JP2015021818A - Surface plasmon enhanced fluorescence measuring apparatus and surface plasmon enhanced fluorescence measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface plasmon resonance fluorescence analysis apparatus using GC-SPFS capable of accurately detecting a substance to be detected even when a nonspecific binding occurs.SOLUTION: The surface plasmon enhanced fluorescence measuring apparatus is mounted with a chip including: a metal film having a diffraction grating; an antibody fixed to the diffraction grating; and a substance to be detected that is bound to the antibody and is marked with a fluorescent material. The measuring apparatus includes: a light source for irradiating the diffraction grating with excitation light; a light detection unit for detecting fluorescence from the fluorescent material; and a vibration applying unit for applying vibration to the substance to be detected bound to the antibody. The light detection unit detects the fluorescence from the fluorescent material in a state in which vibration is applied to the substance to be detected by the vibration applying unit. The measuring apparatus analyzes the existence of the substance to be detected or the amount thereof by frequency-analyzing an output signal from the light detection unit.

Description

  The present invention relates to a surface plasmon enhanced fluorescence measuring apparatus and a surface plasmon enhanced fluorescence measuring method for detecting a detection target substance contained in a specimen using surface plasmon resonance.

  In a clinical test or the like, if a very small amount of a substance to be detected such as protein or DNA can be detected with high sensitivity and quantity, it becomes possible to quickly grasp the patient's condition and perform treatment. For this reason, a method capable of detecting a minute amount of a substance to be detected with high sensitivity and quantity is demanded.

  As a method that can detect a substance to be detected with high sensitivity, surface plasmon-field enhanced fluorescence spectroscopy (hereinafter abbreviated as “SPFS”) is known. SPFS utilizes the fact that surface plasmon resonance (hereinafter abbreviated as “SPR”) occurs when light is irradiated onto a metal film under predetermined conditions. A primary antibody capable of specifically binding to the substance to be detected is immobilized on the metal film to form a reaction field for specifically capturing the substance to be detected. When a specimen containing a substance to be detected is provided in this reaction field, the substance to be detected binds to the reaction field. Next, when a secondary antibody labeled with a fluorescent substance is provided to the reaction field, the target substance bound to the reaction field is labeled with the fluorescent substance. When the metal film is irradiated with excitation light in this state, the fluorescent substance that labels the substance to be detected is excited by the electric field enhanced by SPR and emits fluorescence. Therefore, the presence or amount of the substance to be detected can be detected by detecting fluorescence. In SPFS, a fluorescent substance is excited by an electric field enhanced by SPR, so that a substance to be detected can be detected with high sensitivity.

  SPFS is roughly classified into prism coupling (PC) -SPFS and lattice coupling (GC) -SPFS by means for coupling (coupling) excitation light and SPR. In PC-SPFS, a prism having a metal film formed on one surface is used. In this method, excitation light and SPR are combined by totally reflecting excitation light at the interface between the prism and the metal film. PC-SPFS is the mainstream method at present, but because of the use of prisms and the large incident angle of excitation light on the metal film, PC-SPFS is problematic in terms of miniaturization of the measuring device. have.

  On the other hand, GC-SPFS couples excitation light and SPR using a diffraction grating (see Non-Patent Document 1). Since GC-SPFS does not use a prism and the incident angle of excitation light with respect to the diffraction grating is small, the measuring device can be made smaller than PC-SPFS.

Keiko Tawa, Hironobu Hori, Kenji Kintaka, Kazuyuki Kiyosue, Yoshiro Tatsu, and Junji Nishii, "Optical microscopic observation of fluorescence enhanced by grating-coupled surface plasmon resonance", Optics Express, Vol. 16, pp. 9781-9790.

  SPFS is based on the premise that a substance to be detected is specifically bound on a metal film. However, when a specimen or reagent solution is provided on a metal film, a substance other than the substance to be detected (for example, a secondary antibody labeled with a fluorescent substance) may bind nonspecifically. Since SPFS is highly sensitive, it also detects fluorescence (noise) derived from this non-specific binding. Further, even if the detection sensitivity is improved, the fluorescence derived from non-specific binding also increases, so the detection accuracy (S / N ratio) cannot be improved. As described above, there is room for improvement in detection accuracy in the measurement apparatus and the measurement method using SPFS.

  The present invention has been made in view of the above points, and is a measurement apparatus and a measurement method using GC-SPFS, which can detect a substance to be detected with high accuracy even when non-specific binding occurs. It is an object of the present invention to provide a measuring apparatus and a measuring method that can perform measurement.

  In the measurement apparatus and measurement method using GC-SPFS, the present inventors can solve the above-mentioned problem by applying vibration at a predetermined frequency to a substance to be detected bound to a primary antibody when detecting fluorescence. The present invention was completed by adding a headline and further examination.

  That is, the present invention relates to the following surface plasmon enhanced fluorescence measuring apparatus.

[1] A chip having a metal film on which a diffraction grating is formed, an antibody immobilized on the diffraction grating, and a target substance that is bound to the antibody and is labeled with a fluorescent substance is attached, and excitation light is emitted. By irradiating the diffraction grating, a surface plasmon enhanced fluorescence measuring device that detects the presence or amount of the substance to be detected, in order to excite the fluorescent substance with an enhanced electric field and emit fluorescence, The light source for irradiating the diffraction grating with the excitation light, the light detection unit for detecting the fluorescence from the fluorescent material, and the detection target material bound to the antibody include the surface direction of the metal film at a predetermined frequency. A vibration applying unit that applies a vibration in a direction, and the light detection unit detects fluorescence from the fluorescent substance in a state where the detection target substance is vibrated by the vibration applying unit, and the light detection unit. Output signal from Analyzing the presence or amount of the substance to be detected by frequency analysis, surface plasmon enhanced fluorescence measuring apparatus.
[2] The frequency of the vibration applied by the vibration applying unit is less than the frequency at which the detection target substance bound to the antibody resonates, or the frequency at which the detection target substance bound to the antibody resonates. [1] The surface plasmon enhanced fluorescence measuring apparatus according to [1].
[3] The surface plasmon enhanced fluorescence measurement device according to [1] or [2], wherein the vibration applying unit applies vibration to the substance to be detected by applying an alternating electric field.
[4] The surface plasmon according to [1] or [2], wherein the vibration applying unit applies vibration to the detected substance by vibrating another wall surface of the flow path having the metal film as one wall surface. Enhanced fluorescence measuring device.
[5] The surface plasmon enhancement according to [1] or [2], wherein the vibration applying unit applies vibration to the detected substance by reciprocating a liquid in a flow path having the metal film as one wall surface. Fluorescence measuring device.
[6] The surface plasmon enhanced fluorescence according to [1] or [2], wherein the vibration applying unit applies vibration to the detected substance by irradiating an electromagnetic wave having a frequency of 1 × 10 ⁵ to 1 × 10 ¹³ Hz. measuring device.

  The present invention also relates to the following surface plasmon enhanced fluorescence measurement method.

[7] A surface plasmon-enhanced fluorescence measurement method in which a fluorescent substance that labels a substance to be detected detects fluorescence emitted by being excited by an electric field based on surface plasmon resonance, and detects the presence or amount of the substance to be detected. A step of preparing a chip having a metal film formed with a diffraction grating, an antibody immobilized on the diffraction grating, and a target substance labeled with a fluorescent substance bound to the antibody; A step of irradiating the diffraction grating with excitation light in a state in which a vibration in a direction including a plane direction of the metal film is applied to the detection target substance bound to an antibody at a predetermined frequency; and fluorescence emitted from the fluorescent substance A method for measuring surface plasmon enhanced fluorescence, comprising: obtaining a fluorescence signal by detecting the presence of a substance to be detected or analyzing the frequency of the fluorescence signal.
[8] The frequency of the vibration applied to the detected substance is less than the frequency at which the detected substance bound to the antibody resonates, or the frequency at which the detected substance bound to the antibody resonates. [7] The surface plasmon enhanced fluorescence measurement method according to [7].

  According to the present invention, it is possible to detect a substance to be detected with a high S / N ratio even when non-specific binding occurs.

It is a schematic diagram which shows the structure of the surface plasmon enhanced fluorescence measuring apparatus which concerns on one embodiment of this invention. 2A to 2C are schematic views for explaining the vibration of the detection target substance bound to the reaction field. It is a figure for demonstrating an inverted pendulum model. It is a graph which shows distribution of the electric field strength on a diffraction grating. It is a graph which shows the change of the fluorescence intensity of the composite_body | complex couple | bonded by the composite_body | complex couple | bonded by specific binding (SP), and non-specific binding (US). It is a flowchart which shows operation | movement of a surface plasmon enhanced fluorescence measuring apparatus.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a schematic diagram showing a configuration of a surface plasmon enhanced fluorescence measuring apparatus (hereinafter referred to as “SPFS apparatus”) 100 according to an embodiment of the present invention. As shown in FIG. 1, the SPFS device 100 includes a light source 110, a collimating lens 120, an excitation light filter 130, a first condenser lens 140, a fluorescent filter 150, a second condenser lens 160, a light detector 170, and vibration application. Part 180 and control part 190. The SPFS device 100 is used in a state where the chip 200 is mounted on a chip holder (not shown). Therefore, the chip 200 will be described first, and then the SPFS device 100 will be described.

  The chip 200 includes a substrate 210 and a metal film 220 formed on the substrate 210. A diffraction grating 230 is formed on the metal film 220. The primary antibody 310 is immobilized on the diffraction grating 230, and the surface of the diffraction grating 230 also functions as a reaction field for binding the primary antibody 310 and the substance 320 to be detected. In FIG. 1, the primary antibody 310 and the substance to be detected 320 are omitted. The chip 200 is preferably a structure in which each piece has a length of several millimeters to several centimeters. However, the chip 200 is a smaller structure or a larger structure not included in the category of “chip”. May be.

  The substrate 210 is a support member for the metal film 220. The material of the substrate 210 is not particularly limited as long as it has mechanical strength capable of supporting the metal film 220. Examples of the material of the substrate 210 include inorganic materials such as glass, quartz, and silicon, and resins such as polymethyl methacrylate, polycarbonate, polystyrene, and polyolefin.

  The metal film 220 is disposed on the substrate 210. As described above, the diffraction grating 230 is formed on the metal film 220. When the metal film 220 is irradiated with light, surface plasmons generated in the metal film 220 and evanescent waves generated by the diffraction grating 230 are combined to generate surface plasmon resonance. The material of the metal film 220 is not particularly limited as long as it is a metal that generates surface plasmons. Examples of the material of the metal film 220 include gold, silver, copper, aluminum, and alloys thereof. A method for forming the metal film 220 is not particularly limited. Examples of the method for forming the metal film 220 include sputtering, vapor deposition, and plating. The thickness of the metal film 220 is not particularly limited. For example, the thickness of the metal film 220 is about 30 to 70 nm.

  The diffraction grating 230 generates an evanescent wave when the metal film 220 is irradiated with light. The shape of the diffraction grating 230 is not particularly limited as long as an evanescent wave can be generated. Examples of the cross-sectional shape of the diffraction grating 230 include a rectangular wave shape, a sine wave shape, a sawtooth shape, and the like. The pitch and depth of the concavo-convex shape of the diffraction grating can be appropriately set so that surface plasmon resonance occurs. In the present embodiment, a plurality of concave stripes parallel to each other are formed on the surface of the metal film 220 at a predetermined interval. That is, the diffraction grating 230 is a one-dimensional diffraction grating.

  The method for forming the diffraction grating 230 is not particularly limited. For example, after the metal film 220 is formed on the flat substrate 210, the metal film 220 may be provided with an uneven shape. Alternatively, the metal film 220 may be formed over the substrate 210 that has been previously provided with an uneven shape. In any method, the metal film 220 including the diffraction grating 230 can be formed.

  A primary antibody 310 for capturing the substance to be detected 320 is immobilized on the diffraction grating 230 (reaction field) (see FIG. 2). The primary antibody 310 specifically binds to the substance to be detected. In the present embodiment, the primary antibody is immobilized substantially uniformly on the surface of the diffraction grating 230.

The method for immobilizing the primary antibody 310 is not particularly limited. For example, a self-assembled monomolecular film (hereinafter referred to as “SAM film”) or a polymer film bonded with the primary antibody 310 may be formed on the diffraction grating 230. Examples of SAM films include films formed with substituted aliphatic thiols such as HOOC— (CH ₂ ) ₁₁ —SH. Examples of the material constituting the polymer film include polyethylene glycol and MPC polymer. Alternatively, a polymer having a reactive group that can be bound to the primary antibody (or a functional group that can be converted to a reactive group) may be immobilized on the diffraction grating 230, and the primary antibody may be bound to the polymer.

  As shown in FIG. 1, the excitation light α is applied to the metal film 220 (diffraction grating 230) at a predetermined incident angle θ1. In the irradiation region, the surface plasmon generated in the metal film 220 and the evanescent wave generated by the diffraction grating 230 are combined to generate surface plasmon resonance. When a fluorescent substance is present in the irradiated region, the fluorescent substance is excited by the enhanced electric field formed by surface plasmon resonance, and fluorescent β is emitted.

  Hereinafter, each component of the SPFS device 100 will be described.

  The light source 110, the collimating lens 120, and the excitation light filter 130 constitute an excitation light irradiation unit. The excitation light irradiation unit emits the collimated excitation light α having a constant wavelength and light amount so that the shape of the irradiation spot on the surface of the metal film 220 (diffraction grating 230) of the chip 200 is substantially circular. Further, the excitation light irradiation unit emits only the P wave for the metal film 220 toward the diffraction grating 230 so that diffracted light that can be combined with surface plasmons (SPR) in the metal film 220 is generated in the diffraction grating 230.

  In the present embodiment, light source 110 is a laser diode. The light source 110 emits excitation light α (single mode laser light) toward the diffraction grating 230 of the chip 200. The type of the light source 110 is not particularly limited, and may not be a laser diode. Examples of the light source 110 include a light emitting diode, a mercury lamp, and other laser light sources.

  The collimating lens 120 collimates the excitation light α emitted from the light source 110. The excitation light α emitted from the laser diode (light source 110) has a flat outline shape even when collimated. Therefore, the laser diode is held in a predetermined posture so that the shape of the irradiation spot on the surface of the metal film 220 is substantially circular.

  The excitation light filter 130 includes, for example, a band pass filter and a linear polarization filter, and tunes the excitation light α emitted from the light source 110. Since the excitation light α from the laser diode (light source 110) has a slight wavelength distribution width, the bandpass filter turns the excitation light α from the laser diode into a narrow band light having only the center wavelength. In addition, since the excitation light α from the laser diode (light source 110) is not completely linearly polarized light, the linear polarization filter converts the excitation light α from the laser diode into completely linearly polarized light. The excitation light filter 130 may include a half-wave plate that adjusts the polarization direction of the excitation light α so that the P-wave component is incident on the metal film 220.

  The incident angle θ1 (see FIG. 1) of the excitation light α with respect to the metal film 220 is preferably at or near the resonance angle at which SPR occurs most in the metal film 220. Therefore, the SPFS device 100 preferably has a first angle adjustment unit (not shown) that adjusts the incident angle θ1 by relatively rotating the optical axis of the excitation light α and the chip 200. For example, the first angle adjustment unit may rotate the excitation light irradiation unit or the chip 200 around the intersection between the optical axis of the excitation light α and the metal film 220.

  The 1st condensing lens 140, the fluorescence filter 150, the 2nd condensing lens 160, and the photon detection part 170 comprise a fluorescence detection unit. The fluorescence detection unit is disposed with respect to the excitation light irradiation unit so as to sandwich a straight line passing through the intersection of the optical axis of the excitation light α and the metal film 220 and perpendicular to the metal film 220. The fluorescence detection unit detects fluorescence β emitted from the diffraction grating 230 (reaction field).

  The first condenser lens 140 and the second condenser lens 160 constitute a conjugate optical system that is not easily affected by stray light. The light traveling between the first condenser lens 140 and the second condenser lens 160 becomes substantially parallel light. The first condenser lens 140 and the second condenser lens 160 form a fluorescent image on the metal film 220 on the light receiving surface of the light detection unit 170.

  The fluorescent filter 150 is disposed between the first condenser lens 140 and the second condenser lens 160. The fluorescence filter 150 includes, for example, a cut filter and a neutral density (ND) filter, and removes noise components other than the fluorescence β (for example, excitation light α and external light) from the light reaching the light detection unit 170, The amount of light reaching the light detection unit 170 is adjusted.

  The light detection unit 170 detects a fluorescent image on the metal film 220. For example, the light detection unit 170 is a photomultiplier tube with high sensitivity and a high SN ratio. The light detection unit 170 may be an avalanche photodiode (APD), a photodiode (PD), a CCD image sensor, or the like.

  The angle θ2 (see FIG. 1) of the optical axis of the fluorescence detection unit with respect to the perpendicular of the metal film 220 is an angle (fluorescence peak angle) at which the intensity of the fluorescence β emitted from the diffraction grating 230 (reaction field) is maximized. Is preferred. Therefore, the SPFS device 100 preferably includes a second angle adjustment unit (not shown) that adjusts the angle θ2 by relatively rotating the optical axis of the fluorescence detection unit and the chip 200. For example, the second angle adjustment unit may rotate the fluorescence detection unit or the chip 200 around the intersection between the optical axis of the fluorescence detection unit and the metal film 220.

  The vibration applying unit 180 applies a vibration in a direction including a plane direction at a predetermined frequency to a detection target substance labeled with a fluorescent substance that is coupled to a reaction field (primary antibody) on the diffraction grating 230. Here, “vibration in a direction including the plane direction” means that the vibration direction includes a component in the plane direction (horizontal direction). In the present embodiment, the vibration applying unit 180 includes a high voltage power source 182 and a pair of electrodes 184 connected to the high voltage power source 182. The pair of electrodes 184 are arranged so as to sandwich the diffraction grating 230 (reaction field). The high voltage power source 182 can apply an alternating voltage or a pulsed voltage with alternating polarities between the electrodes 184. Therefore, the vibration applying unit 180 can apply an alternating electric field whose polarity changes at a predetermined frequency to the reaction field on the diffraction grating 230. When the detected substance bonded to the reaction field has a positive or negative charge, the detected substance is vibrated by an alternating electric field. The voltage is, for example, several hundred volts or less, and preferably several tens volts or less. The frequency of vibration applied by the vibration applying unit 180 is preferably a frequency at which the detection target substance bound to the reaction field (primary antibody) resonates or a frequency lower than that. For example, the frequency is about several Hz to several tens Hz.

  FIG. 2 is a schematic diagram for explaining the vibration of the detection target substance bound to the reaction field. In this figure, the surface of the diffraction grating 230 (for example, the bottom surface of the groove) is shown enlarged. In addition, the polarity of the voltage applied to the pair of electrodes 184 is also shown. In the example shown in FIG. 2A, the substance 320 to be detected is bound to the primary antibody 310 immobilized on the metal film 220 (diffraction grating 230). Further, the secondary antibody 330 labeled with the fluorescent substance 340 is bound to the substance to be detected 320. It is assumed that the complex composed of the substance to be detected 320, the secondary antibody 330, and the fluorescent substance 340 has a negative charge. In addition to the complex bound to the metal film 220 by such specific binding SP, there is also a complex bound to the metal film 220 by non-specific binding US. Examples of the non-specific binding US include direct binding of the detected substance 320 or other substance 350 to the metal film 220, direct binding of the secondary antibody 330 to the metal film 220, and the like.

  As shown in FIGS. 2B and 2C, when an alternating electric field is applied to the reaction field, a substance having a positive or negative charge among the substances specifically or non-specifically bound to the reaction field is It is vibrated in the direction including the surface direction. At this time, the length and mass of the complex bound by the specific binding SP and the complex (substance) bound by the non-specific binding US are different. Therefore, the vibration of each component is different. For example, when the secondary antibody 330 is directly bonded to the metal film 220, the secondary antibody 330 hardly vibrates even when an alternating electric field is applied to the reaction field. Further, when the detected substance 320 is directly bound to the metal film 220 and the secondary antibody 330 is bound to the detected substance 320, the complex bound by this non-specific binding US The resonant frequency is greater than the resonant frequency of the complex that is bound by the specific binding SP. Therefore, if the difference between these vibrations can be detected, it is possible to distinguish between the complex bound by the specific binding SP and the complex bound by the non-specific binding US.

  Here, the resonance frequency of the complex bound by the specific binding SP will be described. In order to calculate the resonance frequency of the complex, the equation of motion of the protein is examined using the inverted pendulum model shown in FIG.

As shown in FIG. 3, the mass of the protein is m, the length of the protein is l, the spring constant is k, the length from the protein base point to the spring action point is h, and the viscosity damping coefficient due to the solvent is C. When the moment of inertia, the overturning moment, the restoring force and restoring moment of the spring are as follows:
Moment of inertia: J = ml ²
Falling moment: mg · l · sinθ ≒ mgl · θ
Spring restoring force: -k · hθ
Restoring moment: −k · hθ · h = −k · h ² · θ

In this case, the equation of motion is expressed as the following equation (1).

The above equation (1) can be rewritten as the following equation (2).

Here, when the resonance angular frequency by gravity and spring restoring force is ωn, the following equations (3) and (4) are established. In equation (3), ξ is a damping ratio.

Using the above formulas (3) and (4), the above formula (2) is expressed as the following formula (5).

The overall resonance period T including the viscous damping of the solvent is expressed by the following equation (6).

Here, the length h from the protein base point to the spring action point, the protein length l, the protein mass m, the gravitational acceleration g, and the spring constant k are assumed as follows.
h = l = 1.8 × 10 ⁻⁹ (m)
m = 6.17 × 10 ⁻²² (kg)
g = 9.8 (m / t ² )
k = 2.5 (N / m) = 2.5 (kg / t ₂ )

Then, kt, moment of inertia J, and resonance angular frequency ωn are as follows.
kt = kh ² −mgl≈0.81 × 10 ⁻¹⁵ (kgm ² / t ² )
J = m × l ² = 6.17 × 10 ⁻²² × (18 × 10 ⁻⁹ ) ² = 1.99908 × 10 ⁻³⁷ (kgm ² )
ωn = √ (kt / J) ≈0.636 × 10 ¹¹ (Hz)

The viscosity η of the solvent (water) and the size (radius) r of the protein are assumed as follows.
η = 0.89 (cP) = 0.89 × 10 ⁻³ (kg / (mt))
r = 5 (nm)

Then, the volume V of the protein, the viscous damping coefficient C, the damping ratio ξ, the resonance period T, and the resonance frequency f are as follows.
V = 4 / 3πr ³ = 5.236 × 10 ⁻²⁵ (m ³ )
C = η × V = 0.89 × 10 ⁻³ × 5.236 × 10 ⁻²⁵ ≈4.66004 × 10 ⁻²⁸ (kgm ² / t)
ξ = C / (2ωn × J) = 4.66004 × 10 ⁻²⁸ /(2×0.636×10 ¹¹ × 1.999908 × 10 ⁻³⁷ )
T = 2π / (ωn × √ (1-ξ ² )) ≈9.88 × 10 ⁻¹¹ (t)
f = 1 / T≈10 (GHz)

  By selecting a solvent having a higher viscosity, the resonance frequency f of the complex bound by the specific binding SP can be set to an arbitrary value. The resonance frequency f is preferably about several Hz.

  FIG. 4 is a graph showing the electric field intensity distribution on the diffraction grating 230. As shown in FIG. 4, the electric field strength changes periodically corresponding to the shape of the diffraction grating 230. In such a situation, when an alternating electric field is applied to the reaction field to vibrate a complex specifically or non-specifically bound to the reaction field in a direction including the plane direction of the metal film 220, these complexes The ambient electric field strength changes periodically. Therefore, the fluorescence intensity from the fluorescent material contained in these complexes also periodically changes.

  As described above, the length and mass of the complex bound by the specific binding SP and the complex (substance) bound by the non-specific binding US are different. The resonance frequency of the complex bound by the non-specific binding US is higher than the resonance frequency of the complex bound by the specific binding SP. Thus, as shown in FIG. 5, the frequency of the fluorescent signal from the complex bound by the non-specific binding US is greater than the frequency of the fluorescent signal from the complex bound by the specific binding SP. Become.

  Returning to the description of the SPFS apparatus 100. The control unit 190 controls operations of the excitation light irradiation unit (light source 110), the fluorescence detection unit (light detection unit 170), the excitation light irradiation unit, the angle adjustment unit of the fluorescence detection unit, and the vibration applying unit 180. In addition, the control unit 190 analyzes the frequency of the output signal (fluorescence signal) from the light detection unit 170, so that the fluorescence signal from the complex bound by the specific binding SP is derived from other fluorescence signals (noise). Separate and analyze the presence or amount of the substance to be detected. The control unit 190 is a computer that executes software, for example. As a frequency analysis, a transient response analysis can be used as one form.

  For example, the control unit 190 passes the output signal (fluorescence signal) from the light detection unit 170 through a band-pass filter or a low-pass filter, thereby converting the fluorescence signal from the complex bound by the specific binding SP to other fluorescence. It can be separated from the signal (noise). In addition, the control unit 190 performs frequency conversion processing (for example, fast Fourier transform) on the output signal (fluorescence signal) from the light detection unit 170, and the fluorescence at the vibration frequency of the complex bound by the specific binding SP. By calculating the intensity, the presence or amount of the substance to be detected can be analyzed. Further, the control unit performs frequency conversion processing (for example, fast Fourier transform) on the output signal (fluorescence signal) from the light detection unit 170, passes the obtained signal through a filter that passes a predetermined vibration frequency, It is also possible to separate the fluorescence signal from the complex bound by the specific binding SP from other fluorescence signals (noise) by performing frequency inverse transform processing (for example, fast Fourier inverse transform) on the passed signal. it can.

  Next, the detection operation of the SPFS device 100 will be described. FIG. 6 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 100.

  First, preparation for measurement is performed (step S10). Specifically, the chip 200 is installed at a predetermined position of the SPFS device 100. Further, when a moisturizing agent is present on the metal film 220 of the chip 200, the moisturizing agent is removed by washing the metal film 220 so that the primary antibody 310 can appropriately capture the substance 320 to be detected.

  Next, the target substance 320 in the specimen is reacted with the primary antibody 310 (primary reaction, step S20). Specifically, a specimen is provided on the metal film 220, and the specimen and the primary antibody 310 are brought into contact with each other. When the detected substance 320 is present in the specimen, at least a part of the detected substance 320 is bound to the primary antibody 310. Thereafter, the metal film 220 is washed with a buffer solution or the like to remove substances that have not bound to the primary antibody 310. The types of the specimen and the substance to be detected 320 are not particularly limited. Examples of the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluted solutions thereof. Examples of the substance to be detected 320 include nucleic acids (such as DNA and RNA), proteins (such as polypeptides and oligopeptides), amino acids, carbohydrates, lipids, and modified molecules thereof.

  Next, the target substance 320 bound to the primary antibody 310 is labeled with the fluorescent substance 340 (secondary reaction, step S30). Specifically, a fluorescent labeling liquid containing the secondary antibody 330 labeled with the fluorescent substance 340 is provided on the metal film 220, and the target substance 320 bound to the primary antibody 310 and the fluorescent labeling liquid are brought into contact with each other. . The fluorescent labeling solution is, for example, a buffer solution containing the secondary antibody 330 labeled with a fluorescent substance. When the substance to be detected 320 is bound to the primary antibody 310, at least a part of the substance to be detected 320 is labeled with the fluorescent substance 340 (see FIG. 2A). Thereafter, the metal film 220 is washed with a buffer solution or the like to remove the free secondary antibody 330 and the like. The order of the primary reaction and the secondary reaction is not limited to this. For example, after the substance to be detected 320 is bound to the secondary antibody 330, a liquid containing these complexes may be provided on the metal film 220. Further, the specimen and the fluorescent labeling solution may be provided on the metal film 220 at the same time.

  Next, vibration with a predetermined frequency is applied to the substance 320 to be detected, which is bound to the primary antibody 310 and labeled with the fluorescent substance 340 (step S40). Specifically, the control unit 190 applies an alternating electric field to the vibration applying unit 180 to cause the detection target material 320 to vibrate at a predetermined frequency (see FIGS. 2B and 2C).

  Next, with the vibration of a predetermined frequency applied to the substance to be detected 320, the excitation light α is irradiated onto the metal film 220, and the intensity of the fluorescence β emitted from the fluorescent material 340 is measured (step S50). Specifically, the control unit 190 causes the light source 110 to emit the excitation light α. At the same time, the control unit 190 causes the light detection unit 170 to detect the intensity of the fluorescence β from the metal film 220. The light detection unit 170 outputs the measurement result to the control unit 190.

  Finally, the control unit 190 performs frequency analysis on the output signal (fluorescence signal) from the light detection unit 170, separates the fluorescence signal derived from the specific binding SP, and detects the presence or detection of the detected substance. The amount of the substance is analyzed (step S60).

  By the above procedure, the fluorescence signal derived from the specific binding SP can be separated, and the presence of the substance to be detected or the amount of the substance to be detected in the sample can be detected with high accuracy.

  As described above, in the present embodiment, the primary antibody is immobilized substantially uniformly on the surface of the diffraction grating 230. Therefore, the change in the fluorescence intensity from the substance to be detected 320 located on the bottom surface of the concave portion of the diffraction grating 230 and the change in the fluorescence intensity from the substance to be detected 320 located on the top surface of the convex part may cancel each other. Conceivable. However, since there is a large difference in the electric field strength between the bottom surface of the concave portion of the diffraction grating 230 and the top surface of the convex portion, the change in fluorescence intensity at the top surface of the convex portion is compared with the change in fluorescence intensity at the bottom surface of the concave portion. Very small. For this reason, this is hardly a problem. Further, a mask is arranged in the fluorescence detection unit to shield fluorescence from a specific region (for example, a region having a low electric field intensity), or a specific region (for example, an electric field strength) in a fluorescent image detected by the light detection unit 170. Such a problem may be avoided more reliably by performing a frequency analysis only for a large area).

  As described above, since the SPFS apparatus 100 according to the present embodiment can separate the fluorescent signal derived from the specific binding SP, the substance to be detected can be accurately obtained even when non-specific adsorption occurs. Can be detected.

In the above-described embodiment, the example in which the vibration applying unit 180 applies an alternating electric field to apply vibration to the target substance has been described, but the means for applying vibration to the target substance is not limited to this. For example, in this case, the vibration applying unit 180 may apply vibration to the detection target substance by vibrating the other wall surface of the flow path having the metal film 220 as one wall surface. Further, the vibration applying unit 180 may apply vibration to the detection target substance by reciprocating the liquid in the flow path having the metal film 220 as one wall surface. Furthermore, the vibration imparting unit 180 may apply vibration to the substance to be detected by irradiating an electromagnetic wave having a frequency of 1 × 10 ⁵ to 1 × 10 ¹³ Hz to vibrate molecules.

  In the above-described embodiment, the example in which the vibration applying unit 180 vibrates the target substance mainly in the surface direction of the metal film 220 has been described. However, the vibration applying unit 180 vibrates the target substance in the thickness direction of the metal film 220. (See Japanese Patent Application Laid-Open No. 2011-80935 by the inventors of the present application). Since the enhanced electric field formed by SPR exists only in the vicinity of the metal film 220, the intensity of the fluorescence emitted from the fluorescent material in response to the vibration is increased even if the detection target is vibrated in the thickness direction of the metal film 220. Change. Also, when the substance to be detected is vibrated in the thickness direction of the metal film 220, the frequency of the fluorescence signal differs between the complex bound by the specific binding SP and the complex bound by the non-specific binding US. Therefore, when the detection target substance is vibrated in the thickness direction of the metal film 220, the fluorescence signal derived from the specific binding SP is separated and the presence or detection of the detection target substance in the specimen is separated as in the above embodiment. The amount of the detection substance can be detected with high accuracy. The vibration applying unit 180 may vibrate the substance to be detected in the surface direction of the metal film 220, may vibrate in the thickness direction, or may vibrate in the surface direction and the thickness direction.

  In the above embodiment, the example in which the chip 200 is irradiated with the excitation light α from the metal film 220 side has been described. However, the chip 200 may be irradiated with the excitation light α from the substrate 210 side.

  The surface plasmon enhanced fluorescence measurement apparatus and the surface plasmon enhanced fluorescence measurement method according to the present invention can measure a substance to be detected with high reliability, and thus are useful for clinical examinations, for example.

100 Surface plasmon enhanced fluorescence measuring device (SPFS device)
DESCRIPTION OF SYMBOLS 110 Light source 120 Collimating lens 130 Excitation light filter 140 1st condensing lens 150 Fluorescence filter 160 2nd condensing lens 170 Light detection part 180 Vibration provision part 182 High voltage power supply 184 Electrode 190 Control part 200 Chip 210 Substrate 220 Metal film 230 Diffraction grating 310 Primary antibody 320 Detected substance 330 Secondary antibody 340 Fluorescent substance α Excitation light β Fluorescence

Claims (8)

A chip having a metal film with a diffraction grating formed thereon, an antibody immobilized on the diffraction grating, and a target substance labeled with a fluorescent substance bonded to the antibody is mounted, and excitation light is transmitted to the diffraction grating. Is a surface plasmon enhanced fluorescence measuring device that detects the presence or the amount of the substance to be detected by irradiating
A light source for irradiating the diffraction grating with the excitation light in order to excite the fluorescent material with an enhanced electric field to emit fluorescence;
A light detection unit for detecting fluorescence from the fluorescent material;
A vibration applying unit that applies a vibration in a direction including a surface direction of the metal film to the detection target substance bound to the antibody at a predetermined frequency;
In a state in which the substance to be detected is vibrated by the vibration applying unit, the fluorescence from the fluorescent substance is detected by the light detection unit, and the output signal from the light detection unit is analyzed by frequency analysis. Analyze the presence or quantity,
Surface plasmon enhanced fluorescence measurement device.   2. The frequency of the vibration applied by the vibration applying unit is less than a frequency at which the detection target substance bound to the antibody resonates, or a frequency at which the detection target substance bound to the antibody resonates. The surface plasmon enhanced fluorescence measuring apparatus described in 1.   The surface plasmon enhanced fluorescence measurement device according to claim 1, wherein the vibration applying unit applies vibration to the substance to be detected by applying an alternating electric field.   3. The surface plasmon enhanced fluorescence measurement according to claim 1, wherein the vibration applying unit applies vibration to the detected substance by vibrating another wall surface of the flow path having the metal film as one wall surface. apparatus.   3. The surface plasmon enhanced fluorescence measurement device according to claim 1, wherein the vibration applying unit applies vibration to the detected substance by reciprocating a liquid in a flow path having the metal film as one wall surface. 4. . 3. The surface plasmon enhanced fluorescence measurement device according to claim 1, wherein the vibration applying unit applies vibration to the detection target substance by irradiating an electromagnetic wave having a frequency of 1 × 10 ⁵ to 1 × 10 ¹³ Hz. A fluorescent substance for labeling a substance to be detected is a surface plasmon-enhanced fluorescence measuring method for detecting fluorescence emitted by being excited by an electric field based on surface plasmon resonance, and detecting the presence or amount of the substance to be detected,
Preparing a chip having a metal film formed with a diffraction grating, an antibody immobilized on the diffraction grating, and a detection target substance bound to the antibody and labeled with a fluorescent substance;
Irradiating the diffraction grating with excitation light in a state where a vibration in a direction including a plane direction of the metal film is applied to the detection target substance bound to the antibody at a predetermined frequency;
Detecting fluorescence emitted from the fluorescent material to obtain a fluorescent signal;
Analyzing the presence or amount of the substance to be detected by frequency analysis of the fluorescent signal;
A method for measuring surface plasmon enhanced fluorescence, comprising:   The frequency of the vibration applied to the detected substance is less than a frequency at which the detected substance bound to the antibody resonates, or a frequency at which the detected substance bound to the antibody resonates. 8. The surface plasmon enhanced fluorescence measurement method according to 7.

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