JP5110254B2 - Fluorescence measurement method, measurement chip for fluorescence measurement, and manufacturing method thereof - Google Patents

Description

  The present invention relates to a fluorescence measurement method in which fluorescence intensity is enhanced by a surface enhancement effect, a measurement chip for use in this fluorescence measurement method, and a method for manufacturing the same.

  Conventionally, a fluorescence measurement method is known as one of methods for quantifying a measurement target substance such as DNA or protein contained in a sample. This fluorescence measurement method is a kind of immunological detection method using an antigen-antibody reaction, and is performed using a measurement chip. This measurement chip is formed by arranging an antibody in each of a large number of minute reaction areas formed on a glass substrate. When a sample is added to the surface of this measurement chip, the antibody arranged in each reaction area Supplements the substance to be measured in the sample. Subsequently, when an antibody labeled with a fluorescent dye is added to the surface of the measurement chip, the antibody binds to the measurement target substance captured on the surface of the measurement chip, and the measurement target substance is interposed between the pair of antibodies. To form a sandwich bond. Thereafter, the measurement chip is washed to wash away unbound samples and antibodies, and the measurement chip is irradiated with excitation light such as laser light, whereby fluorescence is generated from the fluorescent dye by excitation of the excitation light. Then, the fluorescence is separated from the excitation light by a spectroscopic element, and the signal intensity (fluorescence signal intensity) of the separated fluorescence is measured with a fluorescence scanner or a fluorescence microscope, thereby quantifying the measurement target substance contained in the sample. And so on.

  In such a fluorescence measurement method, a phenomenon (surface enhancement effect) is known in which the fluorescence intensity of a fluorescent dye is amplified on a noble metal substrate having an uneven structure. The principle of this surface enhancement effect will be explained in more detail. When light is applied to a particle having a diameter much smaller than the wavelength of light, scattered light is generated, and an electromagnetic field localized in the vicinity of the particle (near-field light) Occurs. Both the scattered light and the near-field light are electromagnetic fields generated by the electric dipoles induced inside the sphere when the incident light hits the sphere, but the scattered light travels far away. On the other hand, the energy of the near-field light concentrates along the spherical shape and cannot be observed at a position away from the sphere. Moreover, the near-field light is distributed from the particle at a distance that is about the diameter of the particle. The excitation light is amplified by the near-field light generated in the vicinity of the noble metal (silver) particles, the number of fluorescent molecules that absorb the light increases, and the return from the excited state to the ground state is quick, and it is easy to be excited ( It is believed that the fluorescence intensity increases. According to the surface enhancement effect based on such a principle, by forming fine irregularities made of noble metal on the surface of the glass substrate on which the measurement chip is formed, and performing a fluorescence measurement method using this measurement chip, It becomes possible to perform a measurement with higher sensitivity. For example, even if the number of fluorescent dyes is relatively small, it is possible to quantify the measurement target substance contained in the sample.

  The concavo-convex structure formed on the measurement chip to obtain such a surface enhancement effect ideally has a uniform concavo-convex structure density, high concavo-convex uniformity, and reproduction of the same concavo-convex structure. It is preferable that the structure is high, the uneven structure can be easily manufactured, and the uneven structure has a nano size of 40 to 100 nm. As a method of forming a concavo-convex structure that satisfies such various conditions, a method of arranging nano-sized fine particles made of noble metal (for example, silver) on the surface of a glass substrate has been conventionally proposed.

  For example, Non-Patent Document 1 and Non-Patent Document 2 disclose that a silver island film (SIF: Silver island film) is disposed on a glass substrate. This silver island film is arranged on a glass substrate by, for example, reducing silver ions in a solution. That is, in this technique, nano-sized fine particles are formed of silver, and the silver fine particles are arranged on a glass substrate in a state of being spaced apart from each other (not in contact with each other).

  Further, Patent Document 1 discloses a technique in which a surface enhancement effect is applied to surface plasmon resonance (SPR: Surface Plasmon Resonance). Here, surface plasmon resonance is a phenomenon in which, when light of a specific wavelength is irradiated on the surface of a metal film while changing the incident angle, the energy of the photon is absorbed and not reflected by the specific incident angle. This incident angle is called a resonance angle, and this resonance angle changes according to the surface density (mass) of the metal film. By measuring the change in the resonance angle, the surface density of the metal film can be specified. it can. The principle of the surface plasmon resonance will be described in more detail. Irradiation light on the back surface of the thin metal film is totally reflected and at the same time generates a weak energy wave (evanescent wave) on the metal film side. In addition, a close-packed wave (surface plasmon) is generated on the metal surface in contact with the dielectric, and when the wave numbers of the two coincide, the reflected light attenuates (surface plasmon resonance phenomenon). The dielectric constant (refractive index squared) affects the evanescent wave, so the interaction between the materials caused on the metal surface causes a difference in the dielectric constant, which affects the surface plasmon and can be seen as a change in resonance. it can. This Patent Document 1 discloses that polymer fine particles having a particle diameter of 5 nm to 100 μm are solid-phased on a substrate.

  Alternatively, Non-Patent Document 3 describes a technique related to surface-enhanced Raman spectroscopy (SERS), and silver particles are produced by vapor-depositing silver on fine particles solid-phased on a substrate. The point is disclosed.

"Evgenia G. Matveeva, et al.," Myoglobin immunoassay based on metallic particulate-enhanced flu, United States Journal of Immunology, United States Journal of Immunology. (Journal of Immunological Methods), June 6, 2005, p. 26-35 Joseph R. Lakowicz et al., “Effects of Silver Island Intensiveness, Fluorescence Intensity, Refractory Intensity, Fluorescence Intensity, Fluorescence Intensity, Refractory Intensity, Fluorescence Intensity, Fluorescence Intensity, and Intensity. , "USA, 301, Analytical Biochemistry, January 15, 2002, P.A. 261-277 Tuan Vo-Dinh, “Surface-enhanced Raman spectroscopy using metal nanostructures”, USA, No. 17, Trends of Analytical Chemistry August 9, 1998, p. 557-582 JP 2000-55920 A

  However, the conventional measurement method in which only fine particles are arranged in a non-contact manner on the substrate as described in Patent Document 1 and Non-Patent Documents 1 to 3 can obtain only an enhancement factor of about 10 times. Thus, a more sensitive surface enhancement method has been desired. In addition, with such a conventional surface enhancement effect, no surface enhancement effect has been confirmed for bright fluorescent dyes (fluorescent dyes with high quantum yield), and conversely, the fluorescence intensity is due to the quenching effect of the metal. Had fallen. This means that it is difficult to effectively use bright fluorescent dyes that are important from a practical point of view, and there is a demand for a method that can enhance sensitivity even when bright fluorescent dyes are used. It was. Furthermore, the self-fluorescence of the substrate becomes strong due to the concavo-convex structure, and this auto-fluorescence becomes noise against the fluorescence from the fluorescent dye, so there is a problem that the S / N ratio (Signal / Noise Ratio) in the fluorescence intensity measurement is low. .

  In particular, the surface plasmon resonance described in Patent Document 1 and the surface enhanced Raman spectroscopy described in Non-Patent Document 3 are based on a principle different from fluorescence measurement. The technology cannot be applied immediately to fluorescence measurements, or the characteristics will be quite different when applied. Specifically, surface plasmon resonance is a technique that does not use a label such as a fluorescent dye, and at least in this respect, is different from the above-described fluorescence measurement. Further, the surface-enhanced Raman spectroscopy is a scattering phenomenon, not a fluorescence phenomenon, and differs from the above-described fluorescence measurement at least in this respect. Therefore, the simple prediction that the same excellent characteristics can be obtained even if the measurement method and the measurement chip having excellent characteristics in the surface plasmon resonance and the surface enhanced Raman spectroscopy are directly used for the fluorescence measurement is completely established. First, it is necessary to search for a measurement method and a measurement chip that can obtain desired characteristics in fluorescence measurement. For example, up to five orders of magnitude have been routinely observed so far in Raman spectroscopy, whereas only about 10-fold enhancement has been observed in fluorescence.

  In order to solve such problems, the present invention can improve the enhancement rate of the surface enhancement effect and improve the S / N ratio and the like, and a measurement for use in this fluorescence measurement method. It is an object to provide a chip for use and a method for manufacturing the same.

In order to solve the above-described problems and achieve the object, the present invention according to claim 1, after disposing a porous film on a substrate, at least silver on the surface of the porous film opposite to the substrate. A step of manufacturing a measurement chip by disposing a noble metal layer containing, a step of disposing a binding substance that specifically binds to a substance to be measured in the noble metal layer of the measurement chip, and the measurement Binding a measurement target substance to the binding substance of the chip, binding a binding substance labeled with a fluorescent dye to the measurement target substance bound to the binding substance of the measurement chip, and observing the fluorescent dye And a step of performing.

Further, in the present invention according to claim 2, after the porous film is arranged on the substrate, the measurement is performed by arranging a noble metal layer containing at least silver on the surface of the porous film opposite to the substrate. A step of producing a measurement chip, a step of arranging a measurement target substance on the noble metal layer of the measurement chip, and a binding substance that specifically binds to the measurement target substance of the measurement chip, the fluorescent dye The method includes a step of binding the binding substance labeled with the substance to be measured and a step of observing the fluorescent dye.

The present invention described in claim 3 is characterized in that, in the present invention described in claim 1 or 2, the binding substance is an antigen or an antibody.

Moreover, the present invention according to claim 4 is the present invention according to any one of claims 1 to 3, wherein between the step of manufacturing the measuring chip and the step of observing the fluorescent dye. And a step of reacting the measuring chip with at least one selected from the group consisting of BSA, skim milk, or casein.

The fluorescence measurement method according to claim 1 can improve the fluorescence signal intensity by increasing the surface enhancement effect by executing the fluorescence measurement method, and is several tens higher than the conventional enhancement rate. Double fluorescence signal intensity can be obtained. Further, even in a fluorescent substance having a high quantum yield (bright fluorescent substance), a fluorescent signal intensity that is several tens of times higher than that of the conventional fluorescent substance can be obtained, so that the practicality of the bright fluorescent substance can be improved. Furthermore, since a high fluorescence signal enhancement effect is exhibited even with weak excitation light, autofluorescence can be suppressed and the S / N ratio of the fluorescence signal can be improved. Furthermore, the measurement chip can be applied to almost all analysis methods (plate assays, biochips, DNA chips, etc., nanosensors) that detect fluorescent signals from the substrate, and improve the sensitivity in each analysis method. Can do.

Further, in the fluorescence measurement method according to claim 2 , a sample (which may include cells) whose measurement object is unknown is previously coupled or approached to the measurement chip, and the measurement object contained in the sample is in case of detecting in several fluorescently labeled binding substance (e.g. an antibody), it is possible to obtain the same effect as the first aspect.

  Hereinafter, with reference to the accompanying drawings, a fluorescence measurement method according to each embodiment of the present invention will be described in detail. [I] First, the basic concept common to each embodiment was explained, then [II] the specific contents of each embodiment were explained, and [III] Finally, modifications to each embodiment were explained. explain. However, the present invention is not limited to each embodiment.

[I] Basic concept common to the embodiments First, the basic concept common to the embodiments will be described. Each embodiment relates to a fluorescence measurement method, and this fluorescence measurement method is performed by the same procedure and apparatus as in the prior art, except as noted. Here, one of the features of the present embodiment is the structure of the measuring chip and the manufacturing method thereof. That is, conventionally, when nano-sized fine particles are arranged on the surface of the glass substrate in order to obtain a surface enhancement effect, the fine particles formed of a noble metal (for example, silver) are simply arranged on the surface of the glass substrate. In addition, conventionally, it was thought that high-sensitivity Raman measurement could not be performed unless there was a gap between the fine particles, and it was technically difficult to arrange the fine particles closely. For some reason, a certain amount of space is provided between the fine particles. Conventionally, however, various problems have arisen such that, even when such an interval is provided, only an enhancement factor of about 10 times can be obtained as described above.

  In contrast, in the present embodiment, a concavo-convex structure is formed by disposing a porous film on the substrate instead of disposing silver fine particles in a non-contact manner on the substrate, and the surface of the concavo-convex structure is further formed. A multilayer concavo-convex structure in which a noble metal film is arranged is adopted. According to such a novel structure, it is possible to form a concavo-convex configuration similar to a case where most of the fine particles are slightly in contact with each other, without providing a space between the fine particles, Various advantages can be obtained as compared with the prior art, and the uneven structure can be easily and reliably formed.

  Furthermore, one of the other characteristics of the present embodiment is to devise a specific structure (material, film thickness, porosity, etc.) of the porous film and a specific structure (thickness, etc.) of the noble metal film. As a result, it is possible to achieve further excellent effects. This point will be described later.

[II] Specific Contents of Each Embodiment Next, specific contents of each embodiment will be described.

[Embodiment 1]
First, the first embodiment will be described. In this form, a measurement chip is configured by further arranging a noble metal layer containing silver on a porous film disposed on a substrate. Below, a structure, a manufacturing method, a usage method, and an effect regarding the measuring chip according to the first embodiment will be sequentially described.

(Configuration of measuring chip)
1 is a partially enlarged perspective view of a measurement chip according to Embodiment 1, and FIG. 2 is a partially enlarged side view of the measurement chip according to Embodiment 1. FIG. The measuring chip 1 is generally configured by sequentially arranging a base layer 3, a porous film 4, and a noble metal layer 5 on one side of a substrate 2.

  The substrate 2 is a basic structure of the measuring chip 1, and is formed in a flat plate shape whose planar shape is substantially rectangular, for example, of glass, plastic, or metal.

  The base layer 3 is used to dispose the porous film 4 on the substrate 2 and is fixed to the side surface of the substrate 2 by a method such as vapor deposition or sputtering. The material and thickness of the base layer 3 are basically arbitrary, but in order to adsorb fine particles 4a described later at a high density in a wide range, it is preferable to form a thin film with, for example, gold or silicon. The film thickness is, for example, about 10 to 50 nm, and more preferably about 20 nm. When such a high-density adsorption action is not required, the base layer 3 can be omitted, and the porous film 4 can be disposed directly on the side surface of the substrate 2. When formed of silicon, the base layer 3 may be omitted and the porous film 4 may be directly formed on one side surface of the substrate 2.

  The porous film 4 is a film-like body (for example, a thin film body) for forming an uneven structure, which is one of the conditions for obtaining the surface enhancement effect. Here, as a formation method of the porous membrane 4, at least two methods can be mentioned. The first forming method is a method in which after a smooth film body is formed, a large number of holes are formed in the film body. The second forming method is a method in which a large number of fine particles 4 a are formed and then arranged on the substrate 2 or the base layer 3 at a high density. Hereinafter, in the present embodiment, a case where the second forming method is employed will be described.

In this way, when the porous film 4 is formed from the fine particles 4a, the material of the fine particles 4a is arbitrary. For example, polystyrene, silicon oxide (SiO ₂ ), natural rubber or synthetic rubber (a Generally, what is called latex can be used. However, in addition to this, as long as the fine particles 4a can be adsorbed on gold or silicon contained in the base layer 3, the fine particles 4a can be made of any material.

  The noble metal layer 5 is for improving the surface enhancement effect. Conventionally, gold has been used in SPR to avoid the adverse effects of silver oxidation, but in the present embodiment, a high fluorescence enhancement effect is obtained by forming the noble metal layer 5 from silver. FIG. 3 is a graph showing changes in fluorescence signal intensity depending on the material of the noble metal layer 5, the vertical axis is the fluorescence signal intensity, the horizontal axis is the concentration of fluorescent protein (RPE: R-phycoerythrin), and each plot in the graph. Is a substrate (when a fluorescent material is directly adsorbed without forming a porous film on the substrate 2), gold (when the noble metal layer 5 is formed of gold), silver (the noble metal layer 5 is formed of silver) ), Respectively. Here, the porous film 4 was formed with fine particles 4a having a particle diameter of 100 nm, and the noble metal layer 5 was formed with a thickness of 10 nm. As is apparent from FIG. 3, when the noble metal layer 5 is formed of silver, the fluorescence signal intensity is dramatically increased as compared with other cases. Therefore, the noble metal layer 5 is formed of silver. Is useful for improving the surface enhancement effect. Below, the case where the noble metal layer 5 is formed with silver is demonstrated.

  Next, the configuration of such a measuring chip 1 will be described in more detail. First, a suitable numerical range of the film thickness of the noble metal layer 5 will be described. Each of FIGS. 4 to 8 is a graph showing the correlation between the fluorescence signal intensity and the film thickness of the noble metal layer 5. 4 to 8 show the case where the particle size of the fine particles 4a forming the porous film 4 is set to 50, 100, 200, 300, and 500 nm sequentially, and in each figure, the vertical axis represents the fluorescence signal intensity. The horizontal axis represents the film thickness of the noble metal layer 5, and each plot in the graph represents the case where the concentration of the fluorescent dye is 0, 80, 400, or 2,000 ng / ml. For example, FIG. 4 shows a case where the particle diameter of the fine particles 4a is 50 nm. When the concentration of the fluorescent dye is 2,000 ng / ml, the fluorescence signal is obtained when the noble metal layer 5 has a thickness of 10 nm. The intensity is about 60,000. The particle diameters 50, 100, 200, 300, and 500 nm of the fine particles 4a shown here are approximate values, and the exact particle diameters of the fine particles 4a actually used in the experiment (the particle diameter of each particle used is a normal distribution). (The most common particle diameters) are 50 nm, 108 nm, 202 nm, and 356 nm, respectively. In addition, it is difficult to adjust the particle diameters of all the particles accurately, and some errors may occur.

  Here, as shown in FIGS. 4 to 8, the fluorescence signal intensity is high when the film thickness of the noble metal layer 5 is about 5 to 20 nm in any case where the particle diameter of the fine particles 4a is 50 to 500 nm. In particular, when the film thickness of the noble metal layer 5 is about 10 to 15 nm, a remarkably high value (peak value) is shown. Accordingly, the thickness of the noble metal layer 5 is preferably at least about 5 to 20 nm, and more preferably about 10 to 15 nm.

  Next, a preferable numerical range of the film thickness of the porous film 4 (here, the particle diameter of the fine particles 4a forming the porous film 4) will be described. As shown in FIGS. 4 to 7 above, when the particle size of the fine particles 4a is 50 to 300 nm, a fluorescence signal intensity of 2,000 or more can be obtained when the film thickness of the noble metal layer 5 is in the range of 10 to 20 nm. However, as shown in FIG. 8, when the particle diameter of the fine particles 4a is 500 nm, only a fluorescent signal intensity of 2,000 or less can be obtained in the same range. For these reasons, it is desirable that the particle size of the fine particles 4a be at least about 300 nm (more precisely, 356 nm, that is, about 360 nm) or less. Furthermore, as shown in FIGS. 4 to 6, when the particle diameter of the fine particles 4 a is 50 to 200 nm, a fluorescence signal intensity of 10,000 or more can be obtained in the entire range of the film thickness of the noble metal layer 5. However, as shown in FIG. 7, when the particle diameter of the fine particles 4a is 300 nm, only a fluorescence signal intensity of 3,000 or less can be obtained. For these reasons, the particle size of the fine particles 4a is more preferably about 50 to 200 nm. Furthermore, when the particle size of the fine particles 4a shown in FIGS. 4 and 5 is 50 to 100 nm and when the particle size of the fine particles 4a shown in FIG. There is a difference in fluorescence signal intensity that is twice or more. For these reasons, the particle size of the fine particles 4a is more preferably about 50 to 100 nm. In addition, even when the particle size of the fine particles 4a is about 20 nm to 50 nm, there is a possibility that the same suitable effect may be obtained, but considering the ease of purchase of the fine particles 4a and the uniformity of the particle size, It is preferable to use fine particles 4a having a particle diameter of 50 to 100 nm.

Next, a preferable numerical range of the porosity of the porous film 4 will be described. Since the fine particles 4a forming the porous film 4 are excited by near-field light, it is preferable that the fine particles 4a are close to each other to some extent. For this reason, in conventional resonant plasmons, the closer the metal and biomolecule are to the surface, the greater the effect. However, when the fine particles 4a are too close to each other, energy transfer from the fluorescent dye to the metal surface occurs, and the fluorescence signal intensity decreases. In consideration of these matters, the inventors of the present application have found a porosity having a high excitation effect by near-field light and a small energy transfer as follows (note that the porosity is, for example, an area of 0. 0). When one fine particle having a diameter of 100 nm is arranged in a region of 01 μm ² , the porosity = (0.1 × 0.1−0.05 × 0.05 × circumferential ratio) /0.01×100 = 21.50%).

  FIG. 9 shows the relationship between the fluorescence signal intensity and the concentration of the fluorescent dye for various porosity ratios, (a) is a table showing experimental results, and (b) is a graph. In FIG. 9B, the vertical axis represents the fluorescence signal intensity, the horizontal axis represents the RPE concentration, and each plot in the graph represents the substrate (adsorbing the fluorescent material directly without forming a porous film on the substrate 2). ), A (porosity = 63.2%), B (porosity = 81.1%), C (porosity = 92.4%), D (porosity = 96.5) %). As is clear from this graph, the fluorescence signal intensities are in the order of porosity = 81.1, 63.2, 92.4, 96.5, 0% in descending order. Therefore, the porosity is preferably about 60 to 97%, and more preferably about 81.1% (for example, 75 to 85%).

(Method for manufacturing measurement chip 1)
The measurement chip 1 configured as described above can be manufactured, for example, by the following method. First, a salt solution such as EDC (1-Ethyl-3- [3-dimethylaminopropyl) carbohydrate) or NaCl is added to a fine particle solution (0.5 M or less, preferably 100 mM or less) to prepare a mixed solution. For example, the EDC solution 2 is added to the fine particle solution 1 solution. This mixed solution is applied to the surface of the substrate 2. At this time, when the substrate 2 is formed of glass or plastic, the base layer 3 is formed on the substrate 2 by vapor deposition or sputtering, and the mixed solution is applied to the surface of the base layer 3. Alternatively, when the substrate 2 is formed of a metal such as silicon, the mixed solution is applied to the surface of the substrate 2 without forming the base layer 3. Then, the substrate 2 is allowed to stand for several seconds or more, then washed with distilled water and dried, and the fine particles 4a are adsorbed on the surface of the substrate 2 at a single layer high density. Thereafter, the noble metal layer 5 is formed on the upper surfaces of the fine particles 4a by vapor deposition or sputtering, whereby the measuring chip 1 is completed. As described above, a specific method for adsorbing the fine particles 4a on the surface of the substrate 2 at a high density in a single layer is arbitrary.
H. Takei, “Surface-absorbed polymerized spheres as a template for surface-absorbing polystyrene spheres as a template for forming nano-sized metal spheres” properties of nanosized Au particle ", USA, B 17 (5), Vacuum Science and Technology (September / October 1999, P1906-1911)

Here, the specific method for forming the porous film 4 having the above-described preferable porosity is arbitrary, but for example, the porous film 4 can be produced by changing the EDC concentration. . That is, the EDC concentration is determined so that the fine particles 4a are bonded to the base layer 3 containing gold or silicon but are not bonded to the other fine particles 4a. On the other hand, when the porous film 4 is formed by perforating a large number of holes in a smooth film body, for example, the film body formed by a known method is subjected to nanoimprinting (hot embossing method). Multiple holes can be drilled. Alternatively, the following solvent-resistant membrane with pores is placed on the membrane, and HNO ₃ (dil or conc), HClO ₄ (dil or conc), H ₂ SO ₄ (conc), HCl (conc ) + HNO ₃ (conc) or other solvent may be used to dissolve the exposed silver film to provide a large number of holes.

(How to use the measuring chip 1)
The measuring chip 1 manufactured in this way is used for the fluorescence measuring method by the same method as the conventional one. FIG. 10 is a diagram conceptually showing a fluorescence measurement method using the measurement chip 1 according to the first embodiment, where (a) shows a state in which an antibody is arranged on the measurement chip 1, and (b) shows a sandwich. Shows the state that made up the bond. First, as shown in FIG. 10A, the antibody 10 is arranged on the noble metal layer 5 of the measurement chip 1. Then, as shown in FIG. 10 (b), the sample 13 is added to the surface of the measurement chip 1, whereby the measurement target substance 11 contained in the sample is supplemented with the antibody 10, and the antibody 13 labeled with the fluorescent dye 12 is further added. Is added to form a sandwich binding in which the substance to be measured 11 is bound between the antibodies 10 and 13. Thereafter, as before, after washing the unbound sample and the antibody 13, the excitation light is irradiated to generate fluorescence from the fluorescent dye 12, and the fluorescence intensity of this fluorescence is measured using a fluorescence scanner, a fluorescence microscope, etc. (preferably confocal). The measurement target substance 11 can be quantitatively measured by measuring using a scanner or a confocal microscope capable of measuring near-field. In the measurement chip 1, specificity due to the excitation wavelength is not recognized, and any fluorescent protein or fluorescent substance can be used. In this specification, “observing” includes the meaning of observing with a confocal microscope or the like and the meaning of measuring fluorescence intensity.

  Here, the antibody 13 is used, but any substance that can specifically bind to the substance to be measured may be used. For example, a receptor, DNA, peptide, or the like can be used in addition to the antibody 13 and the antigen. Further, in the claims, the “binding substance specifically binding to the substance to be measured” and the “binding substance labeled with a fluorescent dye” may be either the same binding substance or different binding substances, or It may be the same class or subclass. In addition, as the “binding substance labeled with a fluorescent dye”, one kind of substance or a plurality of kinds of substances may be used. Furthermore, when an antibody is used as the binding substance, those that bind to different antigenic determinants are preferable. In addition, the antigen and antibody used in this way can be obtained by a conventionally known method.

  Moreover, examples of the “measurement object” that can be measured in the present embodiment include an antigen or an antibody. More specifically, a protein, a peptide consisting of at least 4 amino acids, a hapten, a nucleic acid, a nucleotide, a hormone, an oligosaccharide, a polysaccharide, a natural or synthetic drug, a derivative or complex thereof, or an antibody to these, etc. Can do. Furthermore, cells or the like may be arranged on the chip, and proteins (such as receptors) on the cell membrane may be used as the measurement object. Here, the substance to be measured preferably has two or more types of antigenic determinants so that it can be separately bonded to the substance to be measured.

  In addition, fluorescent protein (RPE, etc.) and fluorescent substance (Alexa Fluor (registered trademark: manufactured by Molecular Probes), Cy3, Cy5, fluorescein, rhodamine, umbelliferone, phycoerythrin, etc.) are applied to the measuring chip 1. It can also act directly. In this case, a measurement chip 1 and a control substrate (plastic or glass slide, for example, a microarray slide manufactured by Nunc) are prepared, and an RPE solution (for example, 0) is applied to each surface of the measurement chip 1 and the control substrate. , 0.4, 2 pg / ml) is applied and reacted at room temperature for 30 minutes or more. Thereafter, the measurement chip 1 and the control substrate are washed, blocked with a buffer solution containing 1% BSA (Bovine Serum Albumin) (reaction at room temperature for 30 minutes or more), further washed and dried, Measurement is performed with a focal fluorescent scanner (for example, ScanArray Lite manufactured by PerkinElmer) (for example, excitation wavelength of 633 nm, laser power of 80%, PMT Gain of 80%). (Note that blocking by BSA will be described in detail in Embodiment 2).

  Alternatively, the fluorescent protein or fluorescent substance can be indirectly acted on the measuring chip 1 by labeling the fluorescent substance on the antibody, protein, DNA or the like. In this case, the measurement chip 1 and the control substrate are prepared in the same manner as described above, and 1 μl of the anti-IL-6 antibody solution (6 μg / ml) is reacted on each surface of the measurement chip 1 and the control substrate for 1 hour at room temperature. Let Thereafter, the measurement chip 1 and the control substrate are washed, blocked with a buffer solution containing 1% BSA (reacted at room temperature for 30 minutes or more), further washed, and then washed with an rIL-6 solution (for example, 0, 0). .8, 4, 20, 100, 500, 2500 pg / ml) is reacted at 1 μl at room temperature for 1 hour. Thereafter, the measurement chip 1 and the control substrate are washed again and reacted with 1 μl of an RPE-labeled anti-IL-6 antibody solution (1 μg / ml) at room temperature for 30 minutes. Finally, the measurement chip 1 and the control substrate are washed and dried, and measured with a fluorescence scanner (for example, excitation wavelength 543 nm, laser power 80%, PMT Gain 80%).

  Moreover, in the said usage method, the fluorescent substance made to act directly or indirectly is not specifically limited. Specific examples include the above-described fluorescent proteins (RPE, etc.) and fluorescent substances (Alexa, Cy3, Cy5, fluorescein, rhodamine, umbelliferone, phycoerythrin, etc.). As a method for labeling an antibody or the like with a fluorescent substance or the like in this manner, a conventionally known method in which the fluorescent substance or the like and the antibody or the like are bound via a covalent bond or a non-covalent bond can be exemplified. Furthermore, examples of the method for bonding via a covalent bond include the glutaraldehyde method, the periodic acid method, the maleimide method, the pyridyl disulfide method, and methods using various conventionally known crosslinking agents (for example, , "Protein Nucleic Acid Enzyme", Vol. 31, No. 37-45 (1985)). In addition, the fluorescent substance and the antibody may be bound via a protein such as bovine albumin or a nano fine particle. Moreover, as a method to couple | bond via a non-covalent bond, the physical adsorption method can be mentioned, for example. That is, they are bonded by intermolecular attractive force such as van der Waals force and hydrophobic bond generated between the fluorescent substance and the antibody. Such a non-covalent bond may be formed according to a conventionally known method. Furthermore, as a method of arranging the binding substance on the noble metal layer, for example, a conventionally known physical adsorption method and the like can be mentioned. In addition, a binding substance can be arranged in the noble metal layer by thiolating the silver surface or modifying it to silver oxide like gold.

(Effect of measuring chip 1 according to Embodiment 1)
Next, the effect of the measurement chip 1 according to the first embodiment will be described in comparison with a conventional measurement chip. FIG. 11 shows the effects of the measurement chip 1 according to Embodiment 1 and the conventional measurement chip (control chip), where (a) is a table showing experimental results and (b) is a graph. . In FIG. 11B, the vertical axis indicates the fluorescence signal intensity, and the horizontal axis indicates the concentration of rIL-6. Here, a chip in which an antibody is directly arranged on a substrate is used as a control chip. As is apparent from FIG. 11, the measurement chip 1 of the present embodiment is superior in fluorescence signal intensity and S / N ratio at any concentration of rIL-6, compared to the control chip. For example, when the rIL-6 concentration is 2500 (pg / ml), the fluorescence signal intensity is about 8.7 times (= 47,051 / 5,410) and the S / N ratio is about 1.3 times ( = 20.1 / 15.7), and it can be seen that an extremely high intensity fluorescence signal can be obtained.

  FIG. 12 shows the result of the fluorescence signal enhancement test by physical adsorption, (a) is a graph showing the experimental result, and (b) is a schematic diagram of the structure of each chip used in the experiment of (a). FIG. In FIG. 12A, the vertical axis indicates the concentration of the fluorescent dye RPE, and the horizontal axis indicates the fluorescence signal intensity. Here, five types of chips having different structures are used as the measurement chips. As shown in FIG. 12B, the chip A is obtained by physically adsorbing the fluorescent dye directly on the substrate, and the chip B is A gold base layer is formed on a substrate and a fluorescent dye is adsorbed thereon. Chip C is a silver base layer formed on a substrate and a fluorescent dye is adsorbed thereon. Chip D is a substrate. The gold base layer is formed on the microparticles 4a disposed thereon, and the fluorescent dye is adsorbed on the microparticles 4a, and the chip E is the microparticles disposed on the substrate 2 with the gold base layer 3 formed thereon. 4a is obtained by adsorbing a fluorescent dye (measurement chip 1 according to the first embodiment). As is apparent from FIG. 12A, when the measurement chip 1 (chip E) according to the first embodiment is used, the measurement chips A to D having other structures are used. In any case where the concentration of the fluorescent dye is changed from Bare to 10,000 (pg / ml), a strong fluorescent signal intensity is obtained. In particular, when the concentration of the fluorescent dye is 2,000 or 10,000 (pg / ml), a significant difference is observed in the fluorescence signal intensity. From this, it can be seen that by using the measurement chip 1 according to the embodiment, the enhancement rate of the fluorescence signal can be greatly increased in a wide range of fluorescent dye concentrations.

  Furthermore, in FIG. 13, the table | surface of the result of the verification test of the intensity | strength of a fluorescence signal and S / N ratio is shown. Here, as a control chip, a chip in which a fluorescent dye is physically adsorbed directly on a substrate is used. As apparent from FIG. 13, when the measurement chip 1 according to the present embodiment is used, the fluorescence signal intensity is about 56.5 times (= 65, 335/1, 161) and the S / N ratio is about 43.7 times (= 493.8 / 11.3), indicating that the fluorescence signal enhancement rate and the S / N ratio can be greatly increased. The

[Embodiment 2]
Next, the specific content of Embodiment 2 which concerns on this invention is demonstrated. The second embodiment has a main feature in the method of using the measuring chip 1 configured in the same manner as the first embodiment. By reacting the measuring chip 1 with a BSA solution or the like, autofluorescence or quenching is performed. This is a form in which ching can be suppressed. The configuration of the second embodiment is substantially the same as the configuration of the first embodiment unless otherwise specified, and the configuration substantially the same as the configuration of the first embodiment is used in the first embodiment. The same reference numerals are given as necessary, and the description thereof is omitted.

  In the second embodiment, after the measurement chip 1 is reacted with the sample, it is further reacted with a BSA solution or the like, thereby suppressing autofluorescence and increasing the S / N ratio in the fluorescence intensity measurement. Quenching is suppressed, and a surface enhancement effect can be obtained even for bright fluorescent dyes.

  FIG. 14 is a table showing changes in fluorescence signal intensity and S / N ratio depending on the reaction solution. Here, 10 μl PRE (0.1 M Citrate buffer solution pH 3.5) is reacted at room temperature for 1 hour with respect to the measuring chip 1 configured in the same manner as in Embodiment 1, and then PB-T (0.2 M phosphorus). Washed with acid buffer 0.01% Triton X-10, pH 6.8), then reacted with each 1% solution (at least one selected from the group consisting of BSA, skim milk, and casein) at room temperature for 30 minutes and washed The sample was dried and measured with a confocal fluorescent scanner (ScanArray Express; Perkin Elmer) (excitation wavelength: 543 nm, laser power: 40%, PMT Gain: 80%). Here, the concentration of lPRE is 4 types of substrate, 0, 80, 400, and 2,000 (ng / ml), and at the same time, when a BSA solution or the like is not used for control (only in case of fluorescent dye) Measurements were also made.

  As a result, as is apparent from FIG. 14, in the whole range where the RPE concentration is 80 (ng / ml) or more, the case where BSA, skim milk, or casein is used, compared to the case where only the fluorescent dye is used, It can be seen that both the fluorescence signal intensity and the S / N ratio are excellent, and there is a significant difference between the two. For example, when the RPE concentration is 2,000 (ng / ml), the fluorescence signal intensity is about 1.6 times (= 30,290 / 18,702) when the case of using only the fluorescent dye and the case of using BSA are compared. ), And the S / N ratio is about 2.3 times (= 74.9 / 32.1). In addition, in order to obtain the self-luminescence or quenching suppressing effect in this way, any protein other than BSA, skim milk, or casein can be used. Further, in the test of FIG. 14, a 1% solution of BSA, skim milk, or casein was used, but the concentration of the solution is preferably 0.1% or more and 10% or less. It can be used with appropriate modifications. This is because if the solution concentration is less than 0.1%, the effect is hardly recognized, whereas if it exceeds 10%, the solution is difficult to dissolve and is difficult to process.

(Effect of Embodiment 2)
As described above, according to the second embodiment, it is possible to obtain substantially the same effect as in the first embodiment, and it is possible to suppress the autofluorescence and increase the S / N ratio in the fluorescence intensity measurement. At the same time, quenching can be suppressed and a surface enhancement effect can be obtained even for bright fluorescent dyes.

[Embodiment 3]
Next, the specific content of Embodiment 3 which concerns on this invention is demonstrated. In the third embodiment, an anion is further provided on the measurement chip configured similarly to the first embodiment. The configuration of the third embodiment is substantially the same as the configuration of the first embodiment unless otherwise specified, and the configuration substantially the same as the configuration of the first embodiment is used in the first embodiment. The same reference numerals are given as necessary, and the description thereof is omitted.

(Configuration of measuring chip)
First, the configuration of the measurement chip 6 according to the third embodiment will be outlined. FIG. 15 is a partially enlarged side view of the measurement chip according to the third embodiment. The measurement chip 6 is configured by further providing an anion (anion) 7 on the noble metal layer 5. This anion is specifically a halogen (halogen ion: Cl ⁻ ). For example, a measurement chip is formed in the same manner as in the first embodiment, and after reacting the sample, a predetermined concentration of NaCl is reacted for a predetermined time. Can be arranged. Here, “arranging” means reacting anions with the substrate. By reacting the anion with the substrate in this way, effects such as negative charging of the silver surface can be obtained.

  FIG. 16 is a diagram showing changes in the fluorescence signal intensity and the S / N ratio depending on the presence or absence of halogen ions, (a) is a table showing experimental results, and (b) is a graph. In FIG. 16B, the vertical axis indicates the fluorescence signal intensity, the horizontal axis indicates the concentration of rIL-6, and each plot in the graph indicates the conventional measurement chip for control, the measurement chip 1 according to the first embodiment. 1 shows a measurement chip (measurement chip 6 according to Embodiment 3) in which halogen ions are arranged. Here, 10 μl of anti-IL-6 antibody (for solid phase; 6 μg / ml, 0.1 M Citrate buffer pH 3.5) was reacted for 1 hour at room temperature on each measurement chip, and then PB-T (0 .1M phosphate buffer 0.01% Triton X-10, pH 6.8). Thereafter, 1 mM NaCl was reacted on the measurement chip at room temperature for 15 minutes, washed, then reacted with BSA (1% BSA-PB-T) at room temperature for 30 minutes, washed again, and then rIL-6 (0, 4, 20 , 100, 500, 2,500 pg / ml) solution is allowed to react at room temperature for 1 hour. Next, after washing the measurement chip, the measurement chip is reacted with 10 μl of RPE-labeled anti-IL-6 antibody solution (1 μg / ml) at room temperature for 30 minutes. Finally, the measurement chip was washed and dried, and this measurement chip was measured with a confocal fluorescence scanner (ScanArray Express; Perkin Elmer) (excitation wavelength 543 nm, laser power 40%, PMT Gain 80%).

As a result, as is apparent from FIG. 16, in the entire range where the rIL-6 concentration is 100 (pg / ml) or more, the conventional measurement chip for control, the measurement chip 1 according to the first embodiment, and the present embodiment It can be seen that the fluorescence signal intensity and the S / N ratio are improved in the order of the measurement chip 6 according to the third embodiment. In particular, when the measurement chip 1 according to the first embodiment and the measurement chip 6 according to the third embodiment are compared, for example, when the rIL-6 concentration is 2,500 (pg / ml), the fluorescence signal The strength is about 2.0 times (= 31,476 / 15,532), and the S / N ratio is about 2.0 times (= 74.9 / 38.0). It has been found that the same improvement can be seen when SO ₄ ²⁻ , NO ₃ ⁻ , or CO ₃ ²⁻ is used in addition to the halogen ion as the material constituting the anion. Excellent improvement effect can be obtained.

For example, FIG. 17 shows the result of the fluorescence signal enhancement test, where (a) is a table showing experimental results and (b) is a graph. In FIG. 17B, the vertical axis represents the concentration of the fluorescent dye RPE, and the horizontal axis represents the fluorescence signal intensity. Here, a control chip using buffer, a chip using halogen ions (Cl ⁻ ), and a chip using SO ₄ ²⁻ are used as measurement chips. As can be seen from FIG. 17, the chip using anions such as Cl ⁻ and SO ₄ ²⁻ has improved fluorescence signal intensity and S / N ratio compared to the control chip. In particular, a chip using halogen ions (Cl ⁻ ) has a remarkable effect of improving the fluorescence signal intensity and the S / N ratio, and it can be seen that halogen ions can obtain the most excellent improvement effect. From this, there is a possibility that an appropriate interval is provided between the fine particles due to the anion, particularly the halogen ion, and this may contribute to the improvement effect of the fluorescence signal intensity and the S / N ratio. There is.

(Effect of Embodiment 3)
As described above, according to the third embodiment, substantially the same effect as in the first embodiment can be obtained, and by providing an anion, the fluorescence signal intensity can be further improved, and the fluorescence with higher sensitivity can be obtained. Measurement can be performed.

[III] Modifications to Each Embodiment While the embodiments have been described above, the specific configuration and method of the present invention are within the scope of the technical idea of each invention described in the claims. Modifications and improvements can be made arbitrarily. Hereinafter, such a modification will be described.

(About problems to be solved and effects of the invention)
First, the problems to be solved by the invention and the effects of the invention are not limited to the above-described contents, and the present invention solves the problems not described above or has the effects not described above. There are also cases where only some of the described problems are solved or only some of the described effects are achieved. For example, even when the enhancement rate of the surface enhancement effect is the same as the conventional case, the problem of the present application is solved by achieving the same effect by a means different from the conventional one.

(About configuration, manufacturing method, or usage)
The configuration, manufacturing method, and usage method of each measurement chip shown in the document and the drawings are merely examples, and can be arbitrarily changed unless otherwise specified.

(Resonance effect)
Further, in the present invention, the absorption spectrum of the substrate 2 is changed by changing the size and thickness of the fine particles 4a, and the absorption spectrum of the substrate 2 and the absorption spectrum of the fluorescent label are matched with each other. Can be resonated with each other to obtain a so-called resonance effect. This resonance effect can further amplify the fluorescence signal intensity and increase the measurement sensitivity.

(Enhancing near field, suppressing quenching)
In the present invention, it is possible to enhance the near field and suppress quenching by providing an appropriate interval (gap) between the fine particles 4a.

(Appendix)
Finally, some of the main features of the above embodiments and the effects thereof are shown as additional notes below.

(Appendix 1)
A substrate,
A porous membrane disposed on at least one side of the substrate;
A noble metal layer containing at least silver, disposed on the surface of the porous film opposite to the substrate;
It is provided with.

(Effects of Appendix 1)
According to Supplementary Note 1, it is possible to improve the fluorescence signal intensity by increasing the surface enhancement effect, and it is possible to obtain a fluorescence signal intensity several tens of times higher than the conventional enhancement factor by one digit or more. Further, even in a fluorescent substance having a high quantum yield (bright fluorescent substance), a fluorescent signal intensity that is several tens of times higher than that of the conventional fluorescent substance can be obtained, so that the practicality of the bright fluorescent substance can be improved. Furthermore, since a high fluorescence signal enhancement effect is exhibited even with weak excitation light, autofluorescence can be suppressed and the S / N ratio of the fluorescence signal can be improved. Furthermore, the measurement chip can be applied to almost all analysis methods (plate assays, biochips, DNA chips, etc., nanosensors) that detect fluorescent signals from the substrate, and improve the sensitivity in each analysis method. Can do.

(Appendix 2)
A binding substance that is arranged in the noble metal layer and specifically binds to a substance to be measured;
The measuring chip for fluorescence measurement according to appendix 1, characterized by comprising:

(Effects of Appendix 2)
According to the supplementary note 2, by arranging the binding substance, the user can easily and quickly perform the fluorescence measurement without arranging the binding substance himself.

(Appendix 3)
The binding substance is an antigen or an antibody;
The measurement chip for fluorescence measurement according to Supplementary Note 2, characterized by:

(Effects of Appendix 3)
According to Supplementary Note 3, the effect of Supplementary Note 2 can be obtained using an antigen or antibody as a binding substance.

(Appendix 4)
The noble metal layer has a thickness of about 5 to 20 nm;
The measurement chip for fluorescence measurement according to any one of appendices 1 to 3, characterized by:

(Effects of Appendix 4)
According to Supplementary Note 4, the fluorescence signal intensity can be further increased by setting the thickness of the noble metal layer in a suitable range.

(Appendix 5)
The noble metal layer has a thickness of about 10 to 15 nm;
The measuring chip for fluorescence measurement according to appendix 4, characterized by:

(Effects of Appendix 5)
According to Supplementary Note 5, the fluorescence signal intensity can be further increased by setting the film thickness of the noble metal layer in a more preferable range.

(Appendix 6)
Forming the porous film by perforating a plurality of holes in a flat film body;
The measurement chip for fluorescence measurement according to any one of appendices 1 to 5, characterized by:

(Effects of Appendix 6)
According to Supplementary Note 6, since the porous film is formed by perforating the film body, it is not necessary to form fine particles, and the porous film can be easily formed.

(Appendix 7)
Forming the porous film from a plurality of fine particles;
The measurement chip for fluorescence measurement according to any one of appendices 1 to 5, characterized by:

(Effects of Appendix 7)
According to Supplementary Note 7, since the porous film is formed using the fine particles, the porous film having a desired structure can be easily formed by controlling the particle size and the porosity of each fine particle.

(Appendix 8)
The particle size of the fine particles is about 360 nm or less;
The measurement chip for fluorescence measurement according to appendix 7, characterized by:

(Effects of Appendix 8)
According to Supplementary Note 8, the fluorescence signal intensity can be further increased by setting the particle diameter of the fine particles within a suitable range.

(Appendix 9)
The fine particles have a particle size of about 50 to 200 nm;
Item 9. A measuring chip for measuring fluorescence according to appendix 8, wherein

(Effects of Appendix 9)
According to Supplementary Note 9, the fluorescence signal intensity can be further increased by setting the particle size of the fine particles in a more preferable range.

(Appendix 10)
The fine particles have a particle size of about 50 to 100 nm;
The measurement chip for fluorescence measurement according to appendix 9, characterized by:

(Effects of Appendix 10)
According to the supplementary note 10, the fluorescence signal intensity can be further increased by setting the particle diameter of the fine particles to a more suitable range.

(Appendix 11)
The porosity of the porous membrane is about 60-97%;
The measurement chip for fluorescence measurement according to any one of appendices 1 to 10, characterized by:

(Effects of Appendix 11)
According to Supplementary Note 11, the fluorescence signal intensity can be further increased by setting the porosity of the porous film within a suitable range.

(Appendix 12)
The porosity of the porous membrane is about 75-85%;
The measurement chip for fluorescence measurement according to appendix 11, characterized by:

(Appendix 12 effects)
According to Supplementary Note 12, the fluorescence signal intensity can be further increased by setting the porosity of the porous film to a more preferable range.

(Appendix 13)
A base layer containing at least gold or silicon, disposed between the substrate and the porous film;
13. A measurement chip for measuring fluorescence according to any one of appendices 1 to 12, characterized in comprising:

(Effects of Supplementary Note 13)
According to the supplementary note 13, the provision of the base layer containing gold makes it possible to easily and reliably arrange the porous film (particularly, the porous film formed of fine particles) on the substrate.

(Appendix 14)
After arranging the substrate, the porous membrane, and the noble metal layer, the measurement chip was reacted with at least one selected from the group consisting of BSA, skim milk, and casein,
14. A measurement chip for fluorescence measurement according to any one of appendices 1 to 13, characterized by:

(Effects of Appendix 14)
According to Supplementary Note 14, by allowing the measurement chip to react with BSA or the like, autofluorescence can be suppressed and the S / N ratio in fluorescence intensity measurement can be increased. At the same time, quenching can be suppressed and a surface enhancement effect can be obtained even for bright fluorescent dyes.

(Appendix 15)
An anion disposed on the surface of the noble metal layer opposite to the substrate;
The measurement chip for fluorescence measurement according to any one of supplementary notes 1 to 14, characterized by comprising:

(Effect of Supplementary Note 15)
According to Supplementary Note 15, by arranging anions in the noble metal layer, the fluorescence signal intensity can be further improved, and fluorescence measurement with higher sensitivity can be performed.

(Appendix 16)
The anion is a halogen ion;
The measurement chip for fluorescence measurement according to supplementary note 15, characterized by:

(Effects of Supplementary Note 16)
According to the supplementary note 16, by using halogen ions as anions, the fluorescence signal intensity can be further improved, and fluorescence measurement with higher sensitivity can be performed.

(Appendix 17)
Preparing a substrate;
Disposing a porous film on at least one side of the substrate;
Arranging a noble metal layer containing at least silver on the surface of the porous film opposite to the substrate;
A method for manufacturing a measurement chip for fluorescence measurement, comprising:

(Effect of Supplementary Note 17)
According to the supplementary note 17, by using the measuring chip manufactured by this manufacturing method, the fluorescence signal intensity can be improved by increasing the surface enhancement effect, which is a number higher by one digit or more than the conventional enhancement rate. Ten times the fluorescence signal intensity can be obtained. Further, even in a fluorescent substance having a high quantum yield (bright fluorescent substance), a fluorescent signal intensity that is several tens of times higher than that of the conventional fluorescent substance can be obtained, so that the practicality of the bright fluorescent substance can be improved. Furthermore, since a high fluorescence signal enhancement effect is exhibited even with weak excitation light, autofluorescence can be suppressed and the S / N ratio of the fluorescence signal can be improved. Furthermore, the measurement chip can be applied to almost all analysis methods (biochips, DNA chips, etc., nanosensors) that detect fluorescent signals from the substrate, and sensitivity can be improved in each analysis method.

(Appendix 18)
Placing a binding substance that specifically binds to the measurement target substance on the noble metal layer;
The manufacturing method of the measuring chip according to appendix 17, characterized by comprising:

(Effect of Supplementary Note 18)
According to the supplementary note 18, by arranging the binding substance, the user can easily and quickly perform the fluorescence measurement without arranging the binding substance himself.

(Appendix 19)
The binding substance is an antigen or an antibody;
19. A method for manufacturing a measuring chip according to appendix 18.

(Effect of Supplementary Note 19)
According to Supplementary Note 19, the effect of Supplementary Note 18 can be obtained using an antigen or antibody as a binding substance.

(Appendix 20)
After the step of preparing the substrate, including a step of disposing a base layer containing at least gold on at least one side surface of the substrate, the porous film being disposed on the base layer,
20. A method for manufacturing a measuring chip according to any one of appendices 17 to 19, characterized by:

(Effects of Appendix 20)
According to the supplementary note 20, by providing the base layer containing gold, the porous film (particularly, the porous film formed of fine particles) can be easily and reliably disposed on the substrate.

(Appendix 21)
After the step of arranging the noble metal layer, the measurement chip is reacted with at least one selected from the group consisting of BSA, skim milk, and casein,
The method for manufacturing a measuring chip according to any one of appendices 17 to 20, characterized in that:

(Effects of Appendix 21)
According to Supplementary Note 21, by allowing the measurement chip to react with BSA or the like, autofluorescence can be suppressed and the S / N ratio in fluorescence intensity measurement can be increased. At the same time, quenching can be suppressed and a surface enhancement effect can be obtained even for bright fluorescent dyes.

(Appendix 22)
Disposing anions on the surface of the noble metal layer opposite to the substrate;
The method for manufacturing a measuring chip according to any one of appendices 17 to 21, characterized in that:

(Effects of Supplementary Note 22)
According to the supplementary note 22, by arranging anions in the noble metal layer, the fluorescence signal intensity can be further improved, and fluorescence measurement with higher sensitivity can be performed.

(Appendix 23)
A step of manufacturing a measuring chip by disposing a noble metal layer containing at least silver on the surface of the porous film opposite to the substrate after disposing the porous film on the substrate;
Disposing a binding substance that specifically binds to a measurement target substance on the noble metal layer of the measurement chip;
Binding a substance to be measured to the binding substance of the measurement chip;
Binding a binding substance labeled with a fluorescent dye to a measurement target substance bound to the binding substance of the measurement chip;
Observing the fluorescent dye;
Fluorescence measurement method characterized by including.

(Effects of Appendix 23)
According to Supplementary Note 23, it is possible to improve the fluorescence signal intensity by increasing the surface enhancement effect, and it is possible to obtain a fluorescence signal intensity several tens of times higher than the conventional enhancement rate by one digit or more. Further, even in a fluorescent substance having a high quantum yield (bright fluorescent substance), a fluorescent signal intensity that is several tens of times higher than that of the conventional fluorescent substance can be obtained, so that the practicality of the bright fluorescent substance can be improved. Furthermore, since a high fluorescence signal enhancement effect is exhibited even with weak excitation light, autofluorescence can be suppressed and the S / N ratio of the fluorescence signal can be improved. Furthermore, the measurement chip can be applied to almost all analysis methods (plate assays, biochips, DNA chips, etc., nanosensors) that detect fluorescent signals from the substrate, and improve the sensitivity in each analysis method. Can do.

(Appendix 24)
A step of manufacturing a measuring chip by disposing a noble metal layer containing at least silver on the surface of the porous film opposite to the substrate after disposing the porous film on the substrate;
Placing a measurement target substance on the noble metal layer of the measurement chip;
A binding substance that specifically binds to the measurement target substance of the measurement chip, the binding substance labeled with a fluorescent dye is bound to the measurement target substance; and
Observing the fluorescent dye;
Fluorescence measurement method characterized by including.

(Effects of Appendix 24)
According to Supplementary Note 24, a sample (which may include cells) whose measurement object is unknown is first bound or brought close to the measurement chip, and the measurement object included in this sample is bound with several types of fluorescent labels. Even in the case of detecting with a substance (for example, an antibody), the same effect as in Supplementary Note 23 can be obtained.

(Appendix 25)
The binding substance is an antigen or an antibody;
The fluorescence measurement method according to claim 23 or 24, wherein:

(Effect of Supplementary Note 25)
According to Supplementary Note 25, the effect of Supplementary Note 23 or 24 can be obtained using an antigen or antibody as a binding substance.

(Appendix 26)
A step of reacting the measurement chip with at least one selected from the group consisting of BSA, skim milk, or casein between the step of producing the measurement chip and the step of observing the fluorescent dye;
The fluorescence measurement method according to any one of claims 23 to 25, comprising:

(Effects of Supplementary Note 26)
According to Supplementary Note 26, by allowing the measurement chip to react with BSA or the like, autofluorescence can be suppressed and the S / N ratio in the fluorescence intensity measurement can be increased. At the same time, quenching can be suppressed and a surface enhancement effect can be obtained even for bright fluorescent dyes.

  As described above, the fluorescence measurement method, the measurement chip, and the manufacturing method thereof according to the present invention are useful for quantifying the measurement target substance in the sample, and in particular, the fluorescence signal intensity and the S / N ratio are measured. It is suitable for improving and measuring with high sensitivity.

It is a partial expansion perspective view of the measuring chip concerning Embodiment 1 of the present invention. 2 is a partially enlarged side view of the measurement chip according to Embodiment 1. FIG. It is a graph which shows the change of the fluorescence signal intensity | strength by the difference in the material of a noble metal layer. It is a graph which shows the correlation between a fluorescence signal intensity | strength and the film thickness of a noble metal layer, and is a graph when the particle size of microparticles | fine-particles is 50 nm. It is a graph which shows the correlation between a fluorescence signal intensity | strength and the film thickness of a noble metal layer, and is a graph when the particle size of microparticles | fine-particles is 100 nm. It is a graph which shows the correlation between a fluorescence signal intensity | strength and the film thickness of a noble metal layer, and is a graph at the time of setting the particle size of microparticles | fine-particles to 200 nm. It is a graph which shows the correlation between a fluorescence signal intensity | strength and the film thickness of a noble metal layer, and is a graph when the particle size of microparticles | fine-particles is 300 nm. It is a graph which shows the correlation between a fluorescence signal intensity | strength and the film thickness of a noble metal layer, and is a graph when the particle size of microparticles | fine-particles is 500 nm. The relationship between the fluorescence signal intensity and the concentration of the fluorescent dye is shown for various vacancies, (a) is a table showing experimental results, and (b) is a graph. It is a figure which shows notionally the fluorescence measuring method using the chip | tip for a measurement which concerns on Embodiment 1, (a) shows the state which has arrange | positioned the antibody to the chip | tip for a measurement, (b) shows the state which comprised the sandwich coupling | bonding. . The effect of the chip | tip for a measurement which concerns on Embodiment 1, and the chip | tip for a comparison is shown, (a) is a table | surface which shows an experimental result, (b) is a graph. The result of the fluorescence signal enhancement test by physical adsorption is shown, (a) is a graph showing the experimental result, and (b) is a diagram schematically showing the structure of each chip used in the experiment of (a). It is a table | surface which shows the result of the verification test of the intensity | strength of a fluorescence signal, and S / N ratio. 6 is a table showing changes in fluorescence signal intensity and S / N ratio by a reaction solution according to Embodiment 2. 6 is a partially enlarged side view of a measurement chip according to Embodiment 3. FIG. It is a figure which shows the change of the fluorescence signal intensity | strength and S / N ratio by the presence or absence of a halogen ion, (a) is a table | surface which shows an experimental result, (b) is a graph. The result of the fluorescence signal enhancement test is shown, (a) is a table showing experimental results, and (b) is a graph.

Explanation of symbols

1, 6 Measuring chip 2 Substrate 3 Base layer 4 Porous membrane 5 Noble metal layer 7 Anion

Claims (4)

A step of manufacturing a measuring chip by disposing a noble metal layer containing at least silver on the surface of the porous film opposite to the substrate after disposing the porous film on the substrate;
Disposing a binding substance that specifically binds to a measurement target substance on the noble metal layer of the measurement chip;
Binding a substance to be measured to the binding substance of the measurement chip;
Binding a binding substance labeled with a fluorescent dye to a measurement target substance bound to the binding substance of the measurement chip;
Observing the fluorescent dye;
Fluorescence measurement method characterized by including. A step of manufacturing a measuring chip by disposing a noble metal layer containing at least silver on the surface of the porous film opposite to the substrate after disposing the porous film on the substrate;
Placing a measurement target substance on the noble metal layer of the measurement chip;
A binding substance that specifically binds to the measurement target substance of the measurement chip, the binding substance labeled with a fluorescent dye is bound to the measurement target substance; and
Observing the fluorescent dye;
Fluorescence measurement method characterized by including. The binding substance is an antigen or an antibody;
The fluorescence measurement method according to claim 1 or 2, wherein: A step of reacting the measurement chip with at least one selected from the group consisting of BSA, skim milk, or casein between the step of producing the measurement chip and the step of observing the fluorescent dye;
Fluorometry according to any one of claims 1 to 3, which comprises a.

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