JP2009080011A - Fluorescence detection method - Google Patents

Abstract

In fluorescence detection using total reflection illumination and the electric field enhancement effect by plasmons, quantitative effects and S / N ratio can be improved by reducing the influence of non-specific adsorption.
In a fluorescence detection method using electric field enhancement by plasmons, a sample 1 containing a target substance 2 labeled with a first label 5 is labeled with a second label 4 and is specific to the target substance 2 The specific binding substance 3 that binds to the target is supplied to the fixed detection unit, and the target substance 2 and the specific binding substance 3 are bound to each other, and the donor among the first label 5 and the second label 4 that are close to each other As a result, the label that functions as the other acceptor is excited by the fluorescence resonance energy transfer E, thereby detecting the fluorescence L2 emitted from the label that functions as the acceptor.
[Selection] Figure 1

Description

  The present invention relates to a fluorescence detection method using plasmon enhancement, and more particularly to a fluorescence detection method using fluorescence resonance energy transfer.

  In bio-measurement and the like, the fluorescence method is widely used as a highly sensitive and easy measurement method. The fluorescence method is qualitative or qualitative by irradiating a sample considered to contain a substance to be detected that emits fluorescence when excited by light of a specific wavelength, by irradiating the excitation light of the specific wavelength and detecting the fluorescence emitted at this time. This is a method for quantitatively confirming the presence of a substance to be detected. In addition, when the substance to be detected is not a fluorescent material, the substance to be detected is labeled with a fluorescent label such as an organic fluorescent dye, and then the fluorescence is detected in the same manner. It is a method to confirm.

  In particular, in recent years, the fluorescence method has become an indispensable tool for bioresearch, coupled with the development of high-performance photodetectors such as the development of cooled CCDs. Also in the material used for fluorescent labeling, fluorescent dyes with high fluorescence quantum yield, particularly in the visible region, such as FITC (fluorescence 525 nm, fluorescence quantum yield 0.6) and Cy5 (fluorescence 680 nm, fluorescence quantum yield 0.3). ) And more than 0.2 fluorescent dyes, which are practical guidelines, have been developed and widely used.

  Furthermore, Non-Patent Document 1 reports that high-sensitivity detection of less than 1 pM is possible by increasing the fluorescence signal using the electric field enhancement effect by plasmons.

  The outline is as follows, for example, and will be briefly described with reference to FIG. On the metal thin film 20, a specific binding substance 3 that specifically binds to the substance to be detected 2 contained in the sample 1 is fixed. The sample 1 is supplied into the sample holder 7, and the detection target substance 2 and the specific binding substance 3 labeled with the fluorescent label 5 are specifically bound. Thereafter, the excitation light 9 emitted from the light source 8 is incident on the interface 20a between the dielectric prism substrate 6 and the metal thin film 20 through the dielectric prism substrate 6 so as to satisfy the total reflection condition at the interface 20a. At this time, surface plasmons are generated in the metal thin film 20 due to resonance between the evanescent light 22 generated on the surface of the interface 20 a and the metal thin film 20. Then, the fluorescent label 5 is excited by absorbing the electric field enhanced evanescent light 22 in response to the electric field enhancing effect by the surface plasmon. The excited fluorescent label 5 emits fluorescence of a predetermined wavelength, and the detection target substance 2 is detected by this fluorescence detection.

In the above method, the electric field strength of the evanescent light 22 is enhanced by several tens to several hundreds due to the electric field enhancement effect by the surface plasmon. Further, the evanescent light 22 reaches only a region of about several hundred nm from the interface 20a. Using this characteristic, the amount of the fluorescent label 5 excited by the evanescent light 22 is efficiently increased by concentrating the detection target substance 2, that is, the fluorescent label 5 in the detection unit using the specific binding substance 3. At the same time, it is possible to greatly reduce the influence of scattering from the impurities 90 unintentionally remaining in the sample and light emission from the floating fluorescent label 91. Due to the effects as described above, a high S / N ratio can be realized by increasing the total amount of fluorescence and suppressing noise to enable high sensitivity detection.
JP-T 8-503994 Margarida MLM Vareiro, et al., Analytical Chemistry, Vol. 77, No. 8, p.2426-2431 (2005)

  In practice, however, non-specific adsorption due to electrostatic effects and hydrophobic effects generated between the substance and the substrate surface is a major problem. Non-specific adsorption is, for example, in the above-described case, as shown in FIG. 5b in which the labeled target substance 2 does not bind to the specific binding substance 3 and adsorbs nonspecifically on the surface of the metal thin film 20. Refers to the state. At this time, the fluorescence signal from the fluorescent label 5 ′ caused by non-specific adsorption becomes noise with respect to the fluorescence signal from the fluorescent label 5 caused by the specific binding to be detected, thereby reducing the quantitativeness of the measurement. It becomes a factor. This is less affected when fluorescence detection is simply performed on / off, but becomes a serious problem when quantitative measurement is required. In particular, when the concentration of the substance to be detected in the sample is about 1 pM, the fluorescence signal itself is weak, and this influence is great.

  In the presence of such non-specific adsorption, the fluorescence conversion part and the fluorescence amplification part are improved in order to improve the quantitative detection of fluorescence detection (increase in the quantum yield of the label, electric field enhancement by plasmon, etc., respectively). Included), the noise also increases at the same time, and as a result, the sensitivity does not improve as expected. Thus, in order to perform quantitative fluorescence detection, it is essential to reduce the influence of nonspecific adsorption.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a fluorescence detection method having high quantitativeness by reducing the influence of non-specific adsorption and improving the S / N ratio.

The fluorescence detection method according to the present invention comprises:
The evanescent light generated on the interface surface when the excitation light is incident on the interface between the dielectric prism substrate and the detector including the metal thin film formed on the dielectric prism substrate so as to satisfy the total reflection condition. The surface plasmon is generated by resonance between the surface and the metal thin film, and the substance contained in the sample supplied to the detector absorbs the evanescent light whose electric field is enhanced by the surface plasmon and detects the fluorescence emitted by the photodetector. In the detection method,
A step (A) of preparing a detection target substance labeled with a first label in a sample;
Preparing a specific binding substance that is labeled with the second label and specifically binds to the substance to be detected, and fixing the specific binding substance on the detection unit (B);
After steps (A) and (B), in order to bring the distance between the first label and the second label close to the extent that fluorescence resonance energy transfer occurs, a sample is supplied to the detection unit, A step (C) of binding a substance to be detected to a specific binding substance;
After the step (C), the first and second labels that are close to each other are illuminated with evanescent light, whereby either one of the first and second labels that functions as a donor is labeled. The excitation energy is transferred to the other label that functions as an acceptor by fluorescence resonance energy transfer to excite the other label, thereby detecting the fluorescence emitted from the other label. Is.

  In the present invention, the “detection unit” is a place where a sample or the like is supplied so as to be in contact therewith, a label is fixed to the detection unit, and the fixed label is excited and emitted by the evanescent light to be detected. It is arranged to detect substances.

  The “photodetector” detects fluorescence emitted from a label in a sample. Depending on the measurement, it is possible to measure the fluorescence spectrum in combination with the spectroscopic means.

  In addition, “fluorescence resonance energy transfer (FRET)” means a singlet state when a donor, ie, an energy donor and an acceptor that satisfies a specific condition, ie, an energy acceptor, is in the vicinity. Is transferred to the acceptor, and the acceptor is excited. At this time, if the acceptor is a fluorescent material, fluorescence is observed from the acceptor side.

  The photodetector is preferably arranged on the side where the metal thin film is formed with respect to the dielectric prism substrate.

  Moreover, it is desirable that the detection unit has an inflexible film made of a hydrophobic material and having a thickness of 10 to 100 nm on the surface of the metal thin film. At this time, the inflexible film is more preferably composed of a polymer material.

Furthermore, one of the labels functioning as a donor is any one labeling material selected from organic dyes, fluorescent quantum dots, and metal fine particles,
The other label functioning as an acceptor is desirably one label material selected from organic dyes and fluorescent quantum dots. In this case, the other label that functions as an acceptor is more preferably an organic dye.

  According to the fluorescence detection method of the present invention, in the fluorescence detection method using total reflection illumination, FRET that occurs between the first label and the second label is caused by binding the target substance to the specific binding substance. By detecting the fluorescence emitted after that, it is possible to reduce the influence of non-specific adsorption and improve the quantitativeness.

  In addition, the fluorescence to be detected that is emitted via FRET is greatly Stokes shifted with respect to the wavelength of the excitation light, so that it can be easily separated from incident light and scattered light, and the S / N ratio can be further improved. It becomes possible.

  Furthermore, since the electric field enhancement effect by the surface plasmon generated in the metal thin film is utilized, the weak fluorescence signal amount obtained can be amplified.

  Hereinafter, although the best embodiment in the present invention is described using a drawing, the present invention is not limited to this.

"Fluorescence detection method"
<First Embodiment>
FIG. 1 a is a schematic partial cross-sectional view of a fluorescence detection apparatus used when detecting an antigen 2 from a sample 1 containing an antigen 2 as a substance to be detected using the fluorescence detection method according to the present embodiment.

  As shown in the figure, the fluorescence detection apparatus includes a light source 8 that emits excitation light 9 having a predetermined wavelength that can excite the second label 4, and excitation light 9 that is arranged to transmit the excitation light 9 from one surface. A dielectric prism substrate 6 made of a material to be transmitted, a metal thin film 20 formed on a surface different from the incident surface of the excitation light 9 of the dielectric prism substrate 6, and an inflexible film 21 formed on the metal thin film 20 A primary antibody 3 fixed on the inflexible film 21; a second label 4 that labels the primary antibody 3; and a sample holder that holds the sample 1 so that the sample 1 is in contact with the inflexible film 21 7 and the photodetector 10 disposed on the side on which the metal thin film 20 is formed with respect to the dielectric prism substrate 6. In the figure, the antigen 2 contained in the sample 1 and the first label 5 that labels the antigen 2 are also shown.

  The excitation light 9 may be, for example, single-wavelength light obtained from a laser light source or the like, or broad light obtained from a white light source or the like, and is not particularly limited, but can be appropriately selected according to detection conditions.

  The light source 8 may be, for example, a laser light source and is not particularly limited, but can be appropriately selected according to detection conditions. If necessary, the light source 8 causes the excitation light 9 to enter the interface 20a between the dielectric prism substrate 6 and the metal thin film 20 through the dielectric prism substrate 6 so as to satisfy the total reflection condition at this interface. A light guide system such as a mirror or a lens for guiding the excitation light 9 can be appropriately combined.

  The dielectric prism substrate 6 is made of a transparent material such as transparent resin or glass. The dielectric prism substrate 6 is preferably formed of a resin. In this case, it is more preferable to use a resin such as polymethyl methacrylate (PMMA), polycarbonate (PC), or amorphous polyolefin (APO) containing cycloolefin. desirable.

  The metal thin film 20 is not particularly limited and can be appropriately selected according to detection conditions as the thin film material. However, Au, Ag, Pt, or the like is preferably used from the viewpoint of surface plasmon generation conditions. Moreover, the deposition method of the metal thin film 20 can be formed by various thin film production methods, such as a sputtering method, a vapor deposition method, a plating method, the coating method using a metal colloid, and a spray method, These methods are used. It can select suitably according to the material to do. On the other hand, the film thickness is not particularly limited and can be appropriately selected according to detection conditions. However, from the viewpoint of surface plasmon generation conditions and the like, the film thickness is preferably in the range of 20 nm to 60 nm.

  As the thin film material, for example, a silicon oxide film or a polymer material can be used for the inflexible film 21. In this case, a polymer material is particularly desirable from the viewpoints of film forming conditions and surface treatment conditions. For example, in this case, it can be prepared by a simple method such as spin coating. In addition, when the inflexible film 21 is formed of a hydrophobic material as in the present invention, factors that cause quenching such as metal ions and dissolved oxygen present in the sample liquid are contained in the inflexible film. These quenching factors prevent the excitation energy of the fluorescent label from being deprived.

It is desirable to select a specific material for the inflexible film 21 that has a difference in linear (thermal) expansion coefficient within 35 × 10 ^{−6 as} compared with the material used for the dielectric prism substrate 6. For example, it can be suitably selected from those listed in Table 1 below.

Here, the reason why the difference in coefficient of linear (thermal) expansion is defined to be within 35 × 10 ⁻⁶ as described above is as follows.

In order to increase the stability against environmental fluctuations, particularly temperature fluctuations, it is desirable that the inflexible film 21 and the dielectric prism substrate 6 have close thermal expansion coefficients. That is, if the thermal expansion coefficients of both of them are greatly different from each other, problems such as separation of the two and a decrease in the degree of adhesion tend to be caused when temperature fluctuation occurs. Specifically, it is desirable that the difference between the linear (thermal) expansion coefficients of the two is in the range of 35 × 10 ^{−6 or} less. Note that a metal thin film 20 exists between the inflexible film 21 and the dielectric prism substrate 6, and when the metal thin film 20 has a temperature variation, the upper and lower inflexible films 21 and the dielectric prism are present. Since the film 6 expands and contracts following the substrate 6, it is still desirable that the inflexible film 21 and the dielectric prism substrate 6 have close thermal expansion coefficients. Considering the above points, when the inflexible film 21 is formed from a polymer material, it can be said that it is generally more preferable to select a resin rather than glass as the material of the dielectric prism substrate 6.

  On the other hand, the film thickness of the inflexible film 21 is 10 nm to 100 nm. Here, the reason why the lower limit value and the upper limit value of the film thickness are defined as 10 nm and 100 nm, respectively, is as follows.

  The phosphor existing in the vicinity of the metal is quenched by energy transfer to the metal. The degree of energy transfer is inversely proportional to the third power of the distance if the metal has a semi-infinite thickness, inversely proportional to the fourth power of the distance if the metal is infinitely thin, and It becomes smaller in inverse proportion to the sixth power. In the case of a metal thin film, the distance between the metal and the phosphor is preferably at least several nm or more, more preferably 10 nm or more. Thereby, in this invention, the lower limit of the film thickness of an inflexible film | membrane shall be 10 nm. On the other hand, the phosphor is excited by evanescent light. This evanescent light is at most about the wavelength of the excitation light from the surface of the metal thin film, and the electric field strength is known to attenuate exponentially and exponentially according to the distance from the surface of the metal thin film. Actually, when the relationship between the two is calculated for visible light having a wavelength of 635 nm, the evanescent light reaches only the wavelength (635 nm). However, when it exceeds 100 nm, the electric field strength rapidly attenuates. Since it is desirable that the electric field intensity for exciting the phosphor is large, it is desirable to make the distance between the metal thin film surface and the phosphor smaller than 100 nm in order to perform effective excitation. Thereby, in this invention, the upper limit of the film thickness of an inflexible film | membrane shall be 100 nm.

  When the inflexible film 21 made of a polymer is used, nonspecific adsorption is easily performed if the substance to be detected 2 is a protein or the like. This is due to the hydrophobic effect resulting from the hydrophobic nature of the polymer and protein. In this case, since non-specifically adsorbed protein or the like becomes a factor that impairs the quantitativeness when performing fluorescence detection, it is desirable to apply hydrophilic surface modification to the surface of the inflexible film 21. Furthermore, this surface modification can have a function as a linker for immobilizing a specific binding substance in addition to the above function.

  The primary antibody 3 is a specific binding substance for the antigen 2 and is labeled with the second label 4. The antibody to be used is not particularly limited and can be appropriately selected depending on the detection conditions (particularly the substance to be detected 2). For example, when the antigen 2 is an hCG antigen (molecular weight 38000 Da), a monoclonal antibody or the like that specifically binds to the antigen 2 can be used. For example, an amine coupling method via PEG having a carboxyl group at its end Thus, it can be fixed to the inflexible film 21 made of a polymer. The amine coupling method includes the following steps (1) to (3) as an example. This is an example when a 30 ul (micro liter) cuvette / cell is used.

(1) Activate the -COOH group at the tip (end) of the linker part Add 30 ul of a solution in which an equal volume of 0.1 M NHS and 0.4 M EDC is mixed, and let stand at room temperature for 30 minutes.

NHS: N-hydrooxysuccinimide
EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
(2) Immobilization of primary antibody After washing 5 times with PBS buffer (pH 7.4), 30 ul of primary antibody solution (500 ug / ml) is added and left at room temperature for 30-60 minutes.

(3) After washing unreacted —COOH group 5 times with a blocking PBS buffer (pH 7.4), 30 ul of 1M ethanolamine (pH 8.5) is added, and the mixture is allowed to stand at room temperature for 20 minutes. Further, it is washed 5 times with PBS buffer (pH 7.4).

  The sample holding unit 7 can hold the sample 1 so as to be in contact with the detection unit (more precisely, on the inflexible film 21 in the present embodiment), and prevents detection of fluorescence emitted from the label included in the sample 1. There is no particular limitation as long as the shape or material does not exist. In the case of detecting fluorescence from above, for example, a sample holder 7 having a side surface that does not transmit light and an upper end surface that transmits light well as shown in FIG. 1a can be used. Here, the reason why the side surface that does not transmit light is used is to block unintended light from the outside.

  The photodetector 10 quantitatively detects weak fluorescence emitted from the label contained in the sample 1, and for example, LAS-1000 plus (trade name) manufactured by FUJIFILM Corporation can be suitably used. However, the present invention is not limited to this, and can be appropriately selected according to detection conditions, and a CCD, PD (photodiode), photomultiplier tube, c-MOS, or the like can be used. Further, the photodetector 10 can be used in combination with a spectroscopic means such as a filter or a spectroscope depending on detection conditions.

  In the fluorescence detection method according to the present embodiment, the excitation light 9 is totally reflected on the interface 20a between the dielectric prism substrate 6 and the detection unit including the metal thin film 20 formed on the dielectric prism substrate 6. The surface plasmon is generated by resonance between the evanescent light 22 generated on the surface of the interface 20a and the metal thin film 20, and the substance contained in the sample 1 supplied to the detection unit is caused by the surface plasmon. In the fluorescence detection method in which the fluorescence emitted by absorbing the electric field enhanced evanescent light 22 is detected by the photodetector 10, the step (A) of preparing the antigen 2 labeled with the first label 5 in the sample 1; A primary antibody 3 that is labeled with the second label 4 and specifically binds to the antigen 2 is prepared, and the step (B) of fixing the primary antibody 3 on the detection unit, the steps (A) and ( ), The sample 1 is supplied to the detection unit and included in the sample 1 in order to bring the distance between the first label 5 and the second label 4 so close that the fluorescence resonance energy transfer E occurs. The step (C) of binding the antigen 2 to the primary antibody 3 and the step (C) are followed by illuminating the evanescent light 22 on the first label 5 and the second label 4 that are close to each other. Exciting the label 4 and transferring the excitation energy to the first label 5 by fluorescence resonance energy transfer E to excite the first label 5, thereby detecting the fluorescence emitted from the first label 5. It is characterized by. In the present embodiment, the first label 5 functions as an acceptor, and the second label 4 functions as a donor.

  “Fluorescence Resonance Energy Transfer (FRET)” means that when there is an overlap between the donor's fluorescence spectrum and the absorption spectrum of the acceptor and they are in the vicinity, this excitation energy is transferred to the acceptor when the donor is excited. Thus, the donor returns to the ground state and the acceptor is excited (FIG. 6). For example, the excitation of FITC often used in fluorescence observation has a peak near 490 nm, and the fluorescence is near 520 nm (FIG. 6a). At this time, if a fluorescent probe having an excitation wavelength in the vicinity of 520 nm (fluorescence 560 nm, FIG. 6b) is present adjacent to the FITC, even if the FITC as a donor absorbs and excites the energy, this energy is used by It is sucked out to excite (Figure 6c). As a result, the light intensity at 520 nm, which is the fluorescence of FITC, decreases. On the other hand, the acceptor excited by receiving the donor energy emits fluorescence of a predetermined wavelength (560 nm) and returns to the ground state, and thus the light intensity of 560 nm, which is the fluorescence of the fluorescent probe, increases. Since the rate constant of energy transfer from the donor to the acceptor is inversely proportional to the sixth power of the distance between the donor and the acceptor, FRET actually occurs when the distance between the donor and the acceptor is about 1 to 10 nm. As described above, FRET is characterized in that (1) it occurs when a specific condition is satisfied, and (2) the fluorescence wavelength finally emitted greatly shifts with respect to the excitation wavelength.

  The combination of the first label 5 and the second label 4 is very important for the reasons described above. Combinations of dyes that cause FRET have been studied for a long time. For example, many combinations are shown in Tables 1 and 2 of JP-T-8-503994. However, the combination of labels is not limited to these as long as FRET can be generated. In addition, when labeling the first label 5 to the antigen 2, it is desirable to label directly by chemical bonding rather than labeling via an antibody. As a result, when the antigen 2 is bound to the primary antibody 3, the distance between the first label 5 and the second label 4 can be reduced except for an extra mediator, and FRET can be efficiently generated. . Both the first label 5 and the second label 4 can be donor acceptors. In this case, one of the labels functioning as a donor is any one label material selected from organic dyes, fluorescent quantum dots, and metal fine particles, and the other label functioning as an acceptor is an organic dye and a fluorescent material. It is desirable that it is any one of the labeling materials selected from the quantum dots.

  Hereinafter, the operation in this embodiment will be described with reference to FIG. FIG. 1b is an enlarged view of the vicinity of the detection unit in FIG. 1a.

  A primary antibody 3 that specifically binds to the antigen 2 is immobilized on the inflexible membrane 21. The sample 1 labeled with the first label 5 is supplied into the sample holder 7, and the antigen 2 in the sample 1 is bound to the primary antibody 3 and fixed. Thereafter, the excitation light 9 emitted from the light source 8 is incident on the interface 20a between the dielectric prism substrate 6 and the metal thin film 20 through the dielectric prism substrate 6 so as to satisfy the total reflection condition at the interface 20a. At this time, evanescent light 22 is generated on the surface of the interface 20 a, and surface plasmons are generated in the metal thin film 20 due to resonance between the evanescent light 22 and the metal thin film 20. Then, the first label 5 and the second label 4 which are close to each other due to the binding between the antigen 2 and the primary antibody 3 are illuminated with the evanescent light 22 whose electric field is enhanced by receiving the electric field enhancing effect by the surface plasmon. At this time, the second label 4 functioning as a donor is first excited, and this excitation energy is transferred by FRET to the first label 5 functioning as an acceptor (E in FIG. 1b). The label 5 is excited. As a result, fluorescence L2 having a predetermined wavelength is emitted from the excited first label 5, and the antigen 2 can be detected by detecting the fluorescence L2.

  Here, in the above example, it is the first label 5 that is actually confirmed by fluorescence detection. However, the antigen 2 is bound to the first label 5 basically by pretreatment. The presence of the antigen 2 is indirectly confirmed by confirming the presence of the first label 5.

  Consider the case where the antigen 2 is adsorbed nonspecifically on the inflexible membrane 21. At this time, the primary antibody 3 does not exist in the vicinity of the non-specifically adsorbed antigen 2 ′, that is, in the vicinity of 10 nm or less in the vicinity of the first label 5 ′ (acceptor) that labels the antigen 2 ′. In this case, the second label 4 (donor) is not present. Thereby, since the first label 5 ′ (acceptor) that is not supplied with excitation energy is not excited, the noise of the fluorescence signal with respect to the antigen 2 that is specifically bound to be detected is reduced. Can do. The second label 4 '(donor) that labels the primary antibody 3' that is not bound to the antigen 2 is excited by the excitation light and emits fluorescence L1. However, FRET does not occur because there is no first label 5 (acceptor) that receives this fluorescence L1 nearby. Therefore, by analyzing the fluorescence of each donor and acceptor, the influence of this can be reduced as well.

  As shown in FIG. 2, since the wavelength of the fluorescence L2 emitted from the acceptor is different from the excitation wavelength and the wavelength of the fluorescence L1 emitted from the donor, the fluorescence L2 emitted from the acceptor can be easily dispersed and detected. The ratio can be improved. Further, since the amount of fluorescence emitted by the acceptor varies depending on the amount of antigen 2, it is possible to detect fluorescence with high quantitativeness by analyzing the amount of fluorescence emitted by the acceptor.

  In general, FRET is not suitable for high-sensitivity detection because the generation conditions are severe and the efficiency is remarkably low. However, the fluorescence detection method according to the present invention uses the electric field enhancement effect by plasmons, so that the obtained fluorescence can be obtained. The amount of signal can be amplified.

  Further, in the present embodiment, the formation of the inflexible film 21 prevents the so-called metal quenching in which energy is transferred from the first marker 5 to the metal thin film 20 and energy is lost in the metal. it can. Thereby, fluorescence can be efficiently obtained from the excited first label 5.

  Further, by arranging the photodetector 10 on the side where the metal thin film 20 is formed with respect to the dielectric prism substrate 6, it is not necessary for the fluorescence to be detected to pass through the metal thin film 20, and the fluorescent light is efficiently emitted. Can be detected.

  As described above, by detecting fluorescence with a shifted wavelength, which is emitted only when specific binding occurs using FRET, the influence of non-specific adsorption is reduced and the S / N ratio is improved. Fluorescence detection with quantitative properties is possible.

<Second Embodiment>
The fluorescence detection apparatus used in this embodiment is the same as that used in the first embodiment shown in FIG. 1a. However, in the present embodiment, the first label 5 functions as a donor and the second label 4 functions as an acceptor. That is, it corresponds to an exchange of the function of the first marker 5 and the function of the second marker 4 in the first embodiment. Other configurations are the same as in the case of the first embodiment, and description of elements equivalent to those of the first embodiment shown in FIG. 1a is omitted unless particularly necessary. The excitation light 9 is appropriately selected according to the donor.

  The fluorescence detection method according to the present embodiment is the same as that of the first embodiment until the step (C), and then the evanescent light 22 is illuminated to the first label 5 and the second label 4 that are close to each other. The first label 5 is excited by this, and the excitation energy is transferred to the second label 4 by fluorescence resonance energy transfer E to excite the second label 4, thereby being emitted from the second label 4. It is characterized by detecting fluorescence L2 (FIG. 3).

  In the present embodiment, as shown in FIG. 3, the electric field is enhanced by receiving the electric field enhancement effect by the surface plasmon on the first label 5 and the second label 4 which are approached by the binding of the antigen 2 and the primary antibody 3. When the evanescent light 22 is illuminated, the first label 5 that functions as a donor is first excited, and this excitation energy is transferred to the second label 4 that functions as an acceptor by FRET (E in FIG. 3). Then the second label 4 is excited. As a result, fluorescence L2 having a predetermined wavelength is emitted from the excited second label 4, and the antigen 2 can be detected by spectroscopic detection of the fluorescence L2.

  The case where the antigen 2 is adsorbed nonspecifically on the inflexible film 21 can be considered in the same manner using FIG. At this time, the primary antibody 3 does not exist in the vicinity of the non-specifically adsorbed antigen 2 ′, that is, in the vicinity of 10 nm or less in the vicinity of the first label 5 ′ (donor) that labels the antigen 2 ′. Therefore, the second label 4 (acceptor) is not present. As a result, the first label 5 ′ (donor) that has been provided with energy by the excitation light is excited to emit fluorescence L1, but the second label 4 (acceptor) that receives this fluorescence is present nearby. FRET does not occur. Therefore, by spectroscopically analyzing the fluorescence of each donor and acceptor, it is possible to reduce noise in the fluorescence signal with respect to the antigen 2 that is specifically bound to be detected. Further, the second label 4 ′ (acceptor) that labels the primary antibody 3 ′ not bound to the antigen 2 is not supplied with excitation energy. Thereby, since it is not excited and emits the fluorescence L2, the influence by this can be reduced similarly.

  As described above, the same effects as those of the first embodiment can be obtained.

<Third Embodiment>
The fluorescence detection device used in the present embodiment is a device in which metal fine particles are used as the second label 4 that labels the primary antibody 3 in the device used in the first embodiment shown in FIG. 1a. Also in this embodiment, as in the first embodiment, it is assumed that the first label 5 functions as an acceptor, and the second label 4, that is, metal fine particles function as a donor. Other configurations are the same as in the case of the first embodiment, and description of elements equivalent to those of the first embodiment shown in FIG. 1 is omitted unless particularly necessary. The excitation light 9 is appropriately selected according to the donor.

  Hereinafter, a description will be given with reference to FIG.

  The metal fine particles 4 absorb light of a predetermined wavelength, and Au and Ag nanoparticles are desirable from the viewpoint of FRET generation conditions. In addition, the acceptor that causes FRET can be appropriately selected depending on the predetermined wavelength.

  The fluorescence detection method according to this embodiment is the same as that of the first embodiment, but the only difference is that the metal fine particles 4 absorb energy but do not emit fluorescence.

  In the present embodiment, as shown in FIG. 4, consider a case where the antigen 2 is adsorbed nonspecifically on the inflexible membrane 21. At this time, the primary antibody 3 does not exist in the vicinity of the non-specifically adsorbed antigen 2 ′, that is, in the vicinity of 10 nm or less in the vicinity of the first label 5 ′ (acceptor) that labels the antigen 2 ′. Therefore, the metal fine particles 4 (donor) are not present. Thereby, since the first label 5 ′ (acceptor) that is not supplied with excitation energy is not excited, the noise of the fluorescence signal with respect to the antigen 2 that is specifically bound to be detected is reduced. Can do. In addition, the metal fine particle 4 ′ (donor) labeled with the primary antibody 3 ′ not bound to the antigen 2 is excited because it is supplied with energy by the excitation light, but the metal fine particle 4 ′ itself does not emit light. Therefore, it is not necessary to split the fluorescence, and the influence due to this can be reduced as well.

  As described above, the same effects as those of the first embodiment can be obtained, and fluorescence detection can be facilitated without the need to split the fluorescence signal.

(A) Partial sectional view schematically showing an example of an apparatus for carrying out the fluorescence detection method according to the present invention (b) Schematic showing a fluorescence resonance energy transfer process in the first embodiment Schematic showing the relationship between the amount of substance to be detected and the amount of fluorescence obtained by fluorescence resonance energy transfer Schematic showing the fluorescence resonance energy transfer process in the second embodiment Schematic showing the fluorescence resonance energy transfer process in the third embodiment Partial sectional view schematically showing a fluorescence detection device using general plasmon enhancement Conceptual diagram of fluorescence resonance energy transfer

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample 2 Antigen 3 Specific binding substance 4 2nd label | marker 5 1st label | marker 6 Dielectric prism board 7 Sample holding part 8 Light source 9 Excitation light 10 Photo detector 20 Metal thin film 20a Dielectric prism board and metal thin film Interface 21 Inflexible film 22 Evanescent light E Fluorescence resonance energy transfer L1 Fluorescence emitted from donor L2 Fluorescence emitted from acceptor

Claims (6)

Evanescent light generated on the interface surface when excitation light is incident on the interface between the dielectric prism substrate and the detection unit including the metal thin film formed on the dielectric prism substrate so as to satisfy the total reflection condition. A surface plasmon is generated by resonance with the metal thin film, and a substance contained in the sample supplied to the detection unit absorbs the evanescent light whose electric field is enhanced by the surface plasmon and emits fluorescence. In the fluorescence detection method for detecting by
Preparing a detection target substance labeled with a first label in the sample (A);
Preparing a specific binding substance that is labeled with a second label and that specifically binds to the substance to be detected, and fixing the specific binding substance on the detection unit (B);
After the steps (A) and (B), the sample is supplied to the detection unit in order to bring the distance between the first label and the second label close to the extent that fluorescence resonance energy transfer occurs. A step (C) of binding the substance to be detected contained in the sample to the specific binding substance;
After the step (C), by illuminating the evanescent light on the first label and the second label that are close to each other, it functions as a donor among the first label and the second label. Either one of the labels is excited, and the excitation energy is transferred to the other label that functions as an acceptor by fluorescence resonance energy transfer to excite the other label, thereby detecting the fluorescence emitted from the other label. A fluorescence detection method characterized by that.   2. The fluorescence detection method according to claim 1, wherein the photodetector is disposed on a side where the metal thin film is formed with respect to the dielectric prism substrate.   The fluorescence detection method according to claim 1, wherein the detection unit has an inflexible film having a thickness of 10 to 100 nm made of a hydrophobic material on the surface of the metal thin film.   The fluorescence detection method according to claim 3, wherein the inflexible film is made of a polymer material. Either one of the labels functioning as the donor is any one labeling material selected from organic dyes, fluorescent quantum dots, and metal fine particles,
5. The fluorescence detection method according to claim 1, wherein the other label that functions as the acceptor is any one label material selected from organic dyes and fluorescent quantum dots.   6. The fluorescence detection method according to claim 5, wherein the other label that functions as the acceptor is an organic dye.

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