JP2011080935A - Measuring method and surface plasmon field-enhanced fluorescence measuring device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring method by a surface plasmon field-enhanced fluorescence measuring device capable of separating a signal caused by specific adsorption even when nonspecific adsorption is generated, and improving an S/N ratio. <P>SOLUTION: This measuring method by the surface plasmon field-enhanced fluorescence measuring device includes steps for: applying vibration by a vibration application means to an antibody supplemented onto a reaction field on which a primary antibody with which a specimen is to be reacted specifically is immobilized; irradiating one surface of a metal thin film with excitation light, and enhancing an electric field on the other surface side, to thereby excite a fluorescent material attached to the antibody; and measuring fluorescence from the fluorescent material, and analyzing frequency of an output signal, to thereby analyze the specimen. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a measuring method using a surface plasmon enhanced fluorescence measuring apparatus based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), and a surface plasmon enhanced fluorescence measuring apparatus.

  Conventionally, for example, detection of minute analytes in a living body has been performed based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS). Surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) is a method in which a rough wave (surface plasmon) is generated on the surface of a metal thin film under the condition that the laser light (excitation light) irradiated from a light source attenuates total reflection (ATR) on the surface of the metal thin film. ) To increase the photon amount of the laser light (excitation light) emitted from the light source by several tens to several hundreds times (electric field enhancement effect of surface plasmons), thereby improving the efficiency of the fluorescent material near the metal thin film. It is a method for detecting an extremely small amount and / or an extremely low concentration of analyte by exciting well.

  In recent years, development of a surface plasmon enhanced fluorescence measuring apparatus based on the principle of such surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) has been promoted, and the technical disclosure thereof has been made in, for example, Patent Document 1 and Patent Document 2. Yes.

  Such a surface plasmon enhanced fluorescence measuring apparatus 10 has a basic structure as shown in FIG. 9, and a chip structure in which a metal thin film 102 is provided on the surface of a dielectric member 106 and a reaction field 104 is provided on the surface. 108 is provided.

  The chip structure 108 includes a light source 112 that is incident on the dielectric member 106 and irradiates the excitation light b1 toward the metal thin film 102 on the dielectric member 106 side. The light receiving means 116 for receiving the metal thin film reflected light b2 reflected at is provided.

  On the other hand, on the reaction field 104 side of the chip structure 108, a light detection unit 120 that receives fluorescence b3 emitted from a fluorescent substance labeled with the analyte captured in the reaction field 104 is provided.

  In addition, between the reaction field 104 and the light detection part 120, the light collecting member 122 for efficiently condensing the fluorescence b3 and the light included other than the fluorescence b3 are removed, and only necessary fluorescence is selected. A filter 124 is provided.

  In using the surface plasmon enhanced fluorescence measuring apparatus 10, a primary antibody that specifically binds to an antigen contained in an analyte such as DNA to be detected is immobilized in advance on the surface of the metal thin film 102. . An analyte and a secondary antibody that specifically binds to the analyte are sequentially fed to the reaction field 104 in contact with the metal thin film 102, and the secondary antibody is captured in the reaction field 104. The secondary antibody captured together with the analyte is labeled with a fluorescent substance.

  The trapped reaction field 104 is irradiated with the excitation light b1 from the light source 112 into the dielectric member 106, and the excitation light b1 is incident on the metal thin film 102 at a specific angle (resonance angle) θ1. In this way, a dense wave (surface plasmon) is generated. In addition, when a close-packed wave (surface plasmon) is generated on the metal thin film 102, the excitation light b1 and the electronic vibration in the metal thin film 102 are coupled, resulting in a phenomenon that the light amount of the metal thin film reflected light b2 is reduced.

  The light receiving means 116 and the light source 112 can be paired and rotated around the irradiation area of the metal thin film 102 to change the incident angle to the metal thin film 102. When the incident angle is changed and a point where the signal of the metal thin film reflected light b2 received by the light receiving means 116 is changed (the amount of light is reduced) is found, a resonance angle θ1 at which a dense wave (surface plasmon) is generated is obtained. Can do.

  Then, due to the phenomenon of generating the rough wave (surface plasmon), the fluorescent substance in the reaction field 104 on the metal thin film 102 is efficiently excited, and thereby the amount of fluorescent b3 emitted from the fluorescent substance is increased.

  The increased fluorescence b3 is received by the light detection unit 120 via the light collecting member 122 and the filter 124, so that an extremely small amount and / or extremely low concentration of the analyte can be detected.

  Thus, the surface plasmon enhanced fluorescence measurement device 10 is a high-sensitivity measurement sensor that enables observation of minute molecular activities, particularly between biomolecules.

Japanese Patent No. 3294605 JP 2008-102117 A

  Analytes are not necessarily homogeneous components and are often non-uniform. In such a case, not only specific adsorption but also non-specific adsorption other than this occurs in the reaction field. The analysis sensitivity is increased in order to analyze with a very small amount of sample, but in this case, not only the signal due to specific adsorption but also the signal due to non-specific adsorption is increased, so that the S / N ratio is not improved.

  In view of such problems, the present invention is a measurement method using a surface plasmon enhanced fluorescence measuring apparatus capable of separating a signal due to specific adsorption and improving the S / N ratio even in a state where non-specific adsorption occurs. And a surface plasmon enhanced fluorescence measuring apparatus.

  The above object is achieved by the invention described below.

1. A measurement method using a surface plasmon enhanced fluorescence measuring device having a vibration applying means for applying vibration at a predetermined frequency to a reaction field provided with a metal thin film,
Applying vibration by the vibration applying means to the antibody captured in the reaction field in which a primary antibody that specifically reacts with a sample is immobilized;
Irradiating one surface of the metal thin film with excitation light and exciting the fluorescent substance attached to the antibody by enhancing the electric field on the other surface side;
Measuring the fluorescence from the fluorescent material and analyzing the sample by frequency analysis of the output signal;
A measuring method characterized by comprising:

  2. 2. The measurement method according to 1 above, wherein the vibration applied by the vibration applying means is lower than a frequency at which the antibody immobilized on the reaction field resonates, and the antibody is forced to vibrate by vibration.

  3. 2. The measurement method according to 1 above, wherein the vibration frequency applied by the vibration applying means is a frequency at which the antibody immobilized on the reaction field resonates.

4). The enhanced electric field depends on the distance from the metal film,
The distance of the fluorescent material from the metal thin film is periodically changed by the vibration applied by the vibration applying means, and the fluorescence intensity is changed by the change in the distance of the fluorescent material from the metal thin film. 4. The measurement method according to any one of 1 to 3.

5). A reaction field in which a metal thin film is provided and a primary antibody that specifically reacts with a sample is immobilized;
Vibration applying means for applying vibration to the reaction field at a predetermined frequency;
A light emitting unit that irradiates one surface of the metal thin film with excitation light, enhances an electric field on the other surface side, and excites a fluorescent substance attached to the antibody captured in the reaction field;
A light detection unit for detecting excitation light from the fluorescent material;
Fluorescence from a fluorescent substance attached to the antibody was measured by the light detection unit in a state where the vibration was applied to the antibody captured in the reaction field by the vibration applying means, and the output signal was obtained by frequency analysis. An analysis means for analyzing the sample from the signal intensity;
A surface plasmon enhanced fluorescence measuring apparatus comprising:

  6). 6. The surface plasmon enhanced fluorescence according to 5 above, wherein the vibration applied by the vibration applying means is lower in frequency than the frequency at which the antibody immobilized on the reaction field resonates, and the antibody is forced to vibrate by vibration. measuring device.

  7. 6. The surface plasmon enhanced fluorescence measuring apparatus according to 5 above, wherein the vibration frequency applied by the vibration applying means is a frequency at which the antibody immobilized on the reaction field resonates.

8). The antibody or the fluorescent substance is charged,
The surface plasmon enhanced fluorescence measurement device according to any one of 6 to 7, wherein the vibration applying unit applies an alternating electric field to the reaction field.

  9. 8. The surface plasmon enhancement according to any one of 6 to 7, wherein the vibration applying means vibrates a wall surface other than the reaction field of a flow path having the reaction field as one wall surface. Fluorescence measuring device.

  10. Any one of the above-mentioned 6 to 7, wherein the vibration imparting means reciprocates a liquid in a flow path having the reaction field as one wall surface, and imparts vibration to the specimen by a reciprocating motion. The surface plasmon enhanced fluorescence measuring apparatus according to the item.

  According to the present invention, the specific adsorption sp of the reaction field is vibrated at a predetermined period, the signal from the fluorescence from the fluorescent substance of the specific adsorption sp is frequency-analyzed, and the sample is analyzed from the obtained signal intensity. Even in a state where not only specific adsorption but also non-specific adsorption occurs, signals due to specific adsorption can be separated and the S / N ratio can be improved.

It is the schematic of the surface plasmon enhanced fluorescence measuring apparatus using a microchip liquid feeding system. 2A is a cross-sectional view around the microchip 14, and FIG. 2B is a top view of a part thereof. FIG. 6 is a schematic diagram illustrating a vibration state of a specimen in a reaction field 104. It is a figure which shows the relationship between the distance from the metal thin film 102, and an electric field strength. It is a figure explaining an inverted pendulum model. It is the control flow which the control part 18 performs. This is an example in which frequency analysis is performed on the output signal of the light detection unit 120. As a first modification of the vibration applying means, the wall of the fine channel 143 is mechanically reciprocated to apply vibration to the reaction field. It is the schematic of the conventional surface plasmon enhanced fluorescence measuring apparatus.

  Although the present invention will be described based on an embodiment, the present invention is not limited to the embodiment.

  1 and 2 are schematic views of a surface plasmon enhanced fluorescence measuring apparatus using the microchip liquid feeding system according to the embodiment.

  Surface plasmon-enhanced fluorescence measurement device accurately detects the fluorescence generated by the excited fluorescent material by irradiating a metal thin film with excitation light to generate a rough wave (surface plasmon), and it is extremely accurate even if the detection sensitivity is increased. It is possible to detect fluorescence.

[Surface plasmon enhanced fluorescence measuring apparatus 10]
As shown in FIG. 1, the surface plasmon enhanced fluorescence measuring apparatus 10 of the present invention includes a metal thin film 102, a reaction field 104 formed on one side surface of the metal thin film 102, and a dielectric member formed on the other side surface. 106, and a chip structure 108 having the same.

  Further, the dielectric member 106 side of the chip structure 108 includes a light source 112 that is incident on the dielectric member 106 and functions as a “light emitting unit” that irradiates the excitation light b 1 toward the metal thin film 102. A light receiving means 116 for receiving the metal thin film reflected light b2 irradiated from 112 and reflected by the metal thin film 102 is provided.

  Here, the excitation light b1 emitted from the light source 112 is preferably laser light, and a gas laser or solid-state laser with a wavelength of 200 to 1000 nm and a semiconductor laser with a wavelength of 385 to 800 nm are suitable.

  On the other hand, on the reaction field 104 side of the chip structure 108, a light detection unit 120 that receives fluorescence b3 generated in the reaction field 104 is provided.

  As the light detection unit 120, it is preferable to use an ultrasensitive photomultiplier tube or a CCD image sensor capable of multipoint measurement.

  In addition, between the reaction field 104 of the chip structure 108 and the light detection unit 120, a condensing member 122 for condensing light efficiently, and transmission of light having a wavelength different from that of the fluorescent light b3 in the light. And a filter 124 formed so as to selectively transmit the fluorescence b3.

  As the condensing member 122, any condensing system may be used as long as it aims at efficiently condensing the fluorescence signal on the light detection unit 120. As a simple condensing system, a commercially available objective lens used in a microscope or the like may be used. The magnification of the objective lens is preferably 10 to 100 times.

  On the other hand, as the filter 124, an optical filter, a cut filter, or the like can be used. Examples of the optical filter include a neutral density (ND) filter and a diaphragm lens. Furthermore, the cut filter includes external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), and plasmon scattering light (excitation light originated from plasmon A filter that removes various types of noise light such as scattered light generated by the influence of structures or deposits on the surface of the excitation sensor) and autofluorescence of the enzyme fluorescent substrate, such as an interference filter and a color filter.

  The control unit 18 includes a CPU and a memory, and controls the light source 112, the light detection unit 120, a pump 130 to be described later, and the like by executing a program stored in the memory. The frequency analysis unit 181 of the control unit 18 functions as an “analysis unit” and can perform frequency analysis such as FFT or removal of a specific frequency on the signal from the light detection unit 120.

  In the analyte detection method using such a surface plasmon enhanced fluorescence measuring apparatus 10, a SAM film (Self-Assembled Monolayer: a primary antibody is bound on the surface of the metal thin film 102 in contact with the reaction field 104. (Also referred to as “self-assembled monolayer”) and polymer materials. The primary antibody is bound to one end of the SAM film or the polymer material, and the other end of the SAM film or the polymer material is directly or indirectly fixed to the surface of the metal thin film 102. A plurality of types of polymer materials may be present. Examples of the SAM film include a film formed of a substituted aliphatic thiol such as HOOC- (CH2) 11-SH, and examples of the polymer material include polyethylene glycol (hereinafter referred to as “PEG”) and MPC polymer. Can be mentioned. This may be prepared at the time of use, or a substrate on which these are bonded in advance may be used. Alternatively, a polymer having a reactive group for the primary antibody (or a functional group that can be converted into a reactive group) may be directly immobilized on a gold substrate, and the primary antibody may be immobilized thereon. When an antibody or a polymer is bound using various reactive groups, an amidation condensation reaction through succinimidylation, an addition reaction through maleimidation, or the like is common.

  A solution (hereinafter also referred to as a specimen liquid) containing an analyte (also referred to as a specimen) antigen as a target substance and a reagent liquid containing a secondary antibody are sent to the reaction field 104 thus configured. Antigen can be captured by the immobilized primary antibody. In contrast, the captured antigen is labeled by the action of a reagent solution containing a secondary antibody labeled with a fluorescent substance. The primary antibody may be allowed to act after reacting the antigen and the secondary antibody in advance.

  The detection of the analyte labeled with the fluorescent substance is performed by irradiating the dielectric member 106 with the excitation light b1 from the light source 112 to the reaction field 104 where the analyte is captured, and the excitation light b1 is specific to the metal thin film 102. By entering the metal thin film 102 at an incident angle (resonance angle θ1), a dense wave (surface plasmon) is generated on the metal thin film 102.

  Note that when a close-packed wave (surface plasmon) is generated on the metal thin film 102, the excitation light b1 and the electronic vibration in the metal thin film 102 are coupled, and the signal of the metal thin film reflected light b2 changes (the amount of light decreases). Therefore, it suffices to find a point where the signal of the metal thin film reflected light b2 received by the light receiving means 116 changes (the amount of light decreases).

  Then, due to this rough wave (surface plasmon), the fluorescent material generated in the reaction field 104 on the metal thin film 102 is efficiently excited, thereby increasing the amount of fluorescent b3 emitted from the fluorescent material, and condensing this fluorescent b3. By receiving light at the light detection unit 120 via the member 122 and the filter 124, it is possible to detect an extremely small amount and / or extremely low concentration of the analyte.

  The material of the metal thin film 102 of the chip structure 108 is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably made of gold. It consists of a metal alloy.

  Such a metal is suitable for the metal thin film 102 because it is stable against oxidation and the electric field enhancement due to dense waves (surface plasmons) increases.

  Examples of a method for forming the metal thin film 102 include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Of these, the sputtering method and the vapor deposition method are preferable because the thin film formation conditions can be easily adjusted.

  Further, the thickness of the metal thin film 102 is in the range of gold: 5-500 nm, silver: 5-500 nm, aluminum: 5-500 nm, copper: 5-500 nm, platinum: 5-500 nm, and alloys thereof: 5-500 nm. It is preferable to be within.

  From the viewpoint of the electric field enhancement effect, within the range of gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20-70 nm, and alloys thereof: 10-70 nm More preferably.

  If the thickness of the metal thin film 102 is within the above range, it is easy to generate close-packed waves (surface plasmons). Moreover, as long as the metal thin film 102 has such a thickness, the size (length × width) is not particularly limited.

  The electric field strength decreases as the distance from the metal thin film 102 increases. However, in the vicinity of the surface of the metal thin film (surface to several tens of nm), a quenching phenomenon occurs in which the excited state is quenched even if the electric field is enhanced or energy is transferred to the metal thin film. Therefore, the electric field intensity peak has the peak at the closest distance in a range where the quenching phenomenon does not occur (see FIG. 4 described later).

Further, as the dielectric member 106, a 60-degree prism having a high refractive index can be used. As the material, various optically transparent inorganic substances, natural polymers, and synthetic polymers can be used. From the viewpoint of chemical stability, production stability, and optical transparency, silicon dioxide (SiO ₂ ) or titanium dioxide ( TiO ₂ ) is preferably included.

  Furthermore, such a surface plasmon enhanced fluorescence measurement apparatus 10 adjusts the optimum angle (resonance angle θ1) of surface plasmon resonance by the excitation light b1 irradiated from the light source 112 to the metal thin film 102, so that an angle variable unit (not shown). Z).

  Here, the angle variable unit (not shown) is controlled by the control unit 18, and in the “resonance angle scanning process”, the light receiving means 116 and the light source 116 are used to determine the total reflection attenuation (ATR) condition by the servo motor of the angle variable unit. 112 is synchronized with the irradiation region, and the angle can be changed within a range of 45 to 85 °. The resolution is preferably 0.01 ° or more.

  2A is a cross-sectional view around the microchip 14, and FIG. 2B is a top view of a part thereof. In FIG. 2A, the microchip 14 is attached to the dielectric member 106 shown in FIG. The microchip 14 needs to use a base material that transmits the fluorescence b3 at least around the reaction field 104. In this embodiment, quartz is used as the base material. The quartz substrate 142 is provided with a fine flow path 143. In the state shown in FIG. 2A, the bottom surface of the fine flow path 143 is constituted by the dielectric member 106 having the metal thin film 102 formed on the surface thereof. Yes. The quartz substrate 142, the periphery of which is supported by the fixture 161, is fixed to the dielectric member 106 without a gap.

  The aforementioned reaction field 104 is provided in the path of the fine channel 143. An opening 144a is provided at one end of the microchannel 143, and an opening 144b is provided at the other end. A connection portion 145 is provided on the opening 144a side. A transparent ITO substrate 146A (ITO: Indium-Tin Oxide) is provided on the side of the reaction field 104 facing the metal thin film 102 so as not to hinder the measurement of the fluorescence b3. The ITO substrate 146A will be described later with reference to FIG.

  The pipette 150 can be connected to the connection unit 145. The connection portion 145 is made of an elastic material and functions as a seal member when the pipette 150 is loaded. A reservoir 148 for discarding unnecessary liquid is provided at the end of the other opening 144b. A fine air hole (not shown) is provided in the upper part of the storage part 148.

  The width (Y-direction length) of the microchannel 143 is 1 mm to 3 mm, the height (Z direction) is 50 μm to 500 μm, the width of the reaction field 104 is equal to the width of the microchannel 143, and the length is 1 mm to Although it is 3 mm, it is not necessarily limited. The sizes of the openings 144 a and 144 b at both ends of the fine channel 143 are φ1 mm to φ3 mm, which is equivalent to the width of the fine channel 143. The tip of the pipette 150 has substantially the same shape as the opening 144a, and the root portion has a cylindrical shape with a shaft diameter larger than this. The reservoir 148 has a larger cross-sectional shape than the opening 144b in the flow path direction, and has a shape of 2 to 4 mm square, for example. In addition, although it is set as the substantially square cross-sectional shape in FIG.2 (b), it is good also as a circular cross-sectional shape.

  FIG. 2A shows a state where the pipette 150 is loaded. In addition, although the storage part 148 has shown the example fixed to the microchip 14, it is good also as a structure removable like the pipette 150. FIG. The pipette 150 uses polypropylene as a material and can be easily detached through the connecting portion 145, and the pipette 150 can be easily discarded together with the supplied specimen liquid. The pump 130 is a syringe pump, for example, and can discharge or suck a predetermined amount of liquid. In the present application, the term “discharge” refers to conveying the liquid inside the pipette 150 in the forward direction (rightward in the fine channel 143 in FIG. 2A) by sending liquid or air from the pump 130. Yes, “suction” is the opposite.

  When the control unit 18 controls the pump 130, it is possible to discharge or aspirate the liquid such as the sample liquid stored in the pipette 150 into the microchip 14. In this way, a reagent solution containing a secondary antibody labeled with a fluorescent substance and a sample solution containing an analyte are sent by the pump 130 to the fine channel 143.

  Examples of the specimen include blood, serum, plasma, urine, nasal fluid, saliva, stool, body cavity fluid (eg, cerebrospinal fluid, ascites, pleural effusion). The analyte contained in the sample is, for example, nucleic acid (DNA that may be single-stranded or double-stranded, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., or nucleoside, nucleotide And modified molecules thereof), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules and complexes thereof. Specifically, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signaling substance, a hormone, or the like, and is not particularly limited.

  Furthermore, the fluorescent substance is not particularly limited as long as it is a substance that emits fluorescence b3 by being irradiated with predetermined excitation light b1 or excited by using a field effect. In addition, the fluorescence b3 as used in this specification includes various light emission, such as phosphorescence.

[Vibration applying means]
FIG. 3 is a schematic diagram for explaining the vibration state of the specimen in the reaction field 104. As shown in the figure, the metal thin film 102 and the ITO substrate 146A are connected by a high voltage power source PS. The high-voltage power supply PS can output an alternating voltage or a pulsed voltage with alternating polarities, and can give an alternating electric field whose polarity changes at a predetermined frequency to the reaction field 104. The voltage is, for example, several hundred volts, preferably several tens volts or less. It is. The high-voltage power supply PS and the ITO substrate 146A function as vibration applying means. The predetermined frequency is set to a frequency at which the analyte specifically adsorbed in the reaction field 104 resonates or a frequency lower than this. The frequency is approximately several Hz to several tens Hz. By applying vibration, the specific adsorption sp is resonated or forcedly oscillated.

  In this figure, the primary antibody a1 is fixed in the reaction field 104, and the specimen ay that specifically reacts with the primary antibody a1 and the secondary antibody a2 that reacts specifically with the specimen ay are in this order. It is connected. The secondary antibody a2 is modified with a fluorescent label F. In addition, although the inside of the fine channel 143 is filled with the liquid, illustration is abbreviate | omitted.

  As shown in the figure, mixed with the specific adsorption sp (primary antibody a1, specimen ay, secondary antibody a2 linked), non-specific adsorption us other than specific adsorption (in the example of the figure, specimen ay, secondary antibody) a2) is mixed. In the example shown in the figure, the specific adsorption sp and the non-specific adsorption us are negatively charged, and an alternating electric field is applied at a predetermined frequency to the reaction field 104 and the fine channel 143 around the reaction field 104 by a high-voltage power supply PS. By doing so, these substances are subjected to forces in the vertical direction.

  In the state where the specific adsorption sp receives an upward force as shown in FIG. 3A, the specific adsorption sp is in an extended state, and therefore the distance h1 of the fluorescent label F from the metal thin film 102 becomes long. On the other hand, when the force is applied downward as shown in FIG. 3B, the specific adsorption sp is in a contracted state, so the distance h2 of the fluorescent label F from the metal thin film 102 is shortened.

  Here, as described above, the electric field strength is small in the region close to the metal thin film 102 due to the quenching phenomenon, and increases as the distance from the metal thin film 102 increases. FIG. 4 is a diagram showing the relationship between the distance from the metal thin film 102 and the electric field strength. As shown in the figure, at a distance of zero in contact with the metal thin film 102, the electric field strength is close to zero due to the quenching phenomenon, and the effect decreases as the distance increases. On the other hand, the electric field strength decreases with increasing distance from the metal thin film 102 except in the vicinity. Therefore, the electric field strength gradually decreases with increasing distance from the distance hp. It becomes almost zero at a distance hx (generally 200 to 500 nm) or more.

  Further, the distance hp is, for example, 30 to 50 nm, and the length of the specific adsorption sp is 20 to 30 nm. Therefore, the relationship of distance hp ≧ h1> h2 is established. For this reason, the electric field intensity with respect to the fluorescent label F is stronger at the distance h1 in the state of FIG. 3A than at the distance h2 in the state of FIG. That is, when the distance of the fluorescent label F from the surface of the metal thin film 102 changes within the range of the distance hp or less, the intensity of the fluorescence also changes according to the distance.

  From such characteristics, by giving vibration to the specific adsorption sp, the distance from the metal thin film 102 is periodically changed, and the intensity of the fluorescence emitted from the fluorescent label F is also vibrated by the vibration. On the other hand, the nonspecific adsorption us is different in length and weight from the specific adsorption sp. In particular, non-specific adsorption mainly includes (1) a state where only the fluorescently labeled secondary antibody a2 is lying on the SAM film, and (2) a state where the secondary antibody a2 is directly attached to the specimen ay. . If the non-specific adsorption us is (1) above, even if vibration is applied, the intensity of fluorescence is constant (DC component) without vibration, and if it is (2) above, the length is shorter than the specific adsorption sp, Since the mass is also small, the resonance frequency is larger than that of the specific adsorption sp. Therefore, it is possible to perform an analysis with an improved S / N ratio by extracting the vibration component of the specific adsorption sp by frequency analysis described later.

[Resonance frequency]
Here, the resonance frequency of the specific adsorption sp will be described. In order to calculate the resonance frequency of the specific adsorption sp, first, the equation of motion of the protein is examined using an inverted pendulum model as shown in FIG.

When m is the mass of the protein, l is the length of the protein, k is the spring constant, h is the length to the point of action of the spring, and C is the viscous damping coefficient by the solvent, it is 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 = −kh ² · θ
Based on the above, the equation of motion is expressed by Equation 1 below.

  Formula 1 becomes Formula 2, and when the resonance angular frequency by gravity and spring restoring force is Wn, Formula 3 and Formula 4 are obtained, and Formula 2 is expressed by Formula 5.

  The total resonance period T including the viscous damping of the solvent is expressed by the following formula 6.

h = l = 1.8 × 10 ⁻⁹ (m), M = 6.17 × 10 ⁻²² (kg), g = 9.8 (m / t ² ), and k = 2.5 (N / m) ) = 2.5 (kg / t ² )
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 )
wn = √ (kt / J) ≈0.636 × 10 ¹¹ (Hz)
If the solvent is water, the viscosity η = 0.89 (cP) = 0.89 × 10 ⁻³ (kg / (mt)),
When the size of the protein is a radius r = 5 nm, the volume V = 4 / 3πr ³ = 5.236 × 10 ⁻²⁵ (m ³ )
Viscous damping coefficient: C = η × V = 5.236 × 10 ⁻²⁵ × 0.89 × 10 ⁻³ ≈4.66004 × 10 ⁻²⁸ (kgm ² / t)
Damping ratio: ζ = C / (2Wn × J) = 4.66004 × 10 ⁻²⁸ /(2×0.636×10 ¹¹ × 1.999908 × 10 ⁻³⁷ ) ≈0.0183
Resonance period T = 2π / (Wn × √ (1-ζ ² )) ≈9.88 × 10 ⁻¹¹ (t)
The resonance frequency f = 1 / T≈10 (Ghz).

  The resonance frequency f1 at which the specific adsorption sp resonates can be set to an arbitrary value by selecting the type of solvent and making it higher in viscosity. The resonance frequency f1 is desirably suppressed to about several Hz.

[Control flow]
FIG. 6 is a control flow executed by the control unit 18 of the surface plasmon enhanced fluorescence measurement apparatus. In step S <b> 11, by operating the pump 130, the sample liquid containing the analyte in the pipette 150 is sent to the storage unit 148 through the fine channel 143. By this action, the analyte is supplemented to the primary antibody to which the reaction field 104 is fixed. Subsequently, the pipette 150 is replaced. The exchanged pipette 150 is filled with a secondary antibody solution containing a secondary antibody with a modified fluorescent label, and the secondary antibody solution is sent to the reservoir 148 through the fine channel 143. Since the secondary antibody specifically reacts with the analyte, the reaction field 104 is supplemented with a state in which the primary antibody, the analyte, and the secondary antibody are linked in this order (specific adsorption sp).

  In step S <b> 12, as a cleaning process for the reaction field 104, the pipette 150 is similarly replaced, and a cleaning liquid such as internal water is sent to the storage unit 148 through the fine channel 143.

  By the steps S11 and S12, the specific adsorption sp is formed in the reaction field 104 as shown in FIG. At this time, not a few non-specific adsorption us that could not be removed by the washing process in step S12 are present in the reaction field 104.

  In step S <b> 13, the control unit 18 activates the high-voltage power supply PS as a vibration applying unit and applies an alternating electric field having a predetermined frequency to the reaction field 104 to apply vibration to the specific adsorption sp at a predetermined frequency.

  In step S <b> 14, the fluorescence from the fluorescent label F in the reaction field is measured by the light detection unit 120 by the electric field enhancement generated by the excitation light b <b> 1 from the light source 112.

  In step S15, the frequency analysis unit 181 performs frequency analysis on the output signal obtained in step S14, thereby separating the signals by specific adsorption sp. As frequency analysis, for example, (1) a bandpass filter or a low-pass filter so as to remove signal components other than the frequency at which the specific adsorption sp vibrates due to forced vibration or the resonance frequency f1 (hereinafter collectively referred to as the vibration frequency fsp). And quantifying the analyte from the obtained signal, or (2) performing a frequency conversion process such as FFT (Fast Fourier Transform) and quantifying the analyte from the signal intensity of the vibration frequency fsp. There is. Further, (3) after the FFT, a filter that passes the vibration frequency fsp is applied, and then a frequency inverse transformation process such as IFFT (Fast Fourier Inverse Transform) is performed to perform quantitative determination as in (1) above. Also good.

  FIG. 7 shows an example in which frequency analysis is performed on the output signal of the light detection unit 120. FIG. 7A shows an example in which signal components other than the vibration frequency fsp are removed from the output signal by the band-pass filter. FIG. 7B shows an example in which FFT processing is applied to the output signal.

  In this way, the specific adsorption sp in the reaction field is vibrated at a predetermined period, the signal from the fluorescence from the fluorescent substance of the specific adsorption sp is analyzed in frequency, and the specimen is analyzed from the obtained signal intensity, thereby performing the specific analysis. Not only adsorption but non-specific adsorption occurs, and even when both are mixed, signals due to specific adsorption can be separated and the S / N ratio can be improved.

[Another embodiment of vibration applying means]
Below, the modification of a vibration provision means is demonstrated. In the following, the configuration other than the vibration applying means is the same as the configuration described with reference to FIGS.

  FIG. 8 shows a first modification of the vibration applying means, in which the wall surface of the fine channel 143 is mechanically reciprocated to apply vibration to the reaction field. In the example shown in the figure, the wall surface of the fine channel 143 opposite to the reaction field 104 is a movable wall 146B, and when the piezoelectric element 17 is driven, the movable wall 146B becomes higher than the fine channel 143. It moves in the vertical direction (arrow direction in the figure).

  By driving the piezoelectric element 17 at a predetermined period, the specific adsorption sp of the reaction field 104 can be vibrated.

  Further, as a second modification, by controlling the pump 130, the liquid inside the pipette 150 is discharged or sucked to reciprocate the liquid inside the fine channel 143 at a predetermined cycle. . The specific adsorption sp can be vibrated like an inverted pendulum by the reciprocating motion of the liquid.

  As a third modification, the excitation light b1 and the fluorescence b3 (see FIG. 1) are irradiated with light having, for example, infrared wavelength, around the reaction field 104 by using another light source. By performing the irradiation in a predetermined cycle in a pulsed manner, the specific adsorption sp can be vibrated by heating the periphery of the reaction field 104.

DESCRIPTION OF SYMBOLS 10 Enhanced fluorescence measuring device 14 Microchip 18 Control part 181 Frequency analysis part PS High voltage power supply 146A ITO board | substrate 146B Movable wall 17 Piezoelectric element 142 Quartz substrate 143 Fine flow path 148 Storage part 150 Pipette 161 Fixing tool 102 Metal thin film 104 Reaction field 112 Light source 116 Light-receiving means 120 Photodetector b1 Excitation light b2 Metal thin film reflected light b3 Fluorescence

Claims (10)

A measurement method using a surface plasmon enhanced fluorescence measuring device having a vibration applying means for applying vibration at a predetermined frequency to a reaction field provided with a metal thin film,
Applying vibration by the vibration applying means to the antibody captured in the reaction field in which a primary antibody that specifically reacts with a sample is immobilized;
Irradiating one surface of the metal thin film with excitation light and exciting the fluorescent substance attached to the antibody by enhancing the electric field on the other surface side;
Measuring the fluorescence from the fluorescent material and analyzing the sample by frequency analysis of the output signal;
A measuring method characterized by comprising:   The measurement method according to claim 1, wherein the vibration frequency applied by the vibration applying means is lower than a frequency at which the antibody immobilized on the reaction field resonates, and the antibody is forcibly vibrated by vibration.   The measurement method according to claim 1, wherein the vibration frequency applied by the vibration applying means is a frequency at which the antibody immobilized on the reaction field resonates. The enhanced electric field depends on the distance from the metal film,
The distance of the fluorescent material from the metal thin film is periodically changed by the vibration applied by the vibration applying means, and the fluorescence intensity is changed by the change in the distance of the fluorescent material from the metal thin film. The measuring method as described in any one of Claim 1 to 3. A reaction field in which a metal thin film is provided and a primary antibody that specifically reacts with a sample is immobilized;
Vibration applying means for applying vibration to the reaction field at a predetermined frequency;
A light emitting unit that irradiates one surface of the metal thin film with excitation light, enhances an electric field on the other surface side, and excites a fluorescent substance attached to the antibody captured in the reaction field;
A light detection unit for detecting excitation light from the fluorescent material;
Fluorescence from a fluorescent substance attached to the antibody was measured by the light detection unit in a state where the vibration was applied to the antibody captured in the reaction field by the vibration applying means, and the output signal was obtained by frequency analysis. An analysis means for analyzing the sample from the signal intensity;
A surface plasmon enhanced fluorescence measuring apparatus comprising:   6. The surface plasmon enhancement according to claim 5, wherein a frequency of vibration applied by the vibration applying means is lower than a frequency at which the antibody immobilized on the reaction field resonates, and the antibody is forcibly vibrated by vibration. Fluorescence measuring device.   6. The surface plasmon enhanced fluorescence measuring apparatus according to claim 5, wherein the vibration frequency applied by the vibration applying means is a frequency at which the antibody immobilized on the reaction field resonates. The antibody or the fluorescent substance is charged,
The surface plasmon enhanced fluorescence measurement device according to any one of claims 6 to 7, wherein the vibration applying unit applies an alternating electric field to the reaction field.   The surface plasmon according to any one of claims 6 to 7, wherein the vibration applying unit vibrates a wall surface other than the reaction field of a flow path having the reaction field as a wall surface. Enhanced fluorescence measuring device.   8. The vibration applying unit according to claim 6, wherein the vibration is applied to the flow path having the reaction field as one wall surface, and the liquid is reciprocated and the vibration is applied to the specimen by the reciprocating motion. The surface plasmon enhanced fluorescence measuring apparatus according to one item.

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