JP2006337038A - Analysis method of ligand in sample, and analyzing apparatus of ligands in sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunoassay instrument capable of shortening the measuring time and of more minutely grasping the state of changes due to aging. <P>SOLUTION: Since the change in the intensity of the reflected light of measuring light due to the natural oscillation frequency of a receptor, the ligand in the sample bonded to the receptor and the composite of the receptor and the ligand, which appear in a frequency region lower than the follow-up frequency limit of the ligand, the receptor and the composite of the ligand and the receptor is used, a band of applied external vibration becomes narrower than the conventional bands, applied in order to calculate the follow-up frequency limit, and the measuring time is shortened. <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 an immunoassay method and apparatus for detecting a minute mass change of a liquid sample to be subjected to a biochemical reaction.

  In biochemical reactions, the reaction of binding of a receptor and a ligand is important. For example, in an antigen-antibody reaction, an antibody as a receptor binds to 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 is known in which the change is detected using the surface plasmon resonance phenomenon and the amount of binding between the ligand and the receptor is analyzed (see, for example, 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.

In addition, a technique for controlling the separation and movement of a sample on the 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 (for example, see Patent Document 1) and the refractive index of the metal thin film. Technology that measures a large amount of sample at once using changes (see Patent Document 2) and frequency characteristics of angular fluctuations that cause surface plasmon resonance by applying external vibrations, and measures the amount of ligand-receptor binding. A high-sensitivity and vibration-resistant technique has been proposed by reading the tracking frequency limit that appears as a change in the inflection point.
Anal. Chem. , 1988, 70, 2019-2024. JP 2003-65947 A JP 2003-75336 A

  However, in the conventional method, the frequency characteristic of the angular fluctuation that causes surface plasmon resonance by applying external vibration is measured, and the follow-up frequency limit that appears as the change of the inflection point of the frequency characteristic is read as the binding amount of the ligand and the receptor. Therefore, it is necessary to change the frequency of the external vibration to be applied in a wide band until the tracking frequency limit of the ligand in the sample bound to the receptor and the receptor and the complex of the receptor and the ligand is reached. there were. In addition, since the measurement time is long, there is a problem that it is impossible to grasp a change in state over time.

  The present invention solves the conventional problem by utilizing the change in the intensity of reflected light of the measurement light due to the natural vibration frequency of the ligand and the receptor and the complex of the ligand and the receptor appearing in a frequency region lower than the tracking frequency limit. An object of the present invention is to provide an immunoassay device that can shorten the measurement time and can grasp changes in the state with time in more detail.

  To achieve the above object, the analysis method of the present invention comprises a surface comprising a sample containing a ligand, a receptor capable of specifically binding to the ligand fixed on one side, and an optical prism arranged on the back side. Preparing a metal thin film capable of causing plasmon resonance, irradiation means for irradiating measurement light, light receiving means for receiving reflected light of the measurement light, and analysis means for analyzing a ligand bound to the receptor; And the metal thin film are brought into contact with each other to bind the ligand in the sample to the receptor, and the irradiation means irradiates the surface opposite to the surface of the metal thin film on which the receptor is fixed, The light receiving means receives the reflected light of the measurement light reflected on the surface of the metal thin film, and the analyzing means receives the dielectric near the metal thin film from the reflected light. A method for analyzing the ligand in a sample for detecting a change in a surface plasmon resonance angle caused by a change in the condition, comprising: applying means for applying external vibration to a region where the receptor is fixed; While irradiating the measurement light, an external vibration is applied to the surface of the metal thin film on which the receptor is fixed while irradiating the measurement light, and the analysis means further obtains a frequency characteristic of a surface plasmon resonance angle with respect to the external vibration. The ligand is analyzed from the natural vibration frequency of the receptor and the ligand bound to the receptor included in the frequency characteristic and the complex of the receptor and the ligand.

As described above, in the analysis method and apparatus of the present invention, when an alternating electric field, which is an electric external vibration, is applied to the ligand in the sample bound to the ligand and the receptor and the complex of the receptor and the ligand, the ligand and the ligand The ligand in the sample bound to the receptor and the complex of the receptor and the ligand follow an alternating electric field, the dielectric constant near the metal thin film changes, the surface plasmon resonance angle fluctuates, and the frequency of the applied alternating electric field is At the same natural frequency of the ligand and the complex of the receptor and the ligand in the sample bound to the ligand and the receptor, the ligand in the sample bound to the ligand and the receptor and the complex of the receptor and the ligand is Resonates with the frequency of the applied AC electric field, and the surface It utilizes the fact that amplitude of plasmon resonance angle is amplified. Accordingly, the natural vibration frequency of the ligand and the receptor-ligand complex in the sample bound to the receptor and the receptor appearing in a frequency region lower than the tracking frequency limit of the ligand and the receptor and the ligand-receptor complex. Since the change in the intensity of the reflected light of the measurement light is used, the external vibration band to be applied is narrower than the band applied to obtain the conventional follow-up frequency limit, so that the measurement time can be shortened. In addition, since the measurement time can be shortened, the number of times of measurement within a unit time increases, so that changes in state over time can be grasped in detail.

  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, hormone and hormone receptor, hormone receptor and hormone, polynucleotide and polynucleotide receptor, polynucleotide receptor and polynucleotide, enzyme inhibitor and enzyme, enzyme and Examples include enzyme inhibitors, enzyme substrates and enzymes, enzymes and enzyme substrates, and the like.

  The inventor presumes the mechanism of the change in dielectric constant in the vicinity of the metal thin film as follows. When external vibration is applied to the receptor and the ligand, the external vibration is followed 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. It is presumed that the change in the molecular density changes the dielectric constant in 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. Furthermore, it is preferable to use an alternating electric field.

   The inventor presumes that the ligand is analyzed from the natural vibration frequency of the receptor and the ligand bound to the receptor and the complex of the receptor and the ligand included in the receptor included in the frequency characteristic as follows. ing.

  When an AC electric field, which is an electric external vibration, is applied to the receptor in the previous stage of binding of the ligand and the receptor, the receptor follows the AC electric field, the dielectric constant near the metal thin film changes, and the surface plasmon resonance angle is increased. When the frequency of the alternating electric field to be shaken and applied is the same as the natural vibration frequency of the receptor, the receptor resonates with the frequency of the alternating electric field, and the amplitude of the surface plasmon resonance angle is amplified. When the ligand and the receptor start to bind, the amplitude of the surface plasmon resonance angle is amplified when the frequency of the applied AC electric field is the same as the natural vibration frequency of the receptor and the complex of the ligand and the receptor. . When the ligand and the receptor are sufficiently bound, the amplitude of the surface plasmon resonance angle is amplified when the frequency of the applied AC electric field is the same as the natural frequency of the complex of the ligand and the receptor. The frequency of the applied AC electric field when the amplitude of the surface plasmon resonance angle is amplified depends on the binding state of the ligand and the receptor, and is unique to the ligand, the receptor, and the complex of the receptor and the ligand. Influenced by vibration frequency. The ligand can be analyzed by detecting the frequency of the applied AC electric field when the amplitude of the surface plasmon resonance angle is amplified.

  FIG. 1 shows the best mode of an apparatus using external vibration in the first embodiment of the present invention.

  In FIG. 1, 1 is a light source, 2 is a prism, 3 is measurement light, 4 is a light receiving device, 5 is reflected light, 6 is a reflected light dark portion where a part of the light amount is reduced by plasmon excitation, and 7 is a metal thin film upper side. Electrode, 8 is a lower electrode, 9 is a receptor fixed to the metal film / upper electrode 7, 10 is an AC source, 11 is a frequency divider for converting the frequency of the AC source 10 to 1 / n, and 12 and 16 are specified Active filter that passes only the frequency, 13 is an external vibration, 14 is a capacitor, 15 is an amplifier, 17 is a detection that compares the phase (θ0) of the external vibration with the phase (θ1) of the signal component of the external vibration included in the reflected light , 18 is an A / D converter that converts an analog signal obtained by the detector 17 into a digital signal, 35 is a ligand, and θ is an incident angle.

  The light source 1 is composed of a ROHM AlGaAs double heterojunction visible semiconductor laser, a Matsushita Nitto collimator lens, and a Sigma Kogyo polarization beam splitter, and the measurement light 3 emitted from the beam is split by the beam splitter. Only the polarized light is irradiated to the metal thin film / upper electrode 7 made of gold manufactured by JEOL Laser through the prism 2 manufactured by JEOL Laser while changing the incident angle θ. As a method of changing the incident angle, the light source 1 may be driven and the measurement light 3 may be scanned on the metal thin film / upper electrode 7, or the light source 1 may be fixed and a reflection mirror such as a polygon mirror scanner may be driven. 3 may be scanned.

  The measurement light 3 irradiated to the metal thin film / upper electrode 7 is totally reflected by the metal thin film / upper electrode 7 to generate reflected light 5. When the metal thin film / upper electrode 7 is irradiated with the measurement light 3 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 6 is generated. The intensity of the reflected light 5 is detected by using a light receiving device 4 that converts the intensity of the reflected light 5 into a voltage using an operational amplifier and a resistance element made by National Semiconductor in a Si PIN photodiode manufactured by Hamamatsu Photonics. Although a Si PIN photodiode manufactured by Hamamatsu Photonics was used as the light detection element, a light receiving element such as a CCD may be used.

  A unit composed of an AC source 10 and a frequency divider 11 is connected to the metal thin film / upper electrode 7 and the lower electrode 8. The frequency of nf is generated by the AC source 10, converted to the frequency of f by the frequency divider 11, the external vibration 13 having the frequency of f is applied by the lower electrode 8, and the receptor 9 is vibrated by changing the direction of the molecule. Let Here, since it is the electronic polarization component of the ligand that dominates the dielectric constant for the measurement light in the evanescent wave region, if the receptor 9 in the evanescent region on the surface of the metal thin film / upper electrode 7 changes direction due to the external vibration 13. Since the electron density center also changes, the dielectric constant changes, and the specific angle of the reflected light dark portion 6 where plasmon resonance occurs also changes within the range surrounding the light receiving device 4.

  Further, the receptor 9 and the ligand 35 in the vicinity of the metal thin film / upper electrode 7 and the complex of the receptor 9 and the ligand 35 have a natural vibration frequency that resonates with the external vibration 13. When the frequency of the external vibration 13 to be applied is the natural vibration frequency of the receptor 9, the receptor 9 resonates and the vibration due to the change in direction of the receptor 9 is maximized. Therefore, the dielectric constant for the measurement light in the evanescent wave region also changes, and the change in the angle of the reflected light dark portion 6 where plasmon resonance occurs is maximized. Since the molecular weight increases as the receptor 9 and the ligand 35 are bound, the natural vibration frequency of the complex of the receptor 9 and the ligand 35 changes to a low frequency according to the degree of binding. When the ligand 35 and the receptor 9 are sufficiently bound and the molecular weight becomes constant, the natural vibration frequency of the receptor 9 also becomes constant. The natural vibration frequency of the receptor 9 and the ligand 35 and the complex of the receptor 9 and the ligand 35 appears in a lower frequency region than the tracking frequency. Therefore, the frequency band of the external vibration to be applied is narrowed and the measurement time is shortened. Moreover, since the measurement time can be shortened, the number of measurements in a single time can be increased, so that the state of the ligand 35 over time can be grasped in detail.

  In the embodiment shown in FIG. 1, the metal thin film / electrode is on the upper side and the electrode is on the lower side.

  FIG. 2 shows measured values obtained by measuring the intensity of the reflected light 5 with respect to the incident angle of the measurement light 3 irradiated using the light receiving device 4. 2A and 2B show the results obtained when there is only air without placing a sample on the metal thin film / upper electrode 7 and when water is placed as a sample. As shown in FIGS. 2 (a) and 2 (b), when the sample to be measured changes, the dielectric constant in the vicinity of the metal thin film changes, thereby changing the resonance angle. Therefore, a surface plasmon resonance curve is obtained in the measurement using this apparatus, thereby obtaining a sufficient effect.

  FIGS. 3A to 3C are schematic views showing an example of the state in the vicinity of the metal thin film according to the intensity of external vibration in the apparatus of the present invention. FIG. 3A is a schematic view showing a state in which the external vibration intensity is zero, FIG. 3B is a schematic view in a state in which the external vibration intensity is weak, and FIG. In each case, 19, 24, and 30 are measurement light irradiated by the light source 1, 21, 26, and 32 are metal thin film / upper electrodes, and 20, 25, and 31 are measurement light 19, 24, and 30 are metal thin film / upper electrodes 21. , 26, 32 are reflected light dark portions where plasmon resonance occurs, 22, 27, 33 are receptors fixed to the metal thin film / upper electrodes 21, 26, 32, and 29, 35 are external vibrations. θ11, θ12, and θ13 are surface plasmon resonance angles.

Ligand and receptor have a charge. When an external vibration is applied to the ligand and receptor, the metal follows the metal thin film / upper electrode, and the ligand is concentrated on the surface of the metal thin film / upper electrode. Therefore, the molecular density of the evanescent region in the metal thin film / upper electrode changes. As the molecular density in this region changes, the dielectric constant of the evanescent region also changes, and the angle at which plasmon resonance occurs changes.
Therefore, in the conventional surface plasmon measurement device, plasmon resonance occurs only at one angle, but by applying external vibration, plasmon resonance occurs at a plurality of angles over time instead of one. Therefore, the dark line due to plasmon resonance has a width.

  FIG. 4 shows the relationship between the tracking frequency and the natural vibration frequency of the complex of receptor and ligand. The horizontal axis represents the frequency of the external vibration to be applied, and the vertical axis represents the vibration amplitude of the receptor-ligand complex. When the frequency of external vibration to be applied is at the limit of the tracking frequency of the complex of the receptor and the ligand, the complex of the receptor and the ligand cannot vibrate and the fluctuation width disappears. In the conventional method, the frequency of the external vibration to be applied is swept to the limit of the tracking frequency of the complex of the receptor and the ligand, so that the bandwidth becomes wide and it takes a long time to measure. The natural vibration frequency of the receptor-ligand complex appears in the frequency band in which the receptor-ligand complex vibrates due to external vibration. Therefore, since it appears in a frequency band lower than the tracking frequency limit, the frequency band of the external vibration to be applied becomes narrower than the conventional bandwidth, and the measurement time can be shortened.

  FIGS. 5 (a) to 5 (d) show the state of the receptor and receptor-ligand complex and the width of the dark line due to plasmon resonance when the natural vibration frequency of the complex of the receptor and ligand is applied as the frequency of external vibration.

  In FIG. 5A, 38 is a metal thin film and electrode, 37 is a receptor immobilized on the metal thin film and electrode 38, 39 is a lower electrode, and 40 is a plasmon resonance in a stage before starting binding between the receptor 37 and a ligand. It is a waveform of the width of the dark line by. FIG. 5A shows the waveform 40 of the width of the dark line due to plasmon resonance and the state of the frequency of the external vibration to be applied in the stage before starting the binding between the receptor 37 and the ligand. The waveform 40 of the plasmon resonance dark line width has a maximum value when the frequency of the external vibration to be applied is the natural vibration frequency fa of the receptor 37.

  In FIG. 5B, 43 is a metal thin film and electrode, 41 is a receptor immobilized on the metal thin film and electrode 43, 42 is a ligand bound to the receptor 41, 44 is a lower electrode, and 45 is a bond between the ligand and the receptor. Waveform of dark line width due to plasmon resonance when degree is x%, 46 is a waveform obtained by multiplying waveform 40 of dark line width due to plasmon resonance of FIG. 5A by (100−x)%, and 47 is a waveform of FIG. This is a waveform obtained by multiplying the waveform 52 of the width of the dark line by plasmon resonance by x%. FIG. 5B shows the state of the waveform 45 of the dark line width due to plasmon resonance and the frequency of the external vibration to be applied at the stage where the ligand 42 and the receptor 41 are bonded.

  The natural vibration frequency of the complex of the receptor 41 and the ligand 42 is lower than the natural vibration frequency fa of the receptor 41 before the ligand 42 is bound. Since all of the receptors 41 immobilized on the metal film / upper electrode 43 do not bind to the ligand 42 at the same time, the metal film / upper electrode 43 has a receptor 41 and a complex of the receptor 41 and the ligand 42, and plasmon The frequency at which the width of the dark line due to resonance becomes maximum varies in the low frequency region depending on the degree of binding between the ligand 42 and the receptor 41. The width 45 of the dark line due to plasmon resonance when the binding degree of the ligand 42 and the receptor 41 is x% is the waveform 40 of the dark line width due to plasmon resonance in the previous stage where the binding between the ligand and the receptor is performed as shown in FIG. 100-x)% and the waveform 52 of the width of the dark line due to plasmon resonance at the stage where the binding between the ligand and the receptor described later in FIG.

  In FIG. 5C, 50 is a metal thin film / electrode, 48 is a receptor immobilized on the metal thin film / electrode 50, 49 is a ligand bound to the receptor 48, 51 is a lower electrode, 52 is a binding between the ligand and the receptor. Is a waveform of the dark line width due to plasmon resonance when 53 is sufficiently performed, and 53 is a waveform of the dark line width due to plasmon resonance at the stage before the start of binding between the receptor 48 and the ligand 49. FIG. 5C shows the state of the dark line width due to plasmon resonance and the frequency of the applied external vibration at the stage where the ligand 49 and the receptor 48 are sufficiently bonded. If the binding between the ligand 49 and the receptor 48 is sufficiently performed, the width of the dark line due to the plasmon resonance is maximized at the natural vibration frequency fb of the complex of the ligand 49 and the receptor 48.

  In FIG. 5 (d), 54 indicates the frequency of external vibration when the width of the dark line of plasmon resonance is maximized. FIG. 5D shows the change over time in the frequency of the external vibration when the width of the dark line of plasmon resonance is maximized. The horizontal axis represents time, and the vertical axis represents the frequency of external vibration when the width of the plasmon resonance dark line is maximized. The frequency of the external vibration when the width of the dark line of plasmon resonance is the maximum is the frequency fa before the binding between the ligand and the receptor is performed, but changes to a low frequency as the binding between the ligand and the receptor proceeds. When the ligand and the receptor are sufficiently bonded, the natural vibration frequency is not changed, and the frequency of the external vibration when the width of the plasmon resonance dark line becomes maximum is the natural vibration frequency fb of the complex of the ligand and the receptor. It becomes. The measurement is terminated when there is no change in the frequency of the external vibration when the width of the plasmon resonance dark line becomes maximum.

  6A and 6B, how to change the frequency of the external vibration to be applied will be described.

  In FIG. 6A, a method for obtaining the natural vibration frequency of a complex of a ligand and a receptor will be described.

  First, the natural vibration frequency fa of the receptor in the previous stage before the binding between the ligand and the receptor is performed. The frequency of the external vibration to be applied is swept from 0 Hz to the follower frequency limit of the receptor, and the frequency at which the width of the dark line due to plasmon resonance is maximized is obtained. The obtained frequency becomes the natural vibration frequency fa of the receptor.

  Second, at the stage where the ligand and the receptor are bound, the frequency of the external vibration to be applied is swept between the natural vibration frequency fa of the receptor and 0 Hz, and the frequency at which the width of the dark line due to plasmon resonance is maximized is determined. Ask. While the ligand and receptor are being bound, the frequency at which the width of the dark line due to plasmon resonance becomes maximum changes, so the external vibration frequency is swept between the natural vibration frequency fa and 0 Hz of the ligand until there is no change. Let When there is no change in the frequency at which the width of the dark line due to plasmon resonance becomes maximum, the frequency at which the width of the dark line due to plasmon resonance becomes maximum becomes the natural vibration frequency fb of the complex of the ligand and the receptor.

  In FIG. 6B, the vertical axis represents the frequency of external vibration to be applied, and the horizontal axis represents time. Conventionally, it has been necessary to sweep the frequency of the external vibration to be applied up to the limit of the tracking frequency of the ligand, but this time, the frequency of the external vibration is reduced to the natural vibration frequency fa of the receptor in the previous stage where the ligand and the receptor are combined. Since it is only necessary to sweep, the measurement time can be shortened.

  As described above, in the first embodiment, the change in the width of the dark line of the plasmon resonance depending on the natural vibration frequency of the receptor and the ligand and the ligand-receptor complex that resonate with the frequency of the external vibration to be applied is used. The measurement time of the reaction with the receptor that specifically binds to can be shortened.

  In FIG. 7, it is shown that the number of times of measurement is increased by reducing the measurement time, and the measurement accuracy is improved. The horizontal axis represents time, and the vertical axis represents the width of the plasmon resonance dark line. Previously, measurement was possible only at the location indicated by the white circle ○ 60, but the measurement time can be shortened by the present invention, so that the measurement at the location indicated by the black triangle ▲ 61 can be performed, and more accurate. High measurement is possible.

  According to the measurement method using the intensity change of the reflected light of the measurement light due to the natural vibration frequency of the ligand and the receptor of the immunoassay device of the present invention, the ligand and the receptor appearing in the lower frequency region than the tracking frequency limit, and the ligand and the receptor Since the change in reflected light intensity of the measurement light due to the natural vibration frequency of the composite is used, the measurement time can be shortened because the applied external vibration band becomes narrower than the band applied to obtain the conventional tracking frequency limit. It is useful in fields that require rapid immunoassay.

Schematic diagram showing an example of the apparatus of the present 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 (B) Surface plasmon resonance curve diagram when measured by installing water as a receptor 4 is a schematic diagram showing an example of a state in the vicinity of a metal thin film according to the intensity of external vibration in the apparatus of the present invention, (a) a schematic diagram showing surface plasmon resonance in a state where the external vibration intensity is zero, and (b) external vibration. Schematic diagram showing surface plasmon resonance when the intensity is weak, (c) Schematic diagram showing surface plasmon resonance when the external vibration intensity is strong The figure explaining the relationship between the tracking frequency and the natural vibration frequency of the complex of the receptor and the ligand in the present invention Schematic diagram showing the state of the receptor and the complex of the receptor and ligand and the width of the dark line by plasmon resonance when the natural frequency of the complex of the receptor and ligand in the present invention is applied as the frequency of external vibration, (a) Schematic diagram showing the waveform of the width of the dark line due to plasmon resonance and the state of the frequency of the external vibration to be applied in the stage before starting the binding of the ligand, (b) Dark line due to plasmon resonance in the stage where the ligand and the receptor are bound Schematic diagram showing the state of the waveform of the width and the frequency of the external vibration to be applied, (c) the state of the width of the dark line due to plasmon resonance and the state of the frequency of the external vibration to be applied when the ligand and the receptor are sufficiently bound (D) The frequency of external vibration when the width of the dark line of plasmon resonance is maximized Schematic diagram illustrating a change in The schematic diagram which shows how to change the frequency of the external vibration to be applied in the present invention, (a) the schematic diagram showing the method for obtaining the natural vibration frequency of the complex of the ligand and the receptor, (b) the frequency change of the conventional and the present invention Schematic comparison of how to let The figure explaining the improvement in measurement accuracy due to the increase in the number of measurements in the present invention

Explanation of symbols

1 Light source
2 Prism 3, 19, 24, 30 Measuring light 4 Light receiving device 5 Reflected light
6, 20, 25, 31 Reflected light dark portion in which a part of the light amount is reduced by plasmon excitation 7, 21, 26, 32 Metal thin film / upper electrode 8 Lower electrode,
9, 22, 27, 33 Receptor fixed to metal film and upper electrode 10 AC source,
11 Frequency Divider for Dividing Frequency of AC Source 12, 16 Active Filter Passing Only Specific Frequency 13, 29, 35 External Vibration 14 Capacitor 15 Amplifier 17 Detector 18 for Detecting Surface plasmon Resonance Angle Vibration Width 18 Obtained by Detector 17 A / D converter 36, 42, 49 for converting the analog signal thus obtained into a digital signal Ligand 37, 41, 48 Receptor 38, 43, 50 Metal thin film / electrode 39, 44, 51 Lower electrode
40 Waveform of dark line width by plasmon resonance in the stage before starting binding of receptor 37 and ligand 45 Waveform of dark line width by plasmon resonance when the degree of binding between ligand and receptor is x% 46 Waveform of dark line width by plasmon resonance 40 The waveform obtained by multiplying (100−x)% by 47. The waveform 52 obtained by multiplying the waveform 52 of the dark line width by the plasmon resonance by the binding degree x% of the receptor and the ligand. 52 Plasmon resonance when the ligand and the receptor are sufficiently bound. Waveform of dark line width due to 53 53 Waveform of dark line width due to plasmon resonance before the start of binding of receptor 48 and ligand 49 54 Frequency of external vibration when the width of the dark line of plasmon resonance is maximized 55 Conventional application Frequency band 56 Ligand uniqueness before binding Frequency band applied to search for the dynamic frequency fa 57 Frequency band applied to search for the natural vibration frequency fb at the stage where the ligand and the receptor are performed 58 Conventional frequency band applied 59 Frequency band applied according to the present invention 60 Location measured conventionally 61 Location newly measured in the present invention θ Incident angle θ11, θ12, θ13 Surface plasmon resonance angle

Claims (9)

A sample containing a ligand, a metal thin film capable of causing surface plasmon resonance, in which a receptor capable of binding to the ligand is fixed on one side and an optical prism is arranged on the back side thereof, and an irradiation means for irradiating measurement light And a light receiving means for receiving the reflected light of the measurement light and an analysis means for analyzing the ligand bound to the receptor, and bringing the sample into contact with the metal thin film to bring the ligand in the sample into the receptor. The measurement light reflected on the surface of the metal thin film by the light receiving means is irradiated to the surface opposite to the surface of the metal thin film on which the receptor is fixed by the irradiation means. The reflected light is received, and the analysis means detects the change in the surface plasmon resonance angle caused by the change in the dielectric constant in the vicinity of the metal thin film from the reflected light. The method for analyzing the ligand in a sample, comprising: applying means for applying external vibration to a region where the receptor is fixed; and irradiating the measurement light by the irradiating means, A sample in which external vibration is applied to the surface of the metal thin film on which the receptor is fixed, the frequency characteristic of the surface plasmon resonance angle with respect to the external vibration is obtained by the analyzing means, and the receptor included in the frequency characteristic and the receptor are combined. And analyzing the ligand from the natural vibration frequency of a complex of the ligand and the receptor-ligand complex. Analyzing the ligand based on a change in the fluctuation width of a dark line caused by a change in a natural vibration frequency of a ligand and a complex of the receptor and the ligand in the sample that are bound to the receptor and the receptor included in the frequency characteristic; The analysis method according to claim 1. 3. The analysis method according to claim 2, wherein the frequency band of the external vibration to be applied is set between 0 Hz and the natural vibration frequency that maximizes the fluctuation width of the dark line among the natural vibration frequencies of the receptor. . 4. The analysis method according to claim 1, wherein at least one of the receptor and the ligand is charged. 4. The analysis method according to claim 1, wherein the applying means is means for applying at least one of electrical vibration, magnetic vibration and mechanical vibration. 6. The method according to claim 5, wherein the applying means is means for applying at least electrical vibration, and the analyzing means further includes analyzing physical physical properties of the ligand from the reflected light. Analysis method characterized by The method according to any one of claims 1 to 6, wherein the amount of ligand in the sample bound to the receptor is analyzed. The combination of receptor and ligand is 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 enzyme, The analysis method according to any one of claims 1 to 7, which is an enzyme and an enzyme inhibitor, an enzyme substrate and an enzyme, and an enzyme and an enzyme substrate. An apparatus for analyzing a ligand in a sample using the analysis method according to claim 1.

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