light receiving means. Analysis method of ligands in samples. As a method for analyzing the ligand in the sample, means for applying an external vibration to a region where the receptor is fixed is prepared, and the metal thin film is applied by the application means while irradiating the measurement light by the irradiation means. The external vibration is applied to the surface on which the receptor is fixed, and the frequency characteristic of the surface plasmon resonance angle with respect to the external vibration is further obtained by the analyzing means, and the frequency characteristic in the sample coupled to the receptor is obtained from the frequency characteristic. The method for analyzing a ligand in a sample according to claim 1, further comprising means for analyzing the ligand. The method for analyzing a ligand in a sample according to claim 2, wherein at least one of the receptor and the ligand is charged. 3. The method for analyzing a ligand in a sample according to claim 2, wherein the applying means is means for applying at least one of electrical vibration, magnetic vibration and mechanical vibration. The method for analyzing a ligand in a sample according to claim 2, wherein the amount of the ligand in the sample bound to the receptor is analyzed. 3. The method for analyzing a ligand in a sample according to claim 2, wherein an external vibration having a constant frequency is applied as the means for applying the external vibration. The combination of the receptor and the ligand comprises an antigen and an antibody, an antibody and an antigen, a hormone and a hormone receptor, a hormone receptor and a hormone, a polynucleotide and a polynucleotide receptor, a polynucleotide receptor and a polynucleotide, an enzyme inhibitor The method for analyzing a ligand in a sample according to claim 2, wherein the enzyme is an enzyme, an enzyme inhibitor and an enzyme, an enzyme substrate and an enzyme, or an enzyme and an enzyme substrate. 3. The method for analyzing a ligand in a sample according to claim 2, wherein a maximum value or an average value is used as a signal component of the external vibration included in the reflected light of the measurement light. 3. The method for analyzing a ligand in a sample according to claim 2, wherein a predetermined force due to the external vibration is changed stepwise as the external vibration application method. 3. The method for analyzing a ligand in a sample according to claim 2, wherein a time point at which a stable vibration width is changed from an unstable vibration width is detected as a change in the vibration width of the surface plasmon. An apparatus for analyzing a ligand in a sample using the method for analyzing a ligand in a sample according to claim 1.

Images