JP5622215B2 - Microplate having periodic structure, surface plasmon excitation enhanced fluorescence microscope, fluorescence microplate reader using the same, and method for detecting specific antigen-antibody reaction - Google Patents

Description

  The present invention uses surface plasmon excitation enhanced fluorescence (Surface Plasmon Fluorescence) that excites a fluorescent substance to generate fluorescence by surface plasmon resonance light generated by surface plasmon resonance (hereinafter also referred to as SPR). The present invention relates to a sensitive fluorescence microscope and a microplate reader.

  When performing fluorescence observation of a sample using a general-purpose epifluorescence (epi-illumination) microscope, it is higher when the sample is dark and not sufficiently visible, or when the background is very high (bright) and the sample is not sufficiently visible. Measurement with high sensitivity and S / N is required. Until now, it has been observed using a total reflection fluorescence microscope (TIRF) and a confocal microscope characterized by high resolution.

  On the other hand, regarding the surface plasmon resonance microscope, various papers and patent applications (see Patent Documents 1 to 5 below) have been made, and are commercially available as devices. However, at present, it is impossible to realize SPFM (Surface Plasmon Fluorescence Microscopy) which is a fluorescence microscope by applying the basic optical systems disclosed in Patent Documents 1 to 5. The reason is that a commercially available surface plasmon resonance microscope uses a high refractive index objective lens for the incident optical system, and the wavelength of light that can be used is limited to the near infrared region by limiting the refractive index of the objective lens. This is because it cannot be used as excitation light for general fluorescent molecules.

  On the other hand, the following Patent Documents 6 and 7 disclose development from a surface plasmon resonance microscope having an optical system using a prism to SPFM.

JP 2001-242071 A JP 2001-255267 A JP 2003-83886 A JP 2004-117181 A JP 2006-3282 A JP-A-10-307141 JP 2006-208294 A

  As mentioned above, there are total reflection microscopes and confocal microscopes as microscopes that can detect fluorescence with high sensitivity and high resolution, but they are often equipped with high-power lasers, etc. Therefore, there is a problem that the operation is complicated and the price is high.

  The SPFMs disclosed in Patent Documents 6 and 7 described above also have a problem that the optical system is complicated and the operation is complicated because the Kretschmann type arrangement shown in FIG. 19 is adopted.

  On the other hand, there is a microplate reader as another apparatus for observing fluorescence. In the case of detecting fluorescence with a conventional microplate reader, there is a problem that it is necessary to prepare a sample having a high concentration and not a very small amount in order to generate fluorescence with sufficient intensity.

  An object of the present invention is to provide a surface plasmon excitation enhanced fluorescence microscope that adopts a simple optical system, is easy to operate, and realizes low-cost, highly sensitive fluorescence detection in order to solve the above-described problems. To do.

  Another object of the present invention is to provide a surface plasmon excitation enhanced fluorescent microplate reader that is more sensitive than existing fluorescent microplate readers.

  As a result of intensive research to solve the above problems, the present inventor has made use of surface plasmons generated on a substrate in which a metal and a quenching suppression film are prepared on a periodic structure. It has been found that a microscope and a fluorescent microplate reader can be realized.

  That is, the present invention includes the following aspects.

  The first microplate according to the present invention is used in a fluorescence microscope or a fluorescence microplate reader, carries a sample to be observed, and detects a specific antigen-antibody reaction by enhanced fluorescence using a fluorescently labeled secondary antibody. A base plate having a periodic structure on its surface, a metal layer formed on the periodic structure, and a quenching suppression layer formed on the metal layer, The structure includes a plurality of grooves formed along one direction, the metal layer has a slope on the surface, the metal layer is formed of a metal capable of generating a propagation-type surface plasmon resonance light, and the quenching A step of generating an electric field enhanced by the surface plasmon resonance light, wherein an antibody is adsorbed or bound to the surface of the suppression layer, and light is incident on the microplate. And detecting the enhanced fluorescence from either the base substrate side or the quenching suppression layer side using the generated electric field as an excitation field of a fluorescent molecule, and the enhanced fluorescence from the microplate. The angle formed by the incident direction of the light incident from the base substrate side and the perpendicular of the plane on which the light is incident on the base substrate is not bent by the bending means. The angle is such that the light can enter the microplate and the angle is small enough to generate the surface plasmon resonance light.

  Further, in the second microplate according to the present invention, in the first microplate, the angle formed by the incident direction of the light and the perpendicular of the plane on which the light is incident on the base substrate is from 5 degrees. It is characterized by being within a range of 46 degrees.

  Further, in the third microplate according to the present invention, in the second microplate, the angle formed by the incident direction of the light and the perpendicular of the plane on which the light of the base substrate is incident is from 5 degrees. It is characterized by being within a range of 25 degrees.

  The fourth microplate according to the present invention is characterized in that, in the first microplate, the period of the periodic structure is 10 nm or more and 1 μm or less.

  The fifth microplate according to the present invention is characterized in that, in the fourth microplate, the periodic structure further includes a plurality of groove portions formed along a direction intersecting the one direction.

  The sixth microplate according to the present invention is characterized in that, in any one of the first to fifth microplates, the quenching suppression layer has a thickness of 10 nm to 100 nm.

  The seventh microplate according to the present invention is characterized in that in any one of the first to sixth microplates, the metal layer is formed of a thin film of transition metal having a thickness of 10 to 500 nm.

  Further, an eighth microplate according to the present invention includes an adhesive layer having a thickness of 0.1 to 3 nm between the base substrate and the metal layer in any one of the first to seventh microplates, An adhesion and antioxidant layer having a film thickness of 0.1 to 3 nm is provided between the metal layer and the quenching suppression layer.

The ninth microplate according to the present invention is characterized in that, in the eighth microplate, the metal layer is formed of silver and the quenching suppression layer is formed of SiO ₂ .

  The tenth microplate according to the present invention is the ninth microplate, wherein the quenching suppression layer has a thickness of 10 to 50 nm.

  The eleventh microplate according to the present invention is any one of the first to eighth microplates, wherein the metal layer is formed of gold, and the thickness of the quenching suppression layer is 10 to 70 nm. It is said.

  The twelfth microplate according to the present invention is the fourth microplate, wherein the periodic structure on the surface of the base substrate is a rectangular periodic structure, and the metal layer corresponds to a rectangular step portion. It has the slope on the surface, is formed to a thickness of 200 nm, the ratio of the length of the slope to the period is 10 ± 5%, the depth of the groove is dnm, and the length of the mountain relative to the period When the ratio is M%, 5 ≦ d <15, and 0 <M <70, or 70 <M <100, 15 ≦ d <25, and 0 <M <80, or 80 <M <100, 25 ≦ d <35 and 0 <M <60, or 60 <M <100, or 35 ≦ d <45, and 0 <M <60, or 60 <M <100.

  Further, the thirteenth microplate according to the present invention is the quenching plate according to any one of the first to twelfth microplates, in order to adsorb or bind proteins or cells to the surface of the quenching suppression layer of the microplate. The surface of the suppression layer is subjected to amination or alkylation treatment.

  The fourteenth microplate according to the present invention is the same as the thirteenth microplate, in which the surface of the quenching suppression layer of the microplate is further reacted with biotinylated polyethylene glycol whose end is modified with an active esterified carboxyl group. And is used for detection of binding to a fluorescently labeled antigen or antibody containing streptavidin.

  The first detection method according to the present invention is a method for detecting a specific antigen-antibody reaction by enhanced fluorescence using a fluorescently labeled secondary antibody using a microplate in a fluorescence microscope or a fluorescence microplate reader. The microplate includes a base substrate having a periodic structure on a surface, a metal layer formed on the periodic structure, and a quenching suppression layer formed on the metal layer, wherein the periodic structure A plurality of grooves formed along one direction, the metal layer has a slope on the surface, the metal layer is formed of a metal capable of generating a propagation type surface plasmon resonance light, and the quenching suppression layer An antibody is adsorbed or bound to the surface of the substrate, and the method causes a light incident on the microplate to generate an electric field enhanced by the surface plasmon resonance light. And detecting the enhanced fluorescence from either the base substrate side or the quenching suppression layer side using the generated electric field as an excitation field of fluorescent molecules, and entering from the base substrate side The angle formed by the incident direction of the light and the perpendicular of the plane on which the light of the base substrate is incident is an angle at which the light can be incident on the microplate even if the light is not bent by a bending means, and The angle is so small that the surface plasmon resonance light is generated.

  The second detection method according to the present invention is the first detection method, wherein the angle formed by the incident direction of the light and the perpendicular of the plane on which the light is incident on the base substrate is from 5 degrees. It is characterized by being within a range of 46 degrees.

  According to a third detection method of the present invention, in the second detection method, the angle formed by the incident direction of the light and a perpendicular of the plane on which the light of the base substrate is incident is from 5 degrees. It is characterized by being within a range of 25 degrees.

  The first surface plasmon excitation enhanced fluorescence microscope according to the present invention is a microplate for mounting a sample to be observed and detecting a specific antigen-antibody reaction by enhanced fluorescence using a fluorescently labeled secondary antibody. The microplate includes a base substrate having a periodic structure on a surface thereof, a metal layer formed on the periodic structure of the base substrate, and a quenching formed on the metal layer A suppression layer, the periodic structure includes a plurality of grooves formed along one direction, the metal layer has a slope on the surface, and the metal layer generates propagation-type surface plasmon resonance light. Formed on the surface of the quenching suppression layer, and an antibody is adsorbed or bound to the surface of the quenching suppression layer. Generating an enhanced electric field, and detecting the enhanced fluorescence from either the base substrate side or the quenching suppression layer side using the generated electric field as an excitation field of a fluorescent molecule. Thus, the enhanced fluorescence from the microplate is detected, and an angle formed by an incident direction of the light incident from the base substrate side and a plane perpendicular to the light incident on the base substrate is the light. Is an angle that can be incident on the microplate without being bent by the bending means, and is an angle that is small enough to generate the surface plasmon resonance light.

  In the second fluorescence microscope according to the present invention, in the first fluorescence microscope, the angle formed by the incident direction of the light and a perpendicular of the plane on which the light is incident on the base substrate is from 5 degrees. It is characterized by being within a range of 46 degrees.

  The third fluorescence microscope according to the present invention is the second fluorescence microscope, wherein the angle formed by the incident direction of the light and the perpendicular of the plane on which the light is incident on the base substrate is 5 degrees. It is characterized by being within a range of 25 degrees.

  The first surface plasmon excitation enhanced fluorescence microplate reader according to the present invention is equipped with a sample to be observed and for detecting a specific antigen-antibody reaction by enhanced fluorescence using a fluorescently labeled secondary antibody. A fluorescent microplate reader having a microplate, wherein the microplate has a base substrate having a periodic structure on a surface, a metal layer formed on the periodic structure of the base substrate, and on the metal layer A quenching suppression layer formed, the periodic structure has a plurality of grooves formed along one direction, the metal layer has a slope on the surface, the metal layer is a propagation type surface plasmon resonance It is formed of a metal that can generate light, and an antibody is adsorbed or bound to the surface of the quenching suppression layer, and the microplate makes light incident on the microplate. Generating an electric field enhanced by the surface plasmon resonance light, and using the generated electric field as an excitation field of a fluorescent molecule, the enhanced fluorescence is applied to either the base substrate side or the quenching suppression layer side. Detecting the enhanced fluorescence from the microplate and detecting a direction of incidence of the light incident from the base substrate side and a perpendicular of the plane on which the light of the base substrate is incident Is an angle that allows the light to enter the microplate even if the light is not bent by a bending means, and is an angle that is small enough to generate the surface plasmon resonance light. .

  The second fluorescent microplate reader according to the present invention is the first fluorescent microplate reader, wherein the angle formed by the incident direction of the light and a perpendicular of the plane on which the light is incident on the base substrate is It is characterized by being in the range of 5 to 46 degrees.

  The third fluorescent microplate reader according to the present invention is the second fluorescent microplate reader, wherein the angle formed by the incident direction of the light and a perpendicular of the plane on which the light is incident on the base substrate is It is characterized by being in the range of 5 to 25 degrees.

  According to the present invention, since the incident angle of light can be reduced without using a prism such as a Kretschmann type, any conventional upright or inverted fluorescent microscope employing epi-illumination or transmission illumination can be used. Can be used. Therefore, the operation is simple, an objective lens having a small NA can be used, and bright fluorescence observation is possible.

In addition, it is possible to increase the spatial selectivity in fluorescence observation, that is, to observe only the microplate interface (in the range of less than 200 nm from the microplate surface), so that it is hardly affected by noise due to the background. It is possible to measure without rinsing (cleaning) after injection. Also, the surface plasmon resonance light at the microplate interface is enhanced more than 100 times the incident light intensity, so it is necessary to use a high-power laser. Thus, even a lamp light source can observe fluorescence about 100 times brighter than that of an existing epifluorescence microscope.

  In addition, by using a lamp light source, it becomes possible to select a wavelength in a wide range by selecting an appropriate optical filter, so that it is more compact and less expensive than a microscope equipped with a laser.

  Conventional total reflection microscopes or confocal microscopes are often equipped with high-power lasers, etc., and the optical system is complicated, so the complexity of operation and high cost are problems, and it is widely spread. However, according to the present invention, these problems can be solved, so that it can be expected to spread as a general-purpose high-sensitivity fluorescent microscope. In particular, it can be expected to greatly contribute to the spread of the medical / biological field in which samples are observed by fluorescent labeling.

  In addition, when the present invention is applied to a fluorescent microplate reader, amplification of fluorescence can be expected, and the incident angle of illumination may be low, so even existing microplate readers can measure low-concentration samples and trace amounts of samples. It becomes.

  The microplate of the present invention is effective for detecting a specific antigen-antibody reaction. That is, a specific antigen-antibody reaction can be detected by enhanced fluorescence by using a fluorescently labeled secondary antibody without adsorbing or binding the antibody to the surface of the quenching suppression layer of the microplate and performing a rinsing operation.

It is a figure which shows the possible combination of the incident direction of illumination light, and the observation direction of fluorescence in the fluorescence microscope which is the object of this invention. It is a figure which shows an example of the optical arrangement | positioning of the surface plasmon excitation enhanced fluorescence microscope which concerns on embodiment of this invention. It is sectional drawing which shows the structure of the microplate which concerns on embodiment of this invention. It is a figure which shows the relationship between the polarization direction of incident light, and the direction of a grating | lattice. It is a figure which shows the relationship between the polarization direction of incident light, and the direction of a grating | lattice, and the microplate is rotated 90 degree | times from the state of FIG. 4, and is arrange | positioned. It is a top view which shows a two-dimensional periodic structure. It is sectional drawing and a top view which show the structure of the microplate for fluorescence microplate readers concerning embodiment of this invention. It is a graph which shows the result of the reflectance measurement depending on an incident angle in a periodic structure board | substrate, and has shown the result of both the case where light injects from the quenching suppression layer side, and the case where it injects from the base substrate side. It is a figure which shows the relationship between the polarization plane of incident light, and the direction of a grating | lattice. It is sectional drawing which shows the periodic structure of the base substrate surface. It is a graph which shows the measurement result regarding the board | substrate 1 and the board | substrate 2. FIG. It is sectional drawing which shows the periodic structure which has a slope. It is a graph which shows a simulation result. It is sectional drawing which shows the periodic structure which does not have a slope. It is a microscope picture, (a) shows an upright fluorescence microscope observation result of Cy5-fluorescence labeled protein binding beads by transmitted illumination, and (b) shows an inverted fluorescence microscope observation result of DiI fluorescence labeled cells by epi-illumination. It is a graph which shows the result of having measured the adsorption amount of the protein at the time of chemically processing the substrate surface by the surface plasmon resonance method. It is a graph which shows the measurement result of the reflectance and fluorescence intensity depending on an incident angle in the board | substrate which the fluorescent label protein specifically couple | bonded with the microplate with a periodic structure, and the board | substrate (control) which adsorb | sucked nonspecifically. It is a graph which shows the result of having verified about the difference in the fluorescence intensity observed by the direction of incident light, and the observation direction of fluorescence. It is sectional drawing which shows Kretschmann type | mold optical arrangement | positioning.

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

First, as shown in FIG. 1, there are four types of fluorescence microscopes according to the combination of the incident direction of the illumination light Li with respect to the observation target A and the observation direction of the fluorescence Lp. Specifically, it is as follows.
In the case of the epi-illuminated upright microscope, the incident direction of the illumination light is IN1, and in the case of the epi-illuminated inverted microscope whose observation direction is T, the incident direction of the illumination light is IN2, and the observation direction is B.
In the case of a transmission upright microscope, the incident direction of illumination light is IN2, and the observation direction is T.
In the case of a transmission inverted microscope, the incident direction of illumination light is IN1, and the observation direction is B.
The present invention covers all of these, but in the following, some of these will be described as appropriate for the sake of brevity.

  FIG. 2 is a diagram showing an example of a surface plasmon excitation enhanced fluorescence microscope (hereinafter referred to as SPFM) according to an embodiment of the present invention. The SPFM 1 shown in FIG. 2 includes a microscope main body (hereinafter referred to as “main body”) 2 and a microplate 4 mounted on a microscope stage (hereinafter referred to as “stage”) 3. FIG. 2 shows a state in which the sample A is mounted on the microplate 4, the illumination light Li is incident from below, and the observation light (plasmon excitation enhanced fluorescence) Lp is observed from above. That is, the SPFM 1 in FIG. 2 is classified as a transmission type upright microscope.

  The SPFM 1 is characterized by the microplate 4 and the components of the main body 2 necessary for functioning as the other fluorescence microscope are the same as those of the conventional fluorescence microscope. That is, the light source, the objective lens, the eyepiece lens, the interference mirror (dichroic mirror), the interference filter (excitation filter), the fluorescence filter, and the like, which are constituent elements of the main body 2, are the same as those of a known fluorescence microscope. Therefore, the description regarding them is omitted.

  FIG. 3 is a cross-sectional view showing the configuration of the microplate 4 according to the present embodiment. The microplate 4 includes a base substrate 5, a metal layer 6 and a quenching suppression layer 7 formed on the surface of the base substrate 5. Although omitted in FIG. 3, a layer for adhering two adjacent layers between the base substrate 5 and the metal layer 6 and between the metal layer 6 and the quenching suppression layer 7 ( Hereinafter, it is preferable to include an adhesive layer.

  The base substrate 5 has a lattice having a periodic structure formed on the surface. As long as the base substrate 5 is formed of a material transparent to the incident light Li and the observation light Lp, such as glass or plastic, it can be used in either an epi-illumination or transmission type fluorescence microscope observation. The base substrate 5 does not need to be transparent as long as it is limited to the observation of epi-illumination. The periodic structure has, for example, a shape having a plurality of grooves arranged at approximately equal intervals along one direction, and the grooves are, for example, sawtooth grooves, sinusoidal grooves, and rectangular grooves. The period of the periodic structure, that is, the interval between adjacent grooves is not more than the wavelength used for observation, for example, 10 to 1000 nm (nanometers), and preferably 100 to 600 nm. The periodic structure has a height (groove depth) of 4 to 400 nm and an aspect ratio of 0.005 to 10.

  The periodic structure having a nanoscale periodic structure can be formed using, for example, a method disclosed in Japanese Patent No. 3350711, Japanese Patent No. 2832337, Japanese Patent Application Laid-Open No. 2004-117810, and the like. .

  The metal layer 6 is preferably a transition metal such as gold, silver, copper, platinum, or nickel. The film thickness of the metal layer 6 is preferably 10 to 500 nm, more preferably 50 to 200 nm. However, the metal layer 6 is not limited to a transition metal, and may be any metal that can generate surface plasmons. In this case, the film thickness is preferably 10 to 500 nm, more preferably 50 to 200 nm.

The quenching suppression layer 7 is transparent with no absorption (or less absorption) in the wavelength region of incident light used for observation or generated fluorescence, such as organic polymers such as polycarbonate and polymethyl methacrylate, and silica (SiO ₂ ). Use a thin film. With enhanced fluorescence, which is a feature of the surface plasmon excitation enhanced fluorescence method, when the distance between the fluorescent molecule and the metal is short, the fluorescence excited by a strong excitation field is also transferred to the metal surface and quenched. Therefore, it is necessary to suppress quenching by separating the sample from the metal layer 6 by a predetermined distance. In addition, since the excitation field due to surface plasmon resonance is a near field, the electric field intensity attenuates as the distance from the metal surface increases, so that only fluorescent molecules existing within about 100 nm from the metal surface are efficiently excited. Therefore, the film thickness of the quenching suppression layer 7 is determined in accordance with the type of the metal layer 6 in the range of about 10 nm to 100 nm. For example, the optimum value of the film thickness is 10 to 50 nm when the metal layer 6 is silver, and more preferably 20 to 50 nm. In the case of gold, the optimum value of the film thickness is 10 to 70 nm, more preferably 40 to 70 nm.

It is assumed that an aqueous solution containing a sample (such as a phosphate buffer) is mounted on the microplate 4 for observation. Therefore, when silver is used for the metal layer 6, in order to protect silver which is very unstable in water, between the base substrate 5 and silver and between the silver and quenching suppression layer 7 (for example, SiO ₂ ) It is desirable to form a layer for preventing oxidation (hereinafter referred to as “antioxidation layer”). The antioxidant layer may be at least between silver and the quenching suppression layer 7. For example, a first layer that functions as an adhesive layer is formed between the base substrate 5 and silver, and a second layer that functions as an adhesion and antioxidant layer is formed between the silver and the quenching suppression layer 7. For example, the first layer and the second layer are each formed as a thin film having a thickness of 0.1 to 3 nm. The second layer may be a layer made of a material that can protect silver, and is formed of, for example, chromium (Cr), aluminum, titanium, or palladium. The second layer is preferably made of a material that also serves to enhance the adhesion of the quenching suppression layer 7, and in this sense, chromium (Cr) is suitable.

  Next, the relationship between the polarization direction of incident light and the direction of the periodic structure will be described with reference to FIG. FIG. 4 shows that light is incident from one of the planes on which the periodic structure is formed, and does not mean that the incident is limited to incidence from above. Generation of surface plasmon resonance light requires p-polarized light. Further, in relation to the arrangement of the periodic structure, it is necessary that the p-polarized light includes a component in the direction of the periodic structure (direction perpendicular to the grooves of the grating).

  4A is a perspective view showing a state where light is incident on the microplate 4, and FIG. 4B is a plan view thereof. The planes U1 and U2 are perpendicular to the surface of the microplate 4 and parallel to the direction V1 of the grating 8 formed on the surface of the microplate 4 (direction perpendicular to the grooves of the grating), and a plane orthogonal to the plane U1. U2. Incident light L1 and L2 are light that is output from the light source, condensed in a conical shape by the objective lens, and incident on the microplate 4, respectively, traveling in the planes U1 and U2. Here, it is assumed that the incident lights L1 and L2 are lights that have not been polarized by a polarizing filter or the like. Accordingly, surface plasmon resonance light is generated by a component parallel to V1 among the components of the p-polarized components V2 and V3 of the incident lights L1 and L2 with respect to the microplate 4. As described above, when the incident light on the microplate 4 is not polarized, surface plasmon resonance light is generated by the p-polarized light of the incident light having a component parallel to the direction V1 of the grating 8 formed on the surface of the microplate 4. To do.

  On the other hand, when incident light is polarized in a predetermined direction by a polarizing filter or the like, surface plasmon resonance light may hardly be generated depending on the arrangement of the microplate 4. FIG. 5 is a perspective view similar to FIG. 4 except that the microplate 4 is rotated 90 degrees from the state of FIG. In FIG. 5, incident light of the polarization axis S is collected in a conical shape by the objective lens by a polarizing filter or the like, but the light L1 and L2 propagating in the planes U1 and U2 are in the plane U1. The light is polarized (V2 and V4) in the in-plane direction and in the normal direction to the plane U2. In this case, since the p-polarized component V2 is orthogonal to the grating direction V1, and V4 is s-polarized with respect to the microplate 4, no surface plasmon resonance light is generated by either the light L1 or L2. Even for incident light other than L1 and L2, the surface plasmon resonance light is not generated efficiently because the parallelism between the p-polarization property and V1 is low. Therefore, a bright fluorescent image cannot be obtained with the arrangement of incident light having a polarization axis and a grating as shown in FIG.

  In a conventional fluorescence microscope, if a polarization-dependent element (such as a dichroic mirror) is included in a filter set that is normally incorporated without inserting a polarizer, surface plasmon resonance light is used as described above. Therefore, it is necessary to adjust the arrangement of the microplate 4, that is, the direction of the lattice so as to generate efficiently.

  As described above, the case where the embodiment of the present invention is applied to a fluorescence microscope has been described. However, the present invention is not limited to the above-described embodiment, and various modifications can be made. In particular, it should be noted that the microplate of the present invention can be used in any of the fluorescence microscopes of an episcopic upright microscope, an episcopic inverted microscope, a transmission upright microscope, and a transmission inverted microscope. Further, the present invention can be similarly applied to a fluorescent microplate reader.

  In addition, the shape of the periodic structure is not limited to the shape having grooves in one direction as described above, and a two-dimensional periodic structure in which grooves are formed in two directions intersecting the surface of the base substrate 5 ′ as shown in FIG. It may be a circular (Fresnel) periodic structure. FIG. 6 is a plan view of the base substrate 5 ′ on which the two-dimensional periodic structure is formed. On the surface of the base substrate 5 ′, the plurality of groove portions 9 are arranged in two directions perpendicular to each other, that is, the convex portions 10 are arranged in two directions perpendicular to each other. Note that the two directions in which the grooves 9 are formed do not have to be orthogonal, and may be oblique.

  In the case of using a base substrate on which a two-dimensional periodic structure is formed, the influence on the fluorescence intensity due to the arrangement of the microplate can be reduced even when polarized incident light is used. In addition, when unpolarized light is used, the utilization efficiency of incident light is increased and brighter fluorescence can be observed.

  The microplate according to the present invention used for the fluorescent microplate reader is arranged in an array on the surface of the base substrate 5 as shown in FIG. 7 (the upper side is a longitudinal sectional view and the lower side is a plan view). After forming the periodic structure in the plurality of regions 11, the metal layer 6 and the quenching suppression layer 7 may be sequentially formed on the base substrate 5. At this time, it is desirable to form grooves having a periodic structure in the same direction in all regions 11. When observing with a fluorescent microplate reader, a small amount of sample is spotted on the plurality of regions 11 and observed.

  In FIG. 7, the metal layer 6 and the quenching suppression layer 7 are formed on the entire surface of the base substrate 5. However, the present invention is not limited to this, and at least the metal layer 6 and the region 11 where the periodic structure is formed and It is sufficient that the quenching suppression layer 7 is formed. Further, the two-dimensional periodic structure shown in FIG. 6 may be formed in each region 11.

  Hereinafter, the features of the present invention will be further clarified by showing examples.

A glass substrate is used as a base substrate, and a grating having a periodic structure (period: 480 nm, depth: 31 nm, M: 40% (see FIG. 10, details will be described later)) of the order of the observation wavelength or less is formed on the surface. Furthermore, an adhesive layer (Cr, film thickness 0.3 nm), a metal thin film (silver, film thickness 100 nm), an adhesion and antioxidant layer (Cr, film thickness 1 nm), and a quenching suppression layer (SiO ₂ , film thickness 20 nm) ) By a sputtering method to produce a microplate. As a control (comparative control), a film was similarly formed on a slide glass having no periodic structure.

  FIG. 8 shows the result of the reflectance (that is, observed by epi-illumination) of the microplate observed when p-polarized incident light having a wavelength of 632.8 nm is incident from the base substrate side or the quenching suppression layer side. The relationship between the polarization plane of incident light and the grating direction V1 is parallel. In FIG. 8, the reflectance curve due to incidence from the base substrate side is drawn with a dotted line, and the reflectance curve due to incidence from the quenching suppression layer side is drawn with a solid line.

  From the graph of FIG. 8, it can be seen that the generation of surface plasmon resonance light can be observed both in the incidence from the base substrate side of the periodic structure substrate and in the incidence from the quenching suppression layer side. In FIG. 8, when ψ = 0 (degrees) and φ = 22 (degrees) when incident from the base substrate side, surface plasmon resonance light is most strongly generated, and when incident from the quenching suppression layer side, Ψ = 0 (degrees), φ = 16 (degrees), surface plasmon resonance light is most strongly generated (Ψ is an angle formed by the plane of polarization of incident light and the direction of the grating, as shown in FIG. ). Therefore, it is suggested that sufficiently bright fluorescence observation is possible even with an objective lens having a small NA. On the other hand, such surface plasmon resonance light can be efficiently generated not only in the case of p-polarized incident light but also in the case of incidence using light rotated by an angle Ψ. The substrates without the control periodic structure all showed a reflectivity of 80% or more regardless of the incident angle (thus, it was difficult to observe surface plasmon resonance light).

  Therefore, from the result of FIG. 8, it was found that the surface plasmon resonance field can be generated regardless of which side of the microplate is incident. Actually, it is desirable to determine the optimum state by adjusting the arrangement of the microplate on which the sample is mounted and the angle of the incident light.

  Furthermore, the inventor of the present application has found that there is a substrate that does not generate a surface plasmon resonance field that causes an enhanced fluorescence effect even when a substrate having a similar periodic structure is used. In this regard, the experimental results were compared with the simulation results, and it was found that the duty ratio of the periodic structure, the depth of the groove, and the shape were important as conditions for generating surface plasmon resonance. The desired range of these conditions could be determined by simulation. This will be specifically described below.

(1) First, by using the following two base substrates (SiO ₂ ) having a similar periodic structure, a metal layer and a quenching suppression layer are prepared on the periodic structure of the base substrate by sputtering, and a microplate is formed. Produced. Specifically, a silver thin film was formed on the periodic structure with a thickness of about 200 nm, and further SiO ₂ was formed thereon as a quenching suppression layer with a thickness of about 20 nm. Then, using these microplates, surface plasmon resonance was observed in the same manner as in Example 1.
Substrate 1: Period is 480 nm, groove depth is 31 nm, duty ratio is 0.4
Substrate 2: Period is 480 nm, groove depth is 39 nm, duty ratio is 0.54
Here, the periodic structure on the surface of the base substrate has the rectangular shape shown in FIG. The duty ratio is M / (M + V) where the ratio of the length of the convex portion to the period is M% and the ratio of the length of the concave portion to the period is V%.

  The observation results are shown in FIG. (A) and (b) of FIG. 11 are observation results regarding the substrate 1 and the substrate 2, respectively. As can be seen from FIG. 11, surface plasmon resonance was observed on the substrate 1, but surface plasmon resonance was not observed on the substrate 2.

  When the substrates 1 and 2 are examined with an atomic force microscope (AFM), the base substrate surface has a rectangular periodic structure, but the surface of the metal layer formed thereon has a periodic structure as shown in FIG. It was found that the portion corresponding to the step portion of the slope is inclined (hereinafter, this portion is referred to as a slope). In FIG. 12, the quenching suppression layer on the metal layer is omitted. The ratio of the peak (M%), valley (V%), and slope (SL%) of the periodic structure is V: M: SL = 33: 39: 14 for the substrate 1 and V: M: for the substrate 2. SL = 24: 60: 8.

Therefore, a simulation by a well-known exact coupled wave analysis (RCWA) was performed. The result is shown in FIG. FIGS. 13A and 13B are simulation results for the substrates 1 and 2, respectively, and it can be seen that the experimental data of FIGS. 11A and 11B can be reproduced. Note that, unlike an actual microplate, the simulation was performed under the condition that SiO ₂ formed on the silver thin film exists only in the flat peaks and valleys of the silver thin film and does not exist on the slope.

  (2) Next, in the shape shown in FIG. 12, the ratio of the peak (M%), valley (V%), and slope (SL%) of the periodic structure is changed, and the simulation is performed by rigorous coupled wave analysis (RCWA). Went.

As a result, the following was found.
i) In a periodic structure having a slope with a substantially vertical surface (tilt angle α is 90 degrees) as shown in FIG. 14, surface plasmon resonance depends sensitively on the duty ratio, and conditions for generating a plasmon resonance field are limited. However, in the structure having a slope (height of 10 to 50 nm and SL = 40 to 100 nm, that is, the inclination angle α is 6 to 50 degrees) as shown in FIG. 12, the dependence of the surface plasmon resonance generation on the duty ratio It was found that plasmon resonance does not occur under some limited conditions. In particular, the gentler the slope slope, the smaller the dependence of the surface plasmon resonance on the duty ratio.
ii) Dependence of the surface plasmon resonance on the depth of the groove is reduced by providing a structure having a slope on the surface of the metal layer.
iii) Surface plasmon resonance is not very dependent on the period.

The specific conditions when the thickness of the metal layer (particularly the silver thin film) is about 200 nm are specifically shown as follows.
When the metal layer surface has no slope and a vertical step is formed (α = 90 °), the depth of the recess of the base substrate is d (nm),
d = 10 ± 5 (5 or more and less than 15) and 15 ≦ M <45, 45 <M <85, or 85 <M <100,
d = 20 ± 5 (15 or more and less than 25), and 10 ≦ M <100,
d = 30 ± 5 (25 or more and less than 35), and 10 ≦ M ≦ 40, 60 ≦ M ≦ 70, or 75 ≦ M ≦ 95,
d = 40 ± 5 (35 or more and less than 45) and 0 <M ≦ 40, 45 ≦ M ≦ 55, or 60 ≦ M ≦ 70
It is.
When the surface of the metal layer has a slope (α <90 °) and SL = 10 ± 5 (the base substrate is α = 90 °), the depth of the concave portion of the base substrate is d (nm),
d = 10 ± 5 (5 nm or more and less than 15 nm, that is, α is about 12 degrees), and 0 <M <70, or 70 <M <100
d = 20 ± 5 (15 nm or more and less than 25 nm, that is, α is about 23 degrees), and 0 <M <80, or 80 <M <100
d = 30 ± 5 (25 nm or more and less than 35 nm, that is, α is about 32 degrees), and 0 <M <60, or 60 <M <100
d = 30 ± 5 (35 nm or more and less than 55 nm, that is, α is about 40 degrees), and 0 <M <60, or 60 <M <100
It is.

Adhesive layer (Cr, film thickness 0.3 nm), metal thin film (silver, film thickness 100 nm), adhesion and oxidation prevention on glass with periodic structure (period: 480 nm, depth: 31 nm, M: 40%) A layer (Cr, film thickness 1 nm) and a quenching suppression layer (SiO ₂ , film thickness 20 nm) were formed, and the surface of the quenching suppression layer was aminated with 1% aminopropyltriethoxysilane. As a sample, biotinylated microbeads bound with Cy5 fluorescently labeled protein (streptavidin) were adsorbed on a substrate, and fluorescence was observed with a transmission upright microscope. Further, DiI fluorescently labeled cells were adsorbed on a substrate, and fluorescence was observed with an episcopic inverted microscope. In both fluorescence observations, halogen lamps were used. In the former, a Cy5 filter was inserted on the light emission side and a 633 nm interference filter was inserted on the incident side, and a Cy3 filter cube was used in the latter.

  The observation results are shown in FIG. FIG. 15A is a fluorescence image of a bead observed with a transmission upright microscope, and FIG. 15B is a fluorescence image of a cell with an episcopic inverted microscope. Thus, enhanced fluorescence from the cells adsorbed on the periodic structure microplate was observed.

In Example 3, a metal film or the like was formed on the periodic structure substrate, and the surface thereof was aminated and used as a microscope observation plate. This is a substrate surface treatment for facilitating protein adsorption. . Here, a gold film with a film thickness of 48 nm is formed on a high refractive index glass substrate called LaSFN9 (Herma, Germany), and several μM of the following six kinds of thiol compounds having a terminal mercapto group (micro-molar (molar = mol / mol / L)) The surface of the quenching suppression layer was modified by immersing in an ethanol solution for 15 minutes to 1 hour and then washing. The thiol compounds are as follows.
(1) Mercapto-polyethylene glycol (terminal hydroxyl group)
(2) Mercapto-polyethylene glycol carboxylic acid (terminal carboxyl group)
(3) Mercapto-undecanol (terminal hydroxyl group)
(4) Mercapto-dodecane (terminal methyl group)
(5) Mercapto-aminoundecane (terminal amino group)
(6) Mercapto-biotinylated polyethylene glycol (terminal biotin)
Several μM protein streptavidin solution was injected on the surface, and the amount of adsorption was evaluated by the prism coupling surface plasmon resonance method (Kretschmann type) as shown in FIG. The result is shown in FIG.

  When the surface is a hydroxyl group or a carboxyl group, almost no protein is adsorbed, but the methyl group (alkylated surface) adsorbs more than 60% of the specific bond and more than 90% of the aminated surface. Therefore, it was found that the surface structure can be alkylated, preferably aminated, so that a plate capable of binding or adsorbing proteins and cells to be observed with higher density can be obtained.

A plastic substrate with a thickness of 1 mm is used as the base substrate, and a lattice having a periodic structure (period: 480 nm, depth: 30 nm, M: 45%) of the order of the observation wavelength or less is formed on the surface, and further bonded thereon. A microplate having a layer (Cr, film thickness 0.3 nm), a metal thin film (silver, film thickness 100 nm), an adhesion and antioxidant layer (Cr, film thickness 1 nm), and a quenching suppression layer (SiO ₂ , film thickness 20 nm) Was made.

  The surface of the quenching suppression layer is chemically treated with a 1% aminopropyltriethoxysilane aqueous solution (at room temperature for 40 minutes), and a biotinylated polyethylene glycol 2 mM phosphate buffer solution whose end is modified with an active esterified carboxyl group is added dropwise. It was incubated for 1 hour, reacted with amino groups, and washed with milliQ water.

  A cover glass with a diameter of 15 mm is attached to the surface of this biotinylated substrate with double-sided tape, and 20 μL (microliter) of 8 nM (nanomolar) fluorescently labeled protein (Cy5-streptavidin) phosphate buffer solution is injected to bind the protein to the surface. The apparatus was set in a device for measuring the reflectance and fluorescence intensity with respect to the incident angle without rinsing (cleaning) operation. In addition, as a control, 20 μL of 20 nM fluorescently labeled protein (Cy5-anti-GFP antibody) phosphate buffer aqueous solution is injected, the protein is non-specifically adsorbed, and the reflectance and fluorescence intensity with respect to the incident angle are similarly measured without rinsing. Set.

  A p-polarized light having a wavelength of 632.8 nm was incident from the base substrate side, and the reflectance and fluorescence intensity were measured with respect to the incident angle. The result is shown in FIG.

  As shown in FIG. 17A, 15 minutes after injecting the 8 nMCy5-streptavidin aqueous solution, a surface plasmon resonance angle (minimum point in the reflectance curve) observed at 22 degrees was observed. An enhanced fluorescence peak corresponding to the surface plasmon resonance angle was also observed. Therefore, it was confirmed that the plate was able to detect enhanced fluorescence from the fluorescent molecules adsorbed on the surface of the quenching suppression layer even when light was incident from the base substrate side.

  Further, as shown in FIG. 17B, when a control experiment was performed using a substrate having the same structure, an enhancement corresponding to the surface plasmon resonance angle was performed 15 minutes after injecting the 20 nMCy5-anti-GFP antibody aqueous solution. No fluorescence was seen.

  Therefore, by using this microplate, background light is suppressed in one step of sample injection without rinsing operation, and it is possible to distinguish non-specific adsorption from specific binding even at low concentration and high S / It was shown that the N ratio could be measured.

  The difference in the observed fluorescence intensity between the incident light direction and the fluorescence observation direction was verified. Specifically, as in Example 5, specific adsorption of 5nMCy5-streptavidin was measured from enhanced fluorescence by lattice coupling surface plasmon resonance without washing (rinsing). The result is shown in FIG.

  In FIG. 18, (a) is the result of light entering from the quenching suppression layer side and fluorescence is detected from the quenching suppression layer side, and (b) is the result of light entering from the base substrate side and the quenching suppression layer side. It is the result of having detected fluorescence from. In (a), the fluorescence from unadsorbed fluorescent molecules present in the solution accounts for 30% or more as background light, but in (b), the influence of background light is less than 3%. Thus, since background light is suppressed by irradiation from the base substrate side, S / N in fluorescence observation can be greatly improved compared to irradiation from the quenching suppression layer side. Therefore, a microplate that does not require a rinsing operation can be realized by making light incident from the base substrate side.

1 Surface plasmon excitation enhanced fluorescence microscope (SPFM)
2 Main body 3 Stage 4 Microplate 5, 5 ′ Base substrate 6 Metal layer 7 Quenching suppression layer 8 Periodic structure 9 Groove 10 Protruding portion IN1 Light incident from the quenching suppression layer side IN2 Light incident from the base substrate side T Quenching suppression Detection of fluorescence from the layer side B Detection of fluorescence from the base substrate side Sample Li Illumination light Lp Observation light (plasmon excitation enhanced fluorescence)
L1, L2 Incident light V1 Lattice directions V2, V3 P-polarized component of incident light V4 S-polarized component of incident light φ Incident angles U1, U2 Incident plane

Claims (12)

A method of detecting a specific antigen-antibody reaction with enhanced fluorescence using a fluorescently labeled secondary antibody using a microplate in a fluorescence microscope or a fluorescence microplate reader,
The microplate is
A base substrate having a periodic structure on the surface;
A non-translucent metal layer formed on the periodic structure;
A quenching suppression layer formed on the metal layer,
The periodic structure includes a plurality of grooves formed along one direction, the metal layer has a slope on the surface, and the metal layer is formed of a metal capable of generating a propagation type surface plasmon resonance light, The antibody is adsorbed or bound to the surface of the quenching suppression layer,
The method comprises
Causing light to enter the microplate to generate an electric field enhanced by the surface plasmon resonance light; and
Using the generated electric field as an excitation field of fluorescent molecules, and detecting the enhanced fluorescence from either the base substrate side or the quenching suppression layer side,
The angle formed by the incident direction of the light incident from the base substrate side and the perpendicular of the plane on which the light is incident on the base substrate is equal to the microplate even if the light is not bent by the bending means. A detection method characterized by being an incident angle and an angle that is small enough to generate the surface plasmon resonance light and provide the enhanced fluorescence . 2. The detection according to claim 1 , wherein the angle formed by the incident direction of the light and a perpendicular of a plane on which the light of the base substrate is incident is within a range of 5 degrees to 46 degrees. Method. 3. The detection according to claim 2 , wherein the angle formed by the incident direction of the light and the perpendicular of the plane on which the light of the base substrate is incident is within a range of 5 degrees to 25 degrees. Method.   The period of the periodic structure is 10 nm or more and 1 μm or less,
  The periodic structure on the surface of the base substrate is a rectangular periodic structure;
  The metal layer has the slope on the surface corresponding to the rectangular step portion, and is formed to a thickness of 200 nm;
  The ratio of the length of the slope to the period is 10 ± 5%;
  The depth of the groove is dnm, and the ratio of the peak length to the period is M%.
  5 ≦ d <15, and 0 <M <70, or 70 <M <100,
  15 ≦ d <25 and 0 <M <80, or 80 <M <100,
  25 ≦ d <35 and 0 <M <60, or 60 <M <100, or
  35 ≦ d <45 and 0 <M <60, or 60 <M <100
  The detection method according to claim 1, wherein: A fluorescence microscope equipped with a sample to be observed and having a microplate for detecting a specific antigen-antibody reaction by enhanced fluorescence using a fluorescently labeled secondary antibody,
The microplate includes a base substrate having a periodic structure on a surface thereof, a non-translucent metal layer formed on the periodic structure of the base substrate, and a quenching suppression layer formed on the metal layer. The periodic structure includes a plurality of grooves formed along one direction, the metal layer has a slope on the surface, and the metal layer is a metal that can generate a propagation type surface plasmon resonance light. Formed, the antibody is adsorbed or bound to the surface of the quenching suppression layer,
The fluorescence microscope is
Injecting light into the microplate to generate an electric field enhanced by the surface plasmon resonance light, and using the generated electric field as an excitation field of a fluorescent molecule, the enhanced fluorescence is transmitted to the base substrate side or the by a method comprising the steps of detecting from one of the quenching suppressing layer side, detects the enhanced fluorescence from the microplate,
The angle formed by the incident direction of the light incident from the base substrate side and the perpendicular of the plane on which the light is incident on the base substrate is equal to the microplate even if the light is not bent by the bending means. A surface plasmon excitation-enhanced fluorescence microscope characterized in that the angle is an incident angle and the angle is small enough to generate the surface plasmon resonance light and provide the enhanced fluorescence. 6. The surface according to claim 5 , wherein the angle formed by the incident direction of the light and a perpendicular of a plane on which the light of the base substrate is incident is in a range of 5 degrees to 46 degrees. Plasmon excitation enhanced fluorescence microscope. The surface according to claim 6 , wherein the angle formed by the incident direction of the light and the perpendicular of the plane on which the light of the base substrate is incident is in the range of 5 degrees to 25 degrees. Plasmon excitation enhanced fluorescence microscope.   The period of the periodic structure is 10 nm or more and 1 μm or less,
  The periodic structure on the surface of the base substrate is a rectangular periodic structure;
  The metal layer has the slope on the surface corresponding to the rectangular step portion, and is formed to a thickness of 200 nm;
  The ratio of the length of the slope to the period is 10 ± 5%;
  The depth of the groove is dnm, and the ratio of the peak length to the period is M%.
  5 ≦ d <15, and 0 <M <70, or 70 <M <100,
  15 ≦ d <25 and 0 <M <80, or 80 <M <100,
  25 ≦ d <35 and 0 <M <60, or 60 <M <100, or
  35 ≦ d <45 and 0 <M <60, or 60 <M <100
  The surface plasmon excitation enhanced fluorescence microscope according to claim 5, wherein A fluorescence microplate reader equipped with a sample to be observed and having a microplate for detecting a specific antigen-antibody reaction by enhanced fluorescence using a fluorescently labeled secondary antibody,
The microplate includes a base substrate having a periodic structure on a surface thereof, a non-translucent metal layer formed on the periodic structure of the base substrate, and a quenching suppression layer formed on the metal layer. The periodic structure includes a plurality of grooves formed along one direction, the metal layer has a slope on the surface, and the metal layer is a metal that can generate a propagation type surface plasmon resonance light. Formed, the antibody is adsorbed or bound to the surface of the quenching suppression layer,
The fluorescent microplate reader is
Injecting light into the microplate to generate an electric field enhanced by the surface plasmon resonance light, and using the generated electric field as an excitation field of a fluorescent molecule, the enhanced fluorescence is transmitted to the base substrate side or the by a method comprising the steps of detecting from one of the quenching suppressing layer side, detects the enhanced fluorescence from the microplate,
The angle formed by the incident direction of the light incident from the base substrate side and the perpendicular of the plane on which the light is incident on the base substrate is equal to the microplate even if the light is not bent by the bending means. A surface plasmon excitation-enhanced fluorescence microplate reader having an incident angle and an angle small enough to generate the surface plasmon resonance light and provide the enhanced fluorescence. 10. The surface according to claim 9 , wherein the angle formed by the incident direction of the light and a perpendicular of a plane on which the light of the base substrate is incident is within a range of 5 degrees to 46 degrees. Plasmon excitation enhanced fluorescence microscope. 11. The surface according to claim 10 , wherein the angle formed by the incident direction of the light and a perpendicular of the plane on which the light of the base substrate is incident is within a range of 5 degrees to 25 degrees. Plasmon excitation enhanced fluorescence microplate reader.   The period of the periodic structure is 10 nm or more and 1 μm or less,
  The periodic structure on the surface of the base substrate is a rectangular periodic structure;
  The metal layer has the slope on the surface corresponding to the rectangular step portion, and is formed to a thickness of 200 nm;
  The ratio of the length of the slope to the period is 10 ± 5%;
  The depth of the groove is dnm, and the ratio of the peak length to the period is M%.
  5 ≦ d <15, and 0 <M <70, or 70 <M <100,
  15 ≦ d <25 and 0 <M <80, or 80 <M <100,
  25 ≦ d <35 and 0 <M <60, or 60 <M <100, or
  35 ≦ d <45 and 0 <M <60, or 60 <M <100
  10. The surface plasmon excitation enhanced fluorescence microplate reader according to claim 9.