JP5988239B2 - Fluorescence measurement substrate - Google Patents

Description

  The present invention relates to a fluorescence measurement substrate used for fluorescence analysis and the like.

  Analysis methods using fluorescent labels are used in a wide variety of fields including the field of clinical testing. In order to measure a low-concentration sample in a very small amount of sample by labeling with a fluorescent dye, the brightness of a light source and the sensitivity of a detection apparatus are being increased. Attempts have also been made to select fluorescent dyes with a high quantum yield, use of cluster labels composed of a plurality of dye molecules, removal of excitation light using a high-performance cutoff filter, and the like.

  Another method is a surface-enhanced fluorescence method using a near field. Since the light intensity increases in the near field, it is effective for excitation of the fluorescent dye.

  FIG. 8 shows a conventional fluorescent dye excitation method. FIG. 8A shows the total reflection fluorescence excitation method. By irradiating the surface of the prism 31 with the excitation light 32 from the convex surface side through the inside of the glass under the entire reflection condition, light oozes out on the surface of the prism. A near field is formed by the evanescent light 33 oozing out.

  FIG. 8B shows the surface plasmon fluorescence excitation method. Excitation light 32 is irradiated from the convex surface side of the high refractive index glass 40 to the surface of the high refractive index glass 40 on which the silver or gold thin film 34 having a thickness of about 50 nm is formed under the plasmon resonance excitation condition. Then, a propagation type surface plasmon 35 is generated on the surface of the thin film 34 and a near field is formed. Compared with the total reflection fluorescence excitation method of FIG. 8A, the surface plasmon fluorescence excitation method is considered to be effective because a stronger near field is formed.

  Further, as a method related to plasmon resonance, a method using a localized surface plasmon resonance phenomenon can be mentioned. FIG. 8C shows a fluorescence measurement method using the localized surface plasmon resonance phenomenon. When the metal nanostructure 36 having a size of about 100 nm formed on the glass substrate 30 is irradiated with light, a near field having an electric field that is several tens of times or more stronger than the incident light intensity is generated. When the fluorescent dye is measured in the near field, the fluorescence intensity is enhanced by several tens to one hundred times.

  As a method for producing the metal nanostructure, as shown in Non-Patent Document 1, there is a method of corroding the silver surface with an acid. Further, as shown in Non-Patent Document 2, a method of immobilizing gold and silver colloids on a substrate, and as shown in Non-Patent Document 3, a nano-sized metal pattern is formed by an electron beam drawing apparatus. There is a way.

  FIG. 8D shows another fluorescence measurement method using the localized surface plasmon resonance phenomenon. There is a method in which a metal layer 38 of gold or silver is deposited on a monodisperse nanoparticle 37 such as silica or polystyrene solidified on a glass substrate 30 by vacuum evaporation or sputtering (Patent Document 1, non-patent document). Reference 4). Reference numeral 39 denotes a metal layer deposited on the substrate 30 when the metal layer 38 is formed. In this case, the metal layer 38 formed on the isolated hemispherical nanoparticles 37 has been shown to be effective for surface enhanced fluorescence.

JP 2008-96189 A

P.J.Tarcha et al., Applied Spectroscopy, 53, 43-48, 1999 M. Kawasaki and S. Mine, Journal of Physical Chemistry B, 109, 17254-17261, 2005 K. Alan et al., Journal of Fluorescence, 17, 7-13, 2007 Yamaguchi, T. Kaya, M. Aoyama, H. Takei, Analyst, 134, 776-783, 2009

  However, in the above conventional technique, surface-enhanced fluorescence is confirmed, but in any case, the enhancement rate is about several tens of times, and the enhancement rate is insufficient.

  Further, it has the following problems structurally. In FIG. 8A, since the evanescent light 33 oozing out from the glass interface of the prism is used, it is necessary to strictly construct the optical system, and the prism requires special glass having a high refractive index. Therefore, it is not suitable for building a small and low-cost system. In the surface plasmon fluorescence excitation method of FIG. 8 (b), surface plasmon resonance occurs only when light is incident at an angle satisfying the resonance condition, so it is necessary to construct an optical system very precisely. It is not suitable for building a small and low-cost system.

  On the other hand, as shown in FIGS. 8C and 8D, the method using the localized surface plasmon resonance phenomenon is not strict in the irradiation condition of excitation light, and excites the analyte adsorbed on the noble metal nanoparticles from an arbitrary direction. In addition, since it is only necessary to detect from an arbitrary direction, the optical system has an advantage that strictness is not required.

  Further, as shown in Non-Patent Document 4, the method of FIG. 8D is suitable for low-cost mass production because it is not so affected by the material of the substrate and the flatness of the surface and the material of the fine particles. There is also an advantage that the material can be selected.

  However, in the case of FIGS. 8C and 8D using the localized surface plasmon resonance phenomenon, the metal nanoparticles must be isolated without contacting each other, and the structure is fragile. is there. There is a drawback that it is peeled off or damaged by a slight contact with other objects.

  The present invention has been made in order to solve the above-described problems, and an object of the present invention is to provide a fluorescence measurement substrate having a surface enhancement rate sufficient for fluorescence analysis and a strong structure.

To achieve the above object, the fluorescence measurement substrate of the present invention includes a plurality of fine particles made of a dielectric adsorbed on the substrate, and the surface of the fine particles from the surface of the substrate so as to embed the plurality of fine particles. and a metal layer formed continuously on the substrate subjected to, with some having a plurality of protrusions of the metal layer, wherein the plurality of particles is the particle size 30Nm～1000nm, the from the substrate surface The main feature of the present invention is that the thickness of the metal layer up to the flat portion of the metal layer is 100% or more and 1000% of the particle size .

  According to the present invention, since the metal layer is continuously formed so as to enclose a plurality of fine particles, a surface enhancement effect by propagation type surface plasmon resonance can be obtained. In addition, since the continuously formed metal layer has a plurality of protrusions, a surface enhancement effect by localized plasmon resonance can be obtained. In this way, due to the synergistic effect of the surface enhancement effect by propagation type surface plasmon resonance and the surface enhancement effect by localized type plasmon resonance, even when measuring a low-concentration sample in a very small amount of sample, sufficient signal intensity can be obtained. Can be obtained.

  In addition, since the metal layer is continuously formed on the substrate so as to embed a plurality of fine particles, each metal particle is firmly fixed on the substrate by this metal layer, and the fluorescence measurement substrate having a tough structure Can be provided.

It is sectional drawing which shows the structure of the board | substrate for fluorescence measurement of this invention. It is a figure which shows the preparation methods of the board | substrate for fluorescence measurement of this invention. It is a figure which shows the relationship between the film thickness of the metal layer of a fluorescence measurement board | substrate, and fluorescence intensity. It is a figure which shows the relationship between the film thickness of the metal layer of a fluorescence measurement board | substrate, and fluorescence intensity. It is a figure which shows the comparison of the signal strength from the board | substrate for fluorescence measurement of this invention, and the signal strength from on a glass substrate. It is a figure by the scanning electron micrograph which shows that even when a metal layer is deposited more than the particle size of microparticles | fine-particles, microparticles | fine-particles are completely buried and a metal layer does not become flat. It is a figure by the scanning electron micrograph of the fine particle to which the metal layer peeled from the board | substrate adhered. It is a figure which shows the total reflection type excitation fluorescence measuring method, the propagation type surface plasmon excitation fluorescence measuring method, and the localized type surface plasmon resonance measuring method.

  Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The drawings relating to the structure are schematic and different from the actual ones. In addition, there may be a case where the dimensional relationships and ratios are different between the drawings.

  In the present invention, by using a structure that enables coupling of propagation type surface plasmon resonance and localized type surface plasmon resonance, the enhancement rate is increased and the toughness of the structure is improved. The configuration of this fluorescence measurement substrate is shown in FIG.

  As shown in FIG. 1A, the fluorescence measuring substrate has a metal layer 3 formed on the surface of the substrate 1 with a predetermined thickness. The surface of the metal layer 3 is uneven, and a plurality of protrusions 4 are formed.

  The inside of the metal layer 3 in FIG. 1A is as shown in FIG. A plurality of fine particles 2 are adsorbed and solidified on the surface of the substrate 1, and a metal layer 3 is deposited on the surface of the substrate 1 so as to cover all the fine particles 2. For this reason, the metal layer 3 is formed so as to embed (enclose) all the fine particles 2, but the unevenness due to the arrangement when the fine particles 2 are adsorbed to the substrate 1 is left as it is. Appears as

  As will be described later, the metal layer 3 deposited on the fine particles 2 is not flat even if the thickness of the metal layer 3 is equal to or larger than the particle size of the fine particles 2, for example, several times as shown in FIG. Protrusions 4 are formed, and irregularities are formed. Here, the thickness or vapor deposition thickness of the metal layer 3 extends from the surface of the substrate 1 to the flat surface at the end of the substrate 1 of the metal layer 3 as indicated by h in FIG. It corresponds to the height of. Further, the film thickness or vapor deposition thickness h of the metal layer 3 corresponds to the height from the top of the fine particle 2 to the top of the protrusion 4 as shown in FIG.

  As shown in FIG. 1, by continuously forming the metal layer 3 so as to cover the fine particles 2, propagation plasmon resonance can be generated on the surface of the continuous thin film. On the other hand, by depositing the metal layer 3 so as to cover all the fine particles 2, irregularities are formed in the continuous thin film made of the metal layer 3. This uneven protrusion 4 contributes to the excitation of localized surface plasmons and can generate localized surface plasmon resonance. Thereby, the coupling of the propagation type surface plasmon resonance and the localized type surface plasmon resonance can be realized.

  Here, as the substrate 1, a resin flat substrate such as glass, quartz, silicon, polystyrene, polycarbonate, and polymethyl methacrylate is suitable, but paper and metal may be used. Further, it may be made of a biological material such as wood, skin, horn, or shell that has been waterproofed. Furthermore, when viewed macroscopically, the substrate may not be a flat substrate and may have a fibrous or spherical shape.

  Further, as the fine particles 2 to be adsorbed on the solid surface on the substrate 1, nanoparticles made of a dielectric such as silica, polystyrene, titanium oxide or the like are used. By using a dielectric for the fine particles 2, a higher surface enhancement effect can be expected.

  Gold, silver, platinum, aluminum, copper, or the like can be used for the metal layer 3, but a noble metal such as gold, silver, or platinum is preferable as the material.

  Next, FIG. 2 shows a method for producing the fluorescence measurement substrate of FIG. As shown in FIG. 2A, the fine particles 2 made of a dielectric material such as silica or polystyrene are adsorbed on the solid surface of the substrate 1 as a single layer, and the metal 3A is deposited by vacuum deposition or sputtering as shown in FIG. Deposit on the fine particles 1. As the metal, a noble metal 3A deposited on the fine particles 1 like a hat and a noble metal 3B deposited on the substrate 1 are generated.

  When the metal is further deposited from FIG. 2B, the metal 3A adheres to the lower side of the center of the fine particle 2 as shown in FIG. 2C, and the thickness of the metal 3B increases. . Finally, as shown in FIG. 2D, the metal 3A deposited on each fine particle 2 and the metal 3B deposited on the substrate are connected to each other, and the continuous metal layer 3 and Become. As described above, the metal layer 3 is deposited on the substrate 1 in a state where each fine particle 2 is completely included, and irregularities are formed on the surface of the metal layer 3. The irregularities on the surface of the metal layer 3 appear in a shape corresponding to the irregularities formed by the fine particles 2.

  Next, an example of manufacturing the fluorescence measurement substrate of FIG. 1 will be described. In this embodiment, a method of adsorbing silica nanoparticles on a glass substrate using a glass substrate as the substrate 1 and silica nanoparticles as the fine particles 2 will be described.

  First, silane coupling treatment using hydroxyl groups on the glass substrate surface is performed. An aqueous solution of aminopropyltriethoxysilane (0.1 to 1% by weight) is prepared, and the glass substrate is immersed for 5 seconds to 5 minutes.

  After the treatment of the glass substrate surface, silica nanoparticles (weight ratio 0.1 to 5%) are dropped on the surface. Let stand for 10 seconds to 5 minutes under dust-free conditions. Next, in order to remove an excessive amount of silica nanoparticles, the substrate is sufficiently washed with purified water and dried. Then, a structure in which silica nanoparticles are adsorbed in a single layer on a glass substrate is obtained.

Next, as a method for depositing metal on the surface of the silica nanoparticles, a method using a vacuum deposition method will be described. As the metal, the noble metal silver is most desirable, but gold, copper, or platinum may be used. A glass substrate is placed inside the vacuum deposition apparatus, and the chamber is evacuated. After achieving a vacuum degree of 10 ⁻³ Pascal, deposition is started at a rate of 1 to 10 kg / second. The deposition thickness from the glass substrate surface to the flat portion at the edge of the silver thin film is 30% to 1000% with respect to the particle size of the silica nanoparticles. Thereby, a substrate for fluorescence measurement is obtained.

  Since the fluorescence measurement substrate formed as described above has a configuration as shown in FIG. 1, by using the surface-enhanced fluorescence method, it is possible to realize the coupling between the propagation surface plasmon resonance and the localized surface plasmon resonance, As the most important effect, the enhancement rate can be improved. On the other hand, the noble metal is deposited also in the gaps between the fine particles and becomes a continuous noble metal layer, and is formed on the substrate so as to embed each fine particle, so that a fluorescence measurement substrate having a strong structure is configured.

  In FIG. 3, silica fine particles having a particle diameter of 100 nm are used as the fine particles 2, silver having various thicknesses is deposited on the metal layer 3, 1 mM rhodamine 6G is dropped, and fluorescence is emitted with excitation light having a wavelength of 520 nm. The observation result is shown. The vertical axis represents the fluorescence intensity, and the horizontal axis represents the deposition thickness (nm). The vapor deposition thickness here is a film thickness corresponding to the height h in FIG.

  Incidentally, in Patent Document 1, as shown in FIGS. 4 to 8 in the document, it is shown that the enhancement rate is optimized when a silver thin film having a thickness of 15 nm or less is formed. . The film thickness here indicates the thickness of the hat-shaped silver thin film formed on the fine particles, and is not the deposition thickness h in the present invention.

  On the other hand, as shown in FIG. 3, the substrate for fluorescence measurement in which the silver thin film is formed by vapor deposition so that the film thickness h is about 30 nm to 50 nm is certainly effective for the surface enhanced fluorescence method. In particular, since the entire fine particles 2 are covered and embedded by the continuously formed metal layer 3 with a film thickness of about 30 nm, the effect of surface enhancement is increased. Since the particle diameter of the fine particles is 100 nm, the fine particles are embedded with a metal layer thickness of 30% of the fine particle diameter.

  It can also be seen from FIG. 3 that the enhancement effect is further amplified by increasing the deposition thickness to 80 nm or more. After the deposition thickness is 80 nm to 90 nm and the fluorescence intensity is slightly reduced, when the deposition thickness is 100 nm or more, the fluorescence intensity increases at an accelerated rate. The fluorescence intensity is high up to a film thickness of about 1000 nm, and the fluorescence intensity is particularly high in the film thickness range of 300 nm to 900 nm. From FIG. 3, it can be seen that even when the metal layer thickness is 1000 nm, the fluorescence intensity is strong and effective, so that a film thickness corresponding to 10 times (1000%) the particle diameter of the fine particles of 100 nm may be formed.

  In FIG. 4, silica fine particles having a particle diameter of 50 nm and 150 nm were used as the fine particles 2, respectively. The result of observing fluorescence with excitation light having a wavelength of 520 nm after depositing silver of various thicknesses as the metal layer 3 on each silica fine particle of each particle diameter and dropping 1 mM rhodamine 6G is shown. The vertical axis represents the fluorescence intensity, and the horizontal axis represents the deposition thickness (nm). The vapor deposition thickness here is a film thickness corresponding to the height h in FIG. 1, as in FIG. The shaded bar graph indicates a particle size of 50 nm, and the open bar graph indicates a particle size of 150 nm.

  As shown in FIG. 4, the fluorescence measurement substrate on which a silver thin film having a thickness of 30 nm or more is formed has a strong fluorescence intensity, and is certainly effective for the surface-enhanced fluorescence method. When the particle size is 50 nm, the metal layer thickness is from about 15 nm, and when the particle size is 150 nm, the entire fine particle 2 is covered and embedded by the metal layer 3 formed continuously from the metal layer thickness of about 45 nm. As a result, the effect of surface enhancement is increased. Although not shown in the figure, the fluorescence intensity tends to increase due to the thickness of the metal layer with respect to the fine particles having the respective particle sizes. Further, the ratio of the metal layer thickness to the particle diameter is 30%, and the fine particles are embedded by the metal layer thickness of 30% of the fine particle diameter.

  In addition, as shown in FIG. 4, since the fluorescence intensity is strong even when the metal layer thickness is 500 nm, the fine particle diameter of 50 nm is formed to a film thickness corresponding to 10 times (1000%) of the particle diameter. You can also see that it is good. Similarly, in the case of a particle size of 150 nm, a film thickness corresponding to 10 times the particle size may be formed.

  Next, FIG. 5 shows a state in which the fluorescence signal intensity of the fluorescence measurement substrate of the present invention increases as compared with only the glass substrate on which nothing is formed on the surface. FIG. 5A shows the case where the dye concentration (rhodamine 6G) is 1 mM, and FIG. 5B shows the case where the dye concentration (rhodamine 6G) is 10 μM. The film thickness of the silver thin film was formed at 300 nm. Compared with the case of using only a glass substrate, the fluorescence intensity is greatly increased. In the case of FIG. 5A, the fluorescence intensity is increased several tens of times, and in the case of FIG. 5B, it is increased several times.

  Further, FIG. 6 shows that the metal layer 3 does not become flat in the region where the fine particles 2 exist even if the deposition thickness h is equal to or larger than the particle size of the fine particles 2. (A), (b), and (c) of FIG. 6 show images obtained by depositing silver thin films having film thicknesses h of 100 nm, 500 nm, and 1000 nm on silica nanoparticles having a particle diameter of 100 nm, respectively. As can be seen from these images, even when the film thickness h increases, the metal layer 3 does not become flat and the irregularities due to the silica nanoparticles are not lost.

  In order to show the structure in the present invention more clearly, a scanning electron micrograph of the nanostructure of the fine particles peeled from the substrate is shown in FIG. FIGS. 7A and 7B show cases where the deposition thicknesses are 500 nm and 1000 nm, respectively. It can be seen that the continuous metal thin film and the fine particles are peeled off in an integrated form. It can be seen that the fluorescence measurement substrate of the present invention has a structure that is tough and not easily damaged as compared with conventional metal nanoparticles that are not in contact with each other.

  The spectroscopic substrate of the present invention can be applied to environmental monitoring, quality control, clinical examination, and the like.

1 Substrate 2 Fine particle 3 Metal layer 3A Metal 3B Metal

Claims (3)

A plurality of fine particles made of a dielectric material adsorbed on the substrate, and a metal layer continuously formed on the substrate from the surface of the substrate to the fine particle surface so as to embed the plurality of fine particles, A part of the metal layer has a plurality of protrusions , the plurality of fine particles have a particle size of 30 nm to 1000 nm, and the film thickness of the metal layer from the substrate surface to the flat portion of the metal layer is the particle size A fluorescence measuring substrate having a thickness of 100% to 1000% of the diameter . 2. The fluorescence measurement substrate according to claim 1 , wherein the plurality of fine particles are made of any one of a polymer containing polystyrene, silica, and titanium oxide . The substrate for fluorescence measurement according to claim 1 or 2 , wherein the substrate is made of any material of glass, silicon, polystyrene, polycarbonate, and polymethyl methacrylate resin .