Sensing chip, sensing chip manufacturing method, sensing kit, measurement method and measurement device

Abstract

This chip for sensing comprises a substrate having a plasmon generating region with a periodic structure, and a plurality of capturing molecules for capturing a target substance. The plurality of capturing molecules are bound to the plasmon generating region at a higher density than to a region around the plasmon generating region. This allows for higher detection sensitivity in fluorescence observation than before.

Description

[Technical Field]

[0001] The present invention relates to a sensing chip for detecting a target substance using surface plasmon resonance interactions, a method for manufacturing the sensing chip, a sensing kit, a measurement method, and a measurement device. This application claims priority to Japanese Application No. 2021-029652, filed February 26, 2021, and incorporates by reference all of the contents of said Japanese application. [Background technology]

[0002] High sensitivity of biosensors and immunosensors is a required characteristic for various targets. In particular, with the recent increase in new infectious diseases, there is a demand for highly sensitive immunosensor chips that can easily and quickly detect markers of each disease. To achieve this high sensitivity, efforts are being made to develop high-affinity antibodies and improve the signal-to-noise ratio (S / N ratio).

[0003] As a tool for enhancing signal intensity, a sensor chip capable of enhancing fluorescence using surface plasmon resonance (hereinafter also referred to as a plasmonic chip) is known. For example, Non-Patent Document 1 discloses a chip with a bull's eye structure composed of concentric circles, the cross section of which passing through the center has a periodic structure. Specifically, it discloses that the bull's eye structure enables illumination light having omnidirectional components from an objective lens under a microscope to efficiently couple with surface plasmons (hereinafter referred to as plasmons) to form an enhanced electric field. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Mai Kanda, Eri Fujimoto, Keiko Tawa, "Evaluation of Fluorescence Enhancement of Nanoparticles on a Bull's Eye-Type Plasmonic Chip Using Fluorescence Microscopy," Proceedings of the 67th Spring Meeting of the Japan Society of Applied Physics (published February 28, 2020) [Non-patent document 2] “Light sources and conditions for photoactivation of aryl azide crosslinking and labeling reagents,” [online], Thermo Fisher Scientific Inc., [Retrieved February 1, 2021], Internet <URL:https: / / www.thermofisher.com / document-connect / document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2FApplication-Notes%2FTR0011-Photoactivate-aryl-azides.pdf&title=VGVjaCBUaXA6IExpZ2h0IHNvdXJjZXMgYW5kIGNvbmRpdGlvbnMgZm9yIHBob> [Non-patent document 3] Florian Feist, et al., “Visible Light-InducedLigation via o-Quinodimethane Thioethers”, J. Am. Chem.Soc. 2018, 140, 37, 11848-11854 Summary of the Invention [Problem to be solved by the invention]

[0005] By using plasmonic chips, particularly chips with a bull's-eye structure, the signals of fluorescently labeled molecules can be amplified and detected in optical systems (microscopes, etc.) that use objective lenses for irradiation and detection, thereby improving the sensitivity of biosensors and immunosensors. However, even higher sensitivity is desired. As a result of extensive research, the inventors of the present application came up with the idea of ​​regioselectively binding capture molecules for capturing target substances such as antigens to the chip (i.e., spatially controlling the immobilization of capture molecules to the chip) as an alternative to methods for enhancing the signals of fluorescently labeled molecules. If capture molecules can be regioselectively bound to the chip, it is expected that even higher sensor sensitivity can be achieved.

[0006] Therefore, an object of the present invention is to provide a sensing chip to which capture molecules that capture target substances are reposition-selectively bound, a method for manufacturing the sensing chip, a sensing kit, a measurement method, and a measurement device. [Means for solving the problem]

[0007] (1) A sensing chip according to a first aspect of the present invention includes a substrate having a plasmon generating region and a plurality of capture molecules for capturing a target substance, the plurality of capture molecules being bound to the plasmon generating region at a higher density than to the region surrounding the plasmon generating region.

[0008] (2) Preferably, the plurality of capture molecules are bound to the center of the plasmon generating region at a higher density than in the surrounding region of the center.

[0009] (3) More preferably, the plasmon generating region has a concentric periodic uneven structure.

[0010] (4) More preferably, the predetermined portion including the center of the concentric circle has a convex or concave shape, and the predetermined portion has a circular shape having a diameter equal to or smaller than one period of the concave-convex structure.

[0011] (5) Preferably, the capture molecule contains biotin, which is bound to the plasmon generating region by maleimide and a compound represented by the following general formula (1) or (2), or a compound of TFPA-PEG3-Biotin and 3-aminopropyl triethoxysilane, enabling high-density detection of proteins as target substances.

[0012] [ka] In the general formula (1) and the general formula (2), R is any one of the following compounds A1 to A11.

[0013] [ka]

[0014] (6) A method for manufacturing a sensing chip according to a second aspect of the present invention includes a first step of introducing a photoreactive compound bound to a capture molecule for capturing a target substance onto a substrate having a plasmon generating region, and a second step of irradiating the substrate on the back side with light after the first step, wherein in the second step, the photoreaction of the photoreactive compound is promoted by an enhanced electric field due to plasmons, and the capture molecule is bound to the plasmon generating region. This allows the capture molecule to be selectively bound to the plasmon generating region, enabling highly sensitive detection of the target substance.

[0015] (7) A method for manufacturing a sensing chip according to a third aspect of the present invention includes a first step of introducing capture molecules for capturing a target substance onto a substrate having a plasmon generating region and bound to a photoreactive compound, and a second step of irradiating the substrate with light from the backside on which the first step has been performed, wherein in the second step, the photoreaction of the photoreactive compound is promoted by an enhanced electric field due to the plasmon, and the capture molecules are bound to the plasmon generating region. This allows the capture molecules to be selectively bound to the plasmon generating region, enabling highly sensitive detection of the target substance.

[0016] (8) Preferably, the light irradiated in the second step has a wavelength of 300 nm or more and 550 nm or less, or 600 nm or more and 1100 nm or less.

[0017] (9) More preferably, the photoreactive compound contains a compound represented by the following general formula (1) or (2), or TFPA-PEG3-Biotin. This allows capture molecules to be bound to the plasmon generating region at a higher density than the area around the plasmon generating region, enabling detection of target substances with higher sensitivity.

[0018] [ka] In the general formula (1) and the general formula (2), R is any one of the following compounds A1 to A11.

[0019] [ka]

[0020] (10) More preferably, the light irradiated in the second step has a wavelength of 450 nm or more and 490 nm or less. This suppresses the binding of capture molecules in areas outside the plasmon generating region of the chip. Furthermore, in the plasmon generating region, more capture molecules can be bound to the center than to the periphery. This allows for even higher sensitivity in detecting target substances.

[0021] (11) A sensing kit according to a fourth aspect of the present invention includes a substrate having a plasmon generating region and a photoreactive compound. By adding the photoreactive compound to the substrate and irradiating the substrate with light from the backside, the photoreaction of the photoreactive compound is promoted by the enhanced electric field caused by the plasmon, and the photoreactive compound binds to the plasmon generating region.

[0022] (12) A measurement method according to a fifth aspect of the present invention includes a first step of introducing a target substance bound to a fluorescent substance into the sensing chip, and a second step of irradiating light from the back of the sensing chip on which the first step has been performed, and measuring the fluorescence emitted from the fluorescent substance due to the enhanced electric field caused by plasmons from the front of the sensing chip.

[0023] (13) A measuring device according to a sixth aspect of the present invention includes a light source and a lens that focuses light from the light source, and in a state where a photoreactive compound having capture molecules bound thereto for capturing a target substance is placed on a substrate having a plasmon generating region, the light focused by the lens is irradiated from the backside of the substrate to promote the photoreaction of the photoreactive compound by an enhanced electric field due to the plasmons, and the capture molecules are bound to the plasmon generating region; and in a state where a target substance containing a fluorescent substance is placed on the substrate with the capture molecules bound to the plasmon generating region, the light focused by the lens is irradiated from the backside of the substrate to measure fluorescence emitted from the fluorescent substance by the enhanced electric field due to the plasmons. [Effects of the Invention]

[0024] According to the present invention, by using a sensing chip to which capture molecules are regioselectively bound, detection sensitivity in fluorescence observation can be improved compared to conventional methods. Therefore, by using this sensing chip as a biosensor or immunosensor, a highly sensitive measuring instrument that can easily and quickly detect various disease markers can be realized. Furthermore, the electric field strength is enhanced by grating-coupled plasmon resonance among the propagating plasmons generated in the plasmon generating region, and the enhancement degree depends on the lattice structure, which promotes photoreactions (i.e., photochemical reactions) and enables the efficient production of sensing chips to which capture molecules are regioselectively bound.

[0025] Furthermore, it is possible to easily create regions with and without binding of capture molecules within the same chip, and the fluorescence intensity of the region without binding of capture molecules can be used as the base intensity, thereby enabling the original signal intensity to be evaluated with higher accuracy. Furthermore, unlike manufacturing methods that use a mask to cause a local photoreaction, the manufacturing method of the present invention does not require the complicated process of attaching a mask to the chip (including alignment) and removing the mask after the reaction, thereby simplifying the manufacturing process of the sensing chip. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 is a plan view schematically showing the structure of a sensing chip according to an embodiment of the present invention. [Figure 2] FIG. 2 is a diagram showing an AFM (Atomic Force Microscope) image showing a concentric bull's-eye structure, which is an example of a plasmon generating region. [Figure 3] FIG. 3 is a cross-sectional view showing the structure of the sensing chip shown in FIG. [Figure 4] FIG. 4 is a diagram showing the chemical formula of APTES (3-Aminopropyl triethoxysilane). [Figure 5] FIG. 5 is a diagram showing the state in which the surface (SiO 2 ) of the chip body is modified with APTES. [Figure 6] FIG. 6 is a diagram showing the chemical formula of o-methylbenzaldehydes, which is an example of a photoreactive compound. [Figure 7] FIG. 7 is a diagram showing the state in which o-Methylbenzaldehydes shown in FIG. 6 is bonded to the chip body in the state shown in FIG. [Figure 8] FIG. 8 is a diagram showing the state in which a maleimide compound is introduced into the chip body in the state shown in FIG. [Figure 9] FIG. 9 is a diagram showing the state in which a photoreactive compound and a maleimide compound are bonded together. [Figure 10]FIG. 10 is a diagram showing another method for manufacturing a sensing chip. [Figure 11] FIG. 11 shows the chemical formula of succinimidyl PEG. [Figure 12] FIG. 12 is a block diagram showing a schematic configuration of the measurement device. [Figure 13] FIG. 13 is a plan view showing an example of a periodic structure of a plasmon generating region that is different from the bull's eye structure. [Figure 14] FIG. 14 is a plan view showing an example of a periodic structure of a plasmon generating region different from that shown in FIG. [Figure 15] FIG. 15 is a diagram showing the chemical formula of TFPA-PEG3-Biotin, which is an example of a photoreactive compound. [Figure 16] FIG. 16 shows the photoreaction of TFPA-PEG3-Biotin shown in FIG. [Figure 17] FIG. 17 is a plan view showing the configuration of a prototype sensing chip. [Figure 18] FIG. 18 is a photograph showing the experimental results. [Figure 19] FIG. 19 relates to Example 3 and shows a state in which Cy5-streptavidin as a target substance is introduced onto a substrate to which biotin-maleimide containing a capture molecule is bound. [Figure 20] FIG. 20 shows the state in which the introduced Cy5-streptavidin is bound to the biotin-maleimide bound to the substrate. [Figure 21] FIG. 21 relates to Example 3 and shows the results of observing fluorescence using a chip prepared by irradiating it with UV light to cause a photoreaction. [Figure 22] FIG. 22 shows the results of Example 3, in which UV light was irradiated to cause a photoreaction, and fluorescence was observed using a chip fabricated under conditions different from those of the chip from which the results of FIG. 21 were obtained. [Figure 23] FIG. 23 is a photograph showing the results of observing fluorescence without irradiating the chip with UV light for causing a photoreaction, as a comparative example. [Figure 24]FIG. 24 relates to Example 4 and shows the results of observing fluorescence after irradiating the chip with visible light to cause a photoreaction. [Figure 25] FIG. 25 is a photograph showing the results of observing fluorescence using a chip prepared without irradiation with light to promote the photoreaction or the addition of Cy5-maleimide. [Figure 26] FIG. 26 is a photograph showing the results of observing fluorescence using a chip prepared by adding Cy5-maleimide without irradiating it with light to promote the photoreaction. [Figure 27] FIG. 27 is a photograph showing the results of observing fluorescence using a chip prepared by irradiating it with visible light to promote a photoreaction and adding Cy5-maleimide, relating to Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0027] In the following embodiments, the same components are denoted by the same reference numerals, and have the same names and functions. Therefore, detailed description thereof will not be repeated.

[0028] (Structure of sensing chip) Referring to Fig. 1, a sensing chip 100 according to an embodiment of the present invention includes a chip body 102 and a plasmon generating region 104 formed on the chip body 102. In Fig. 1, a plurality of plasmon generating regions 104 are arranged in a hexagonal lattice pattern. The lower right of Fig. 1 shows an enlarged view of one plasmon generating region 104. Here, the plasmon generating region 104 has a bull's-eye structure (see the AFM image shown in Fig. 2), in which periodic concave and convex portions are formed concentrically within a circular region of diameter φ.

[0029] FIG. 3 shows a cross-sectional view of the plasmon generating region 104 shown in the lower right of FIG. 1 , taken along line III-III passing through the center of the region. Referring to FIG. 3 , the plasmon generating region 104 includes a base substrate 106 having the periodic structure (i.e., bull's-eye structure) described above, and a multilayer film formed on the base substrate 106, the multilayer film including a first adhesive layer 110, a metal layer 112, a second adhesive layer 114, and a quenching suppression layer 116. The plasmon generating region 104 further includes a binding compound 200 disposed on the multilayer film, and a capturing molecule 202 bound to the binding compound 200. The binding compound 200 is a compound in which multiple substances are bound by a photoreaction (i.e., a photochemical reaction) using an enhanced electric field due to plasmon resonance, as described below. The capturing molecule 202 may also be present in the surrounding area of ​​the plasmon generating region 104, but is concentrated above the plasmon generating region 104. That is, the capture molecules 202 are bound to the plasmon generating region 104 at a higher density than in the region surrounding the plasmon generating region 104 .

[0030] The base substrate 106 is formed of, for example, glass or plastic (e.g., polymethyl methacrylate (PMMA)). The base substrate 106 may be transparent or opaque. The periodic structure can be formed by a known method (e.g., nanoprinting, press molding using a stamper (mold), or injection molding).

[0031] In the bullseye structure, the period L1 (the sum of the widths of adjacent concave and convex portions) of the concentric periodic structure is constant. The bullseye structure shown in FIG. 3 has a central convex shape with a diameter L2 (a circle centered on the center of the concentric circles of the periodic structure). In many commonly used bullseye structures, the central portion of the bullseye structure penetrates the entire structure, including the substrate, but the central portion of the plasmon generating region 104 does not penetrate. The period L1 is preferably equal to or less than the wavelength of the light used in fluorescence observation, or is approximately the same as the wavelength. The period L1 is, for example, 100 to 1000 nm, and preferably 200 to 600 nm.

[0032] To obtain maximum fluorescence intensity, the diameter L2 of the central portion of the bull's-eye structure is preferably equal to half the period L1 as shown in FIG. 3, but it need not be equal as long as plasmons can be generated by light irradiation. For example, as will be described later as experimental results, the diameter L2 may be equal to or less than the period L1. The central portion of the bull's-eye structure may have a concave shape. The diameter L2 of the concave shape may or may not be equal to half the period L1.

[0033] The first adhesive layer 110 is a layer for bonding the base substrate 106 and the metal layer 112. If the base substrate 106 itself is made of a material that can stably bond to the metal layer 112, the first adhesive layer 110 may be omitted. The second adhesive layer 114 is a layer for bonding the metal layer 112 and the quenching suppression layer 116. If the quenching suppression layer 116 itself is made of a material that can stably bond to the metal layer 112, the second adhesive layer 114 may be omitted. As will be described later, the quenching suppression layer 116 may also be omitted. The first adhesive layer 110 and the second adhesive layer 114 are preferably as thin as possible, and are formed as titanium (Ti) thin films with a thickness of, for example, 0.1 to 3 nm. The use of titanium can improve the chip's resistance to a surfactant (Tween 20) contained in PBS (phosphate buffer solution), which is used for cleaning in bioassays, etc. The first adhesive layer 110 may be made of chromium (Cr).

[0034] The metal layer 112 is made of, for example, silver (Ag) and is formed by sputtering or the like. When light is incident from the backside, the film thickness of the metal layer (Ag) 112 is preferably 10 to 100 nm, and more preferably 30 to 65 nm. Note that in FIG. 3, the shape of the metal layer 112 is shown to be the same as the uneven shape of the base substrate 106, but when the metal layer 112 is formed by sputtering or the like, the portions corresponding to the stepped portions of the periodic structure of the base substrate 106 are inclined. Therefore, the second adhesive layer 114 and the quenching suppression layer 116 described later can also have inclined shapes.

[0035] The quenching suppression layer 116 is also a layer for binding capture molecules (e.g., antibodies) and is preferably formed of silicon dioxide (SiO2) so that commercially available bioassay kits (e.g., pharmaceuticals) can be used. Many commercially available pharmaceuticals are intended for use with SiO2. SiO2 has no absorption (or little absorption) in the wavelength ranges of incident light and emitted fluorescence typically used in observation, so it can be formed as a transparent thin film. The quenching suppression layer 116 can be formed, for example, by sputtering.

[0036] In the enhanced fluorescence technique, which is a feature of surface plasmon excitation-enhanced fluorescence, if the distance between the fluorescent molecules and the metal layer 112 is short, the fluorescence excited by a strong excitation field will be quenched due to energy transfer to the metal surface. Therefore, it is preferable to prevent quenching by separating the fluorescent molecules by a predetermined distance from the metal layer 112. For this reason, the quenching suppression layer 116 shown in FIG. 3 may be omitted if the molecular layer of the binding compound 200 and the capturing molecule 202 is of an appropriate thickness. Furthermore, because the excitation field caused by surface plasmon resonance is a near-field, its electric field strength attenuates with increasing distance from the metal surface. Therefore, only fluorescent molecules present within approximately 100 nm of the surface of the metal layer 112 are efficiently excited. Therefore, the thickness of the quenching suppression layer 116 is determined within a range of approximately 10 nm to 100 nm depending on the type of metal layer 112, the refractive index of the quenching suppression layer 116, the wavelength of the incident light, and other factors. The multilayer film formed on the base substrate 106 may also include a protective layer in addition to the above. For example, when a protective layer is disposed between the metal layer 112 and the quenching suppression layer 116, it is preferable to determine the total film thickness of the protective layer and the quenching suppression layer 116 within a range of approximately 10 nm to 100 nm depending on the type of metal layer 112, the refractive index of the quenching suppression layer 116, the wavelength of the incident light, etc.

[0037] The sensing chip 100 is used to detect antigen-antibody reactions. The capture molecules 202 are preferably selected to correspond to the antigen to be captured, i.e., to undergo an antigen-antibody reaction with the antigen to be captured. When a solution containing an antigen is dropped onto the sensing chip 100, an antigen-antibody reaction occurs between the capture molecules 202 bound to the surface of the plasmon generating region 104 (i.e., the quenching suppression layer 116) via the binding compound 200 and the introduced antigen. A fluorescently labeled protein (i.e., a fluorescent molecule) is bound to the antigen or antibody (i.e., the capture molecules 202) in advance. In this state, light is irradiated from the back surface of the sensing chip 100 (i.e., the surface on which a periodic structure is not formed), as described below. This generates surface plasmon resonance in the plasmon generating region 104, allowing enhanced fluorescence from the fluorescently labeled protein bound to the antigen or antibody to be detected.

[0038] As will be described later, the binding compounds 200 and the capture molecules 202 may be nonspecifically adsorbed around the plasmon generating region 104, and therefore fluorescence is also emitted from these. Although such fluorescence may become noise, the intensity of the enhanced fluorescence emitted from the plasmon generating region 104 is small. Therefore, the sensing chip 100 enables highly sensitive detection.

[0039] (Method for manufacturing sensing chips) The method for manufacturing the sensing chip 100 includes the following steps 1 to 4. Step 1: APTES (see FIG. 4) is placed on the chip body 102 by silane coupling. The end of APTES that is not bonded to the substrate is an amino group. FIG. 5 shows the state in which the surface of the quenching suppression layer 116 of the chip body 102 and APTES 210 are silane coupled.

[0040] Step 2: A photoreactive compound is added to the chip body 102 on which step 1 has been performed, and the photoreactive compound is bonded to the amino group of APTES (amide bond). For example, o-methylbenzaldehydes (hereinafter also referred to as benzaldehyde) shown in FIG. 6 is used as the photoreactive compound. The photoreactive compound is added by preparing a DMF solution. That is, a solution is prepared by adding TEA (triethylamine), EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), and the photoreactive compound to DMF (N,N-dimethylformamide). This solution is added to the substrate on which step 1 has been performed and allowed to stand for a predetermined time (e.g., 2 hours). Due to a dehydration reaction, APTES and o-methylbenzaldehyde form an amide bond, as shown in FIG. 7.

[0041] Step 3: As shown in FIG. 8, a maleimide compound modified with a capture molecule (antibody) is introduced into the chip body 102 on which step 2 has been performed, and light (e.g., ultraviolet light (hereinafter referred to as UV light)) is irradiated from the backside of the substrate for a predetermined time (e.g., 5 minutes). In FIG. 8, N-succinimidyl-3-maleimidopropionate 214 modified with a capture molecule 202 is shown as the maleimide compound. By irradiating light, an enhanced electric field is formed by plasmons in the plasmon generating region 104. This promotes the photoreaction between the photoreactive compound (i.e., benzaldehyde 212) and the maleimide compound (i.e., N-succinimidyl-3-maleimidopropionate 214). As a result, as shown in Fig. 9, the photoreactive compound (i.e., benzaldehyde 212) and the maleimide compound (i.e., N-succinimidyl-3-maleimidopropionate 214) are bonded in the plasmon generating region 104, resulting in a state in which the binding compound 200 is intensively bound to the plasmon generating region 104. Fig. 9 shows the state in which the o-methylbenzaldehyde group and the maleimide group are bonded.

[0042] Step 4: The chip body 102 after step 3 is washed. A surfactant, such as a mixed phosphate buffer solution (i.e., Tween 20), can be used as the washing solution. In step 3, the photoreaction between the photoreactive compound (i.e., benzaldehyde 212) and the maleimide compound (i.e., N-succinimidyl-3-maleimidopropionate 214) is not promoted in the area surrounding the plasmon generating region 104. Any unreacted maleimide compound (i.e., N-succinimidyl-3-maleimidopropionate 214) is removed from the chip body 102 by washing.

[0043] As a result, a sensing chip 100 is realized in which the capture molecules 202 are site-selectively bound to a specific region (i.e., the plasmon generating region 104) of the chip body 102. That is, in the sensing chip 100, the capture molecules 202 are bound to the plasmon generating region 104 at a higher density than to the region surrounding the plasmon generating region 104. A distinctive feature is that light is irradiated to the plasmon generating region 104, and a photoreaction is promoted using an enhanced electric field caused by plasmons, causing the capture antibodies to bind intensively to the plasmon generating region 104. This results in a significant fluorescence enhancement effect, as will be described later. Since the binding of the capture antibodies can be suppressed around the plasmon generating region 104, detection sensitivity is improved.

[0044] In the above-described manufacturing method, a compound of APTES 210 and benzaldehyde 212 is bound to the chip body 102, and then N-succinimidyl-3-maleimidopropionate 214 modified with a capture molecule 202 is introduced to promote the chemical reaction by plasmon resonance. However, this is not limited to this. As shown in FIG. 10 , a compound of APTES 210 and N-succinimidyl-3-maleimidopropionate 214 may be bound to the chip body 102, and then a photoreactive compound (i.e., benzaldehyde 212) modified with a capture molecule 202 may be introduced, and UV light may be irradiated. This forms an enhanced electric field due to plasmons in the plasmon generating region 104, promoting the photoreaction between the photoreactive compound (i.e., benzaldehyde 212) and the maleimide compound (i.e., N-succinimidyl-3-maleimidopropionate 214). Therefore, as in Figure 9, a photoreactive compound (i.e., benzaldehyde 212) and a maleimide compound (i.e., N-succinimidyl-3-maleimidopropionate 214) are bonded in the plasmon generating region 104, and a sensing chip 100 is produced in which capture molecules 202 are bonded position-selectively to a specific region of the chip body 102 (i.e., the plasmon generating region 104).

[0045] Alternatively, in step 2 above, a photoreactive compound (i.e., benzaldehyde 212) and succinimidyl PEG with a carboxyl end group, as shown in FIG. 11, may be mixed and then introduced into the chip body 102 on which step 1 has been performed. The maleimide compound modified with a capture molecule introduced in step 3 may not only bind to the photoreactive compound but also nonspecifically adsorb to the chip body 102. When a fluorescently labeled antigen is introduced into such a chip, an antigen-antibody reaction occurs with the capture molecule (e.g., an antibody) nonspecifically adsorbed to the chip body 102, causing fluorescence to be emitted from the area surrounding the plasmon generating region 104, resulting in a decrease in detection sensitivity. The introduction of succinimidyl PEG is expected to suppress nonspecific adsorption of the maleimide compound modified with a capture molecule to the chip body 102 in step 3.

[0046] If an enhanced electric field due to plasmons localized in a portion of the plasmon generating region 104 can be formed, it is expected that the above-mentioned photoreaction will be promoted intensively in that portion of the plasmon generating region 104. By employing a bull's-eye structure in the plasmon generating region 104, an enhanced electric field due to localized plasmons can be formed in the center of the bull's-eye structure due to the optical antenna effect. Therefore, the capture molecules can be bound to the center of the plasmon generating region 104 at a higher density than in the surrounding regions. In order to bind the capture molecules to the center of the plasmon generating region 104 at a higher density, it is preferable to irradiate the plasmon generating region 104 with light in the visible light range (380 nm to 780 nm) instead of UV light, for example. Normally, irradiating the plasmon generating region 104 with light in the visible light range has little effect on promoting the photoreaction. In contrast, by irradiating the plasmon generating region 104 with light in the visible light range, plasmons can be localized in the center of the plasmon generating region 104, and photoreactions can be promoted intensively in the center of the plasmon generating region 104. For example, a solution containing microspheres (e.g., silica beads with a diameter of 1 μm or less) whose surfaces are modified with a capture antibody, a biotin compound, or streptavidin bound to a capture antibody is introduced into the chip body 102, and visible light of 300 nm to 550 nm or 600 nm to 1100 nm is irradiated. This allows the silica beads or biotin compound to be bound intensively in the center of the plasmon generating region 104. Visible light of 300 nm to 550 nm (e.g., 450 nm) can be used to promote one-photon photoreactions, and visible light of 600 nm to 1100 nm (e.g., 720 nm) can be used to promote two-photon photoreactions. By using the sensing chip thus fabricated, for example, by adding a fluorescently labeled antigen, it becomes possible to observe the antigen-antibody reaction by fluorescence with even higher sensitivity.

[0047] The chip body 102 having the above-described plasmon generating region 104 can be used together with a photoreactive compound to form a sensing kit. Using such a sensing kit, as described above, the photoreactive compound is placed on the chip body 102 and irradiated with light, thereby promoting the photoreaction by the enhanced electric field caused by plasmons and allowing the photoreactive compound to bind intensively to the plasmon generating region 104.

[0048] (Measuring equipment) The following describes an apparatus used for manufacturing the sensing chip 100 and for fluorescence observation using the sensing chip 100. Referring to Fig. 12, a measuring apparatus 400 includes the sensing chip 100, a light source 402, an optical filter 404, a first lens 406, a second lens 408, and a camera 410. When the measuring apparatus 400 is used for manufacturing the sensing chip 100, the sensing chip 100 shown in Fig. 12 is replaced with a chip body 102 that is not modified with capture molecules 202.

[0049] Light source 402 is a mercury lamp or a halogen lamp. Optical filter 404 passes light of a specific wavelength among the light emitted from light source 402 and blocks other light. For example, a Cy5 filter (i.e., a bandpass filter that passes excitation light of the fluorescent substance Cy5) or a NUA filter (i.e., a bandpass filter that passes wavelengths of 370 to 380 nm) can be used as optical filter 404.

[0050] The first lens 406 is an objective lens for focusing light that has passed through the optical filter 404. When fluorescence observation is performed using the sensing chip 100, a 20x objective lens, for example, is used for the first lens 406. In this case, a halogen lamp is used as the light source 402, and if the sensing chip 100 is modified with molecules fluorescently labeled with, for example, Cy5, a Cy5 filter is used for the optical filter 404. When the chip body 102 is used instead of the sensing chip 100 and light from the optical filter 404 is irradiated from the back of the chip body 102 to perform the above-mentioned step 3, a 100x objective lens is used for the first lens 406. In this case, a mercury lamp is used as the light source 402, and a NUA filter is used for the optical filter 404.

[0051] The second lens 408 is a lens that focuses the fluorescence emitted from the sensing chip 100 and outputs it to the camera 410. The second lens 408 is, for example, a lens with a magnification of 10x. The camera 410 is an imaging device (for example, a CCD camera). Note that the measuring device 400 may include an optical system (for example, a prism, a mirror, etc.) other than the configuration shown in FIG. 12.

[0052] By using the sensing chip 100 described above, it is possible to promote a local photoreaction within the pattern in the plasmon generating region 104 of the concentric periodic structure. This photoreaction between a compound having a maleimide group and a photoreactive compound can be achieved with light in the wavelength range from UV light to visible light. In particular, a two-photon reaction can be expected in the near-infrared region. Furthermore, since a strong electric field is formed particularly in the central portion of the concentric structure, it is possible to achieve localized electric field formation, especially in the central portion of the pattern. It is believed that this local photoreaction can achieve highly sensitive detection in the construction of immunoassays.

[0053] Although the above description has been given of a case where the light source for promoting the photoreaction (a mercury lamp) and the light source for enhanced fluorescence observation (a halogen lamp) are different, this is not limiting. Photoreaction promotion and enhanced fluorescence observation may be performed using a single light source whose emission band includes both the wavelength of light for promoting the photoreaction and the wavelength of light for fluorescence observation.

[0054] Although the case where the periodic structure of the plasmon generating region 104 is a bull's-eye structure has been described above, the present invention is not limited to this. The periodic structure of the plasmon generating region 104 may be one in which periodic concaves and convexes are formed parallel to one direction (i.e., a line and space pattern) as shown in FIG. 13. In FIG. 13, convex portions 182 are formed parallel to one direction on the surface of a base substrate 180, and concave portions 184 are formed around the convex portions 182. Alternatively, it may be a two-dimensional periodic structure as shown in FIG. 14. In FIG. 14, convex portions 192 are formed in two directions intersecting the surface of a base substrate 190, and concave portions 194 are formed around the convex portions 192. Alternatively, it may be one in which the convex and convex portions in FIG. 13 are inverted, or a hole array in which the convex and convex portions in FIG. 14 are inverted.

[0055] The cross-sectional shape of the recesses (grooves) of the periodic structure of the plasmon generating region 104 is not limited to the rectangular shape shown in FIG. 3, but may be, for example, a sawtooth shape, a sinusoidal shape, or the like.

[0056] The metal layer 112 is not limited to silver (Ag) and may be made of any metal that generates surface plasmon resonance, and may be made of gold (Au), aluminum (Al), or the like.

[0057] Although o-Methylbenzaldehydes (see Figure 6) were used as the photoreactive compound above, this is not limiting. Commercially available reagents can also be used to prepare interfaces by photoreaction with the APTES surface. For example, TFPA-PEG3-Biotin, shown in Figure 15, can be used as the photoreactive compound. In this case, it is preferable to use an avidin-modified antibody or an avidin + biotin-modified antibody as a reactant instead of the maleimide compound (N-succinimidyl-3-maleimidopropionate 214).

[0058] With APTES210 bound to the surface of the chip body 102, TFPA-PEG3-Biotin is introduced and UV light is irradiated. This promotes the photoreaction shown in FIG. 16 (see Non-Patent Document 2) in the plasmon generating region 104 due to the enhanced electric field caused by plasmons. Therefore, TFPA-PEG3-Biotin can be intensively bound to the APTES210 bound to the plasmon generating region 104. Then, an avidin-modified antibody or an avidin+biotin-modified antibody is bound to the TFPA-PEG3-Biotin, thereby producing the sensing chip 100 having the structure shown in FIG. 3.

[0059] Furthermore, as the photoreactive compound, a compound represented by the following general formula (1) or (2) may be used.

[0060] [ka]

[0061] In general formula (1) and general formula (2), R is any one of the following compounds A1 to A11. When R is A5, general formula (1) is 3-((2-formyl-3-methylphenyl)thio)propanoic acid, that is, o-Methylbenzaldehydes shown in FIG. 6.

[0062] [ka] The compounds represented by general formula (1) and general formula (2) (R is any one of A1 to A11) can be prepared by the method disclosed in Non-Patent Document 3.

[0063] The binding compound 200 that binds the capturing molecule 202 to the chip body 102 is not limited to one containing APTES 210 bound to the quenching suppression layer 116. The binding compound 200 is formed by photoreaction of a photoreactive compound during the manufacturing process of the sensing chip 100, and may be any compound bound to the quenching suppression layer 116. When the photoreactive compound photoreacts, it is concentrated in the plasmon generating region 104, where it can bind the binding compound 200 and thus the capturing molecule 202. Furthermore, the object (target substance) captured by the capturing molecule 202 is not limited to an antigen, but may also be DNA, etc. The capturing molecule 202 may be any molecule that can capture a target substance. The capturing molecule 202 may be, for example, a compound having a moiety that specifically adsorbs to a target substance. [Example]

[0064] The experimental results below demonstrate the effectiveness of the present invention. A prototype chip with the structure shown in Figure 17 was fabricated. Approximately 2,000 plasmon generating regions 104 were formed only in the upper left region of the chip body 102. The plasmon generating regions 104 had an outer diameter of 20 μm and a period of 480 nm (i.e., the spacing between adjacent convex portions was 240 nm), with the central convex portion having a diameter of 480 nm. The multiple plasmon generating regions 104 were arranged in a hexagonal lattice pattern, with an adjacent spacing of 5 μm (hence, a center-to-center spacing of 25 μm). A multilayer film was formed on the base substrate on which such plasmon generating regions 104 were formed, as described above. Specifically, the first and second adhesive layers were formed using Ti with a thickness of less than 1 nm, the metal layer was formed using Ag with a thickness of 45 nm, and the quenching suppression layer was formed using SiO2 with a thickness of 20 nm.

[0065] A chip body 102 with the plasmon generating region 104 formed as described above was prepared, and a chip modified with a compound was fabricated using the same manufacturing method as described above. Specifically, APTES (see Figure 5) was bonded to the chip body, and then o-Methylbenzaldehydes (see Figure 6) was added as a photoreactive compound and allowed to stand for 2 hours, resulting in the bonding shown in Figure 7. The DMF solution was prepared using 2 mL of DMF, 15 μL of TEA, 11.5 mg of EDC, and 11.2 mg of o-Methylbenzaldehydes. This procedure was performed in a dark room or in a room lit by a yellow lamp. Then, 3.12 nM of Cy5-maleimide, a maleimide compound and fluorescent substance, was added, and UV light was irradiated from the back of the chip body 102 to promote the photoreaction of o-Methylbenzaldehydes. The light source and optical filter used were the above-mentioned mercury lamp and NUA filter (that is, a passing wavelength of 370 to 380 nm), respectively, and the light passing through the NUA filter was focused using a 100x objective lens and irradiated onto the back surface of the chip body 102.

[0066] Fluorescence observation was performed using the fabricated chip. For fluorescence observation, the above-mentioned halogen lamp and Cy5 filter were used as the light source and optical filter, respectively, and the light passing through the Cy5 filter was focused using a 20x objective lens and irradiated onto the back of the chip. The fluorescence emitted from the chip was focused using a 20x objective lens and observed with a CCD camera.

[0067] An image captured by a CCD camera is shown in Figure 18. In Figure 18, the circle shown in the center (i.e., the white dashed line) indicates the boundary of the area irradiated with light to promote the photoreaction during chip fabrication. In Figure 17, the dashed circle shown in the center corresponds to the circle shown in the center of Figure 18. In Figure 18, almost no fluorescence is observed from the area where the plasmon generating region 104 is not formed. In the plasmon generating region 104, fluorescence can be observed due to the enhanced electric field caused by plasmons. It can be seen that the fluorescence intensity from the plasmon generating region 104 formed inside the dashed circle (see Figure 17) is stronger than that of the plasmon generating region 104 formed around it. This confirms that irradiation with light to promote the photoreaction during chip fabrication promoted the binding of Cy5-maleimide to the plasmon generating region 104.

[0068] To confirm the effect of light irradiation to promote the photoreaction during chip fabrication, quantitative evaluation was performed. As shown in Figure 17, the fluorescence intensities measured for the four regions Birr, Bout, Firr, and Fout are shown in Table 1.

[0069] [Table 1]

[0070] Using the measured values ​​shown in Table 1, the fluorescence enhancement factor Ef and the chemical reaction promotion rate Rp were calculated according to the following formula: For convenience, the measured values ​​in each region are indicated by the symbol representing that region. Ef=Bout / Fout (Equation 1) Rp=(Birr-Bout) / (Firr-Fout)×1 / Ef (Formula 2)

[0071] Since neither region Fout nor Bout is irradiated with light for promoting the photoreaction, Ef represents the effect of fluorescence enhancement by the plasmon generating region 104. Region Birr is irradiated with light for promoting the photoreaction, and region Bout is not irradiated with light for promoting the photoreaction, so Birr-Bout includes the effect of promoting the photoreaction and the effect of fluorescence enhancement. On the other hand, Firr-Fout represents only the effect of promoting the photoreaction. Therefore, the effect of promoting the chemical reaction can be evaluated by dividing (Birr-Bout) / (Firr-Fout) by the fluorescence enhancement Ef, as in Equation 2 above.

[0072] Using the values ​​shown in Table 1, a fluorescence enhancement factor Ef of approximately 4.3 and a chemical reaction promotion rate Rp of approximately 10.8 were obtained. In other words, it is believed that by irradiating light to promote the photoreaction, Cy5-maleimide can be intensively bound to the plasmon generating region 104 at a density approximately 10 times higher than that of the surrounding area of ​​the plasmon generating region 104. Therefore, the detection sensitivity of fluorescence observation is significantly improved by approximately 46.5 times (Rp × Ef) due to the synergistic effect of the promotion of the photoreaction and the fluorescence enhancement. This, along with the results of a comparative experiment described below as Example 2, clearly demonstrates the great effectiveness of the present invention.

[0073] On the surface of an actual sensing chip, the plasmon generating region 104 within the region Birr in Figure 17 is expected to be formed over the entire surface. In other words, a sensing chip that does not include the regions Bout and Fout shown in Figure 17 but includes the region Birr and a region corresponding to the region Firr (a region where the plasmon generating region 104 is not formed) is expected. The performance of such a chip can be evaluated by measuring the fluorescence intensity in the regions Birr and Firr. Using the values ​​in Table 1, Birr / Firr is approximately 13.9. This value (Birr / Firr) may vary depending on the light irradiation conditions (including the structural conditions of the plasmon generating region 104) used to promote the photoreaction. However, it is believed that the concentrated binding of Cy5-maleimide to the region Birr will achieve a fluorescence enhancement factor Ef greater than Ef (approximately 4.3 using the values ​​in Table 1). [Example]

[0074] As a comparative example, a chip was fabricated using the same chip body as in Example 1, following the same steps, but without the step of irradiating UV light to promote the photoreaction, and fluorescence observation was performed. That is, as in Example 1, APTES (see FIG. 5) and o-Methylbenzaldehydes (see FIG. 6) were added to the chip body 102 on which the plasmon generating region 104 was formed, and bonded as shown in FIG. 7. In this state, 3.12 nM of Cy5-maleimide was added without promoting the photoreaction (irradiating UV light from the back of the chip body 102), and the chip was left to stand for a predetermined period of time and then washed to fabricate a chip. Fluorescence observation was performed using the fabricated chip in the same manner as in Example 1. The results are shown in Table 2.

[0075] [Table 2]

[0076] Using the measured values ​​shown in Table 2, the fluorescence enhancement factor Ef and the chemical reaction promotion rate Rp were calculated using the above-mentioned formulas 1 and 2. As a result, the fluorescence enhancement factor Ef was approximately 4.7, and the chemical reaction promotion rate Rp was approximately 1. The fact that Rp was approximately 1 indicates that the photoreaction was not promoted because UV light was not irradiated. Therefore, Ef of approximately 4.7 indicates that the fluorescence intensity was enhanced by the amount of binding due to nonspecific adsorption. This demonstrates the effectiveness of the present invention shown in Example 1. [Example]

[0077] Using a chip fabricated in the same manner as in Example 1, an experiment was conducted to confirm that target substances could be captured by capture molecules bound to the chip. Specifically, a chip body (see FIG. 17) with the same structure, material, and dimensions as in Example 1 was prepared. As in Example 1, APTES (see FIG. 5) was bound to the chip body, and then o-Methylbenzaldehydes (see FIG. 6) was added as a photoreactive compound and bound as shown in FIG. 7. Next, UV light was irradiated to the center of the chip body (corresponding to the dashed circle in FIG. 17) for 30 seconds to activate the photoreactive substance. Then, biotin-maleimide prepared at approximately 1 μM was added and maintained, and biotin as a capture molecule was bound to the chip. As a result, as shown in FIG. 19, a compound consisting of APTES 210, benzaldehyde 212, and biotin-maleimide 300 was bound to the plasmon generating region 104. Of the biotin moiety 302 and maleimide moiety 304 that make up the biotin-maleimide 300, the biotin moiety 302 functions as a capture molecule. Next, a protein fluorescently labeled with Cy5 (specifically, streptavidin), i.e., Cy5-streptavidin 312 prepared at two concentrations of approximately 10 nM and 1 nM, was added as a target substance to the chip to which the biotin moiety 302 was bound (see FIG. 19). As a result, as shown in FIG. 20, a complex is formed in which the biotin moiety 302 binds to the Cy5-streptavidin 312 due to the interaction between biotin and avidin. In other words, the Cy5-streptavidin 312 is bound to the plasmon generating region 104.

[0078] Fluorescence observation was performed using the fabricated chips in the same manner as in Example 1. Images captured by a CCD camera are shown in Figures 21 and 22. Figures 21 and 22 correspond to chips fabricated by adding Cy5-streptavidin 312 adjusted to approximately 10 nM and 1 nM, respectively. The bar in the lower right corner of Figure 22 represents a length of 100 μm. In both Figures 21 and 22, almost no fluorescence was observed from areas where the plasmon generating region was not formed. Fluorescence was observed in the plasmon generating region due to the enhanced electric field generated by plasmons. It can be seen that the fluorescence intensity from the plasmon generating region in the center of each chip was stronger than that of the plasmon generating region formed around it. This indicates that more Cy5-streptavidin is bound to the center of the chip (i.e., the area irradiated with UV light to promote the photoreaction) than to the outside of the center. In other words, it can be seen that more biotin, a capture molecule, is bound to the center of the chip than to the outside of the center. 21 and 22, a fluorescence intensity dependent on the concentration of Cy5-streptavidin was observed in the center of the chip, indicating that biosensing was possible using this chip.

[0079] As in Example 1, for a chip fabricated by adding approximately 10 nM Cy5-streptavidin 312, the fluorescence enhancement factor Ef and chemical reaction promotion rate Rp were calculated from the above formulas 1 and 2 using measurements taken inside and outside the area irradiated with UV light to promote the photoreaction during chip fabrication. The results were Ef = 15 and Rp = 1.2. The detection sensitivity of fluorescence observation was significantly improved by 18 times (Rp × Ef) due to the synergistic effect of photoreaction promotion and fluorescence enhancement.

[0080] In a comparative experiment, the same chip body as above was used. After APTES was bound to the chip body, o-Methylbenzaldehydes (see Figure 6) was added, and biotin-maleimide was added without irradiating it with UV light to promote the photoreaction. As a result, biotin as a capture molecule was bound to the chip by nonspecific adsorption. Fluorescence observation was performed using the fabricated chip in the same manner as in Example 1. Figure 23 shows an image captured by a CCD camera. In Figure 23, fluorescence was observed almost uniformly in the plasmon generation region due to the enhanced electric field caused by plasmons. Comparing Figures 21 and 23 demonstrates the effectiveness of irradiating UV light to promote the photoreaction during chip fabrication. In other words, capture molecules can be spatially selectively bound to the chip, improving the accuracy of target substance detection by the capture molecules. [Example]

[0081] The chip interface was prepared using visible light to promote the photoreaction. Specifically, a chip body (see FIG. 17) was prepared with the same materials and dimensions as in Example 1, except for the central structure of each plasmon generating region. The central portion had a half-pitch recessed structure (i.e., a well structure), which was the inverse of the recessed and protruding structure shown in FIG. 3. Similar to Example 1, o-Methylbenzaldehydes (see FIG. 6) was bound to the chip (see FIG. 7) using visible light (specifically, wavelengths of 450 nm to 490 nm) instead of UV light. For light irradiation, a mercury lamp was used as the light source 402 and a GFP filter (i.e., a bandpass filter that passes wavelengths of 450 to 490 nm) was used as the optical filter 404 in the configuration shown in FIG. 12 to generate visible light (hereinafter referred to as GFP light). Subsequently, Cy5-maleimide prepared at 9.36 nM was introduced and bound to the chip. Using the fabricated chip, fluorescence observation was performed in the same manner as in Example 1. Images captured by a CCD camera are shown in FIG. 24. The bar shown in the lower right of Fig. 24 represents a length of 50 µm. Fluorescence observation was performed using the configuration shown in Fig. 12, with a halogen lamp used as light source 402 and objective lenses with magnifications of 20x and 10x used as first lens 406 and second lens 408, respectively. The fluorescence intensities measured for the four regions Birr, Bout, Firr, and Fout shown in Fig. 24 are shown in Table 3.

[0082] [Table 3]

[0083] Using the values ​​in Table 3, the fluorescence enhancement factor Ef was calculated using Equation 1 above, resulting in Ef = 12.9. The chemical reaction promotion rate Rp calculated using Equation 2 above cannot be calculated from the values ​​in Table 3 because both Firr and Fout have the same value of "8." Specifically, by fabricating a chip using GFP light as light to promote the photoreaction, the fluorescence intensity in regions other than the plasmon generating region was similar regardless of whether GFP light was irradiated. This indicates that the use of GFP light prevented the binding of capture molecules to regions other than the plasmon generating region and enabled spatially selective binding of capture molecules only to the plasmon generating region. This is because GFP light with wavelengths of 450 nm to 490 nm corresponds to the absorption edge of o-methylbenzaldehydes (see Figure 6), which was used as the photoresponsive substance, and almost no photoreaction occurred.

[0084] In contrast, when UV light was used, as shown in Table 1, a fluorescence intensity of 31.5 was measured in the region irradiated with UV light (e.g., Firr) even in the region other than the plasmon generating region. This value is clearly greater than the fluorescence intensity of 24.2 in the region not irradiated with UV light (e.g., Fout). This means that the photoreaction proceeds in the region irradiated with UV light even in the region other than the plasmon generating region, and some of the capture molecules bind; in other words, the binding of the capture molecules cannot be sufficiently suppressed. [Example]

[0085] An experiment was conducted to confirm that the use of visible light to promote the photoreaction allows capture molecules to be densely bound to the center of each plasmon generating region. Specifically, a chip body (see FIG. 17) with the same structure, material, and dimensions as in Example 4 was prepared. The center of each plasmon generating region had a recessed structure with a size of 1 / 2 pitch. As in Example 4, o-Methylbenzaldehydes (see FIG. 6) was bound to the chip using GFP light (wavelength 450 nm to 490 nm) as the light to promote the photoreaction. Next, Cy5-maleimide adjusted to 9.36 nM was added, and the chip was washed with PBS (phosphate buffer solution). The prepared chip is referred to as the chip of Example 5.

[0086] As a first comparative example, the same chip body as above (see Figure 17) was prepared, and o-Methylbenzaldehydes (see Figure 6) was added to the chip without irradiating it with light to promote the photoreaction. Cy5-maleimide was not added. The fabricated chip is referred to as the chip of the first comparative example. Furthermore, as a second comparative example, the same chip body as above (see Figure 17) was prepared, and o-Methylbenzaldehydes (see Figure 6) was added to the chip without irradiating it with light to promote the photoreaction. Subsequently, Cy5-maleimide adjusted to 9.36 nM was added, and the chip was then washed with PBS. The fabricated chip is referred to as the chip of the second comparative example.

[0087] Fluorescence observation was performed in the same manner as in Example 1 using the three types of chips prepared as described above. Fluorescence images captured by a CCD camera are shown in Figures 25 to 27. Figures 25 to 27 are images of the chip of Comparative Example 1, the chip of Comparative Example 2, and the chip of Example 5, respectively, and corresponding regions were imaged. The bar shown in the lower right of each figure represents a length of 20 μm. Fluorescence observation was performed using the configuration shown in Figure 12, using a mercury lamp as the light source 402 and objective lenses with magnifications of 20x and 100x as the first lens 406 and the second lens 408, respectively.

[0088] The plasmon generating region can be seen slightly in the fluorescence image in Figure 25. The measured values ​​for the chip of the first comparative example were the fluorescence intensity B(BKG) in the plasmon generating region and the fluorescence intensity F(BKG) outside the plasmon generating region, which were 540 and 523, respectively. The measured values, including the values ​​shown below, are relative values ​​expressed using the same standard. "BKG" means background, and B(BKG) and F(BKG) are the background noise levels inside the plasmon generating region and outside the plasmon generating region, respectively.

[0089] In the fluorescence image of Figure 26, plasmon generating regions can be clearly confirmed compared to the fluorescence image of Figure 25, and it can be seen that within each plasmon generating region, the fluorescence intensity in the center tends to be stronger than the fluorescence intensity in the peripheral region. For the chip of the second comparative example, the fluorescence intensity Bc (unirradiated) in the center of the plasmon generating region, the fluorescence intensity Be (unirradiated) in the peripheral region of the plasmon generating region, and the fluorescence intensity F (unirradiated) outside the plasmon generating region were 760, 670, and 543, respectively. Note that "unirradiated" indicates that light to promote the photoreaction was not irradiated.

[0090] The fluorescence image in Figure 27 shows a clear increase in fluorescence intensity in the plasmon generating region compared to the image in Figure 26. It can also be seen that within each plasmon generating region, the fluorescence intensity in the center is clearly stronger than that in the periphery. For the chip of Example 5, the fluorescence intensity Bc (irradiated) in the center of the plasmon generating region, the fluorescence intensity Be (irradiated) in the periphery of the plasmon generating region, and the fluorescence intensity F (irradiated) outside the plasmon generating region were 890, 710, and 543, respectively. Note that "irradiated" indicates that no light was irradiated to promote the photoreaction.

[0091] Using the above measurements, the corresponding background noise (B(BKG) or F(BKG)) is subtracted to obtain ΔB and ΔF, which are used to evaluate the noise-free fluorescence intensity. Specifically, for the chip of Comparative Example 2, ΔBc (unirradiated) = 220 (= 760 - 540) at the center of the plasmon generating region, and ΔBe (unirradiated) = 130 (= 670 - 540) at the periphery. Therefore, for the chip of Comparative Example 2, the fluorescence intensity obtained at the center of the plasmon generating region was 1.69 times (= 220 / 130) higher than that at the periphery. Because the chip of Comparative Example 2 was not irradiated with light to promote the photoreaction, it is believed that Cy5-maleimide is primarily bound to the chip by nonspecific adsorption, and there is no difference in the number of Cy5-maleimide molecules bound at the center and periphery of the plasmon generating region. Therefore, the 1.69-fold increase is due to the optical antenna effect during fluorescence observation (i.e., the formation of an enhanced electric field due to localized plasmons in the center of the bull's-eye structure). Outside the plasmon generation region, ΔF(unirradiated) = 20 (= 543 - 523).

[0092] Similarly, the fluorescence intensity with noise removed can be evaluated for the chip of Example 5. That is, for the chip of Example 5, ΔBc(irradiation) = 350 (= 890 - 540) at the center of the plasmon generating region, and ΔBe(irradiation) = 170 (= 710 - 540) at the periphery. Therefore, for the chip of Example 5, the fluorescence intensity obtained at the center of the plasmon generating region was approximately 2.05 times (= 350 / 170) that at the periphery. Also, outside the plasmon generating region, ΔF(irradiation)=20(=543−523).

[0093] The chip of Example 5 contains Cy5-maleimide bound by photoreaction in addition to Cy5-maleimide bound by nonspecific adsorption. The reason why ΔBc (irradiated) and ΔBe (irradiated) for the chip of Example 5 are larger than ΔBc (unirradiated) and ΔBe (unirradiated) for the chip of Comparative Example 2, respectively, is due to photoreaction. To evaluate the influence of photoreaction, the difference R (= ΔB (irradiated) - ΔB (unirradiated)) between the measured values ​​of corresponding regions for the chip of Example 5 and the chip of Comparative Example 2 was calculated. Using the above calculated values, the difference Rc (= ΔBc (irradiated) - ΔBc (unirradiated)) between the chip of Example 5 and the chip of Comparative Example 2 for the center of the plasmon generation region is obtained as Rc = 130 (= 350 - 220). The difference Re (= ΔBe (irradiated) - ΔBe (unirradiated)) for the peripheral portion of the plasmon generation region between the chip of Example 5 and the chip of Comparative Example 2 is obtained as Re = 40 (= 170 - 130). Therefore, Rc / Re = 3.25 (= 130 / 40). Rc / Re represents the ratio of the fluorescence intensity at the center of the plasmon generating region to the fluorescence intensity at the periphery of Cy5-maleimide bound by photoreaction. That is, for the chip of Example 5, it can be seen that the fluorescence intensity observed at the center of the plasmon generating region was 3.25 times higher than at the periphery due to the capture molecules bound to the chip by photoreaction. As mentioned above, this center magnification of 3.25 also includes the influence of the optical antenna effect during fluorescence observation. Therefore, by dividing this by the center magnification of 1.69 for the chip of Comparative Example 2, the influence of the optical antenna effect during fluorescence observation can be eliminated. The calculated value is approximately 1.9 (= 3.25 / 1.69). Therefore, by irradiating light to promote the photoreaction, 1.9 times more capture molecules were bound to the center of the plasmon generating region than to the periphery. That is, it was confirmed that by using visible light as light for promoting the photoreaction, it is possible to bind the capture molecules to the center of each plasmon generating region at high density.

[0094] Although the present invention has been described above by explaining the embodiments, the above-described embodiments are merely examples, and the present invention is not limited to the above-described embodiments. The scope of the present invention is defined by the claims in the scope of the claims, taking into consideration the description of the detailed description of the invention, and includes all modifications within the meaning and scope equivalent to the wordings described therein. [Explanation of symbols]

[0095] 100 Sensing Chip 102 Chip body 104 Plasmon generation region 106, 180, 190 base board 110 1st adhesive layer 112 Metal layer 114 Second adhesive layer 116 Quenching suppression layer 182, 192 convex part 184, 194 recess 200 Binding Compounds 202 Capture molecules 210 APTES 212 Benzaldehyde 214 N-Succinimidyl-3-maleimidopropionate 300 Biotin-Maleimide 302 Biotin section 304 Maleimide part 312 Cy5-Streptavidin 400 Measuring Equipment 402 Light source 404 Optical Filter 406 First Lens 408 Second lens 410 Camera Birr, Bout, Firr, Fout area L1 period L2, φ diameter

Claims

1. a substrate having a plasmon generating region that generates propagating plasmons; a plurality of capture molecules for capturing the target substance; the plasmon generating region has a concentric periodic uneven structure and includes a metal layer having the same periodicity as the uneven structure; the plurality of capture molecules are bound to the plasmon generating region at a higher density than in a region surrounding the plasmon generating region by a compound containing a photoreactive compound; the plasmon generating region includes a central portion of the plasmon generating region and a peripheral region that is a region outside the central portion, The plurality of capture molecules are bound to the central portion at a higher density than to the surrounding region.

2. The sensing chip according to claim 1 , wherein the plurality of capture molecules are bound to the plasmon generating region at a density that is 1.2 times or more higher than that of the region surrounding the plasmon generating region.

3. A sensing chip as described in claim 1 or claim 2, wherein the multiple capture molecules are dispersed in the plasmon generating region and the region surrounding the plasmon generating region.

4. the predetermined portion including the center of the concentric circle has a convex or concave shape, 3. The sensing chip according to claim 1, wherein the predetermined portion is a circle having a diameter equal to or smaller than one period of the concave-convex structure.

5. a substrate having a plasmon generating region including a metal layer with a periodic structure that generates propagating plasmons; a plurality of capture molecules for capturing the target substance; the plurality of capture molecules are bound to the plasmon generating region at a higher density than in a region surrounding the plasmon generating region by a compound containing a photoreactive compound; the capture molecule comprises biotin; The photoreactive compound includes a compound represented by the following general formula (1) or general formula (2), or TFPA-PEG3-Biotin, The sensing chip, wherein the biotin is bound to the plasmon generating region via a compound of maleimide, the photoreactive compound, and 3-aminopropyl triethoxysilane. 【Chemistry 1】 In the general formula (1) and the general formula (2), R is any one of the following compounds A1 to A11. 【Chemistry 2】

6. A method for producing a photoreactive compound, comprising: a first step of introducing a photoreactive compound having a capture molecule bound thereto for capturing a target substance onto a substrate having a plasmon generating region including a metal layer having a periodic structure for generating propagating plasmons; a second step of irradiating light from the back surface of the substrate on which the first step has been performed, In the second step, the photoreaction of the photoreactive compound is promoted by an enhanced electric field due to plasmons, and the capture molecules are bound to the plasmon generating region.

7. A method for producing a substrate having a plasmon generating region including a metal layer with a periodic structure that generates propagating plasmons, the substrate having a photoreactive compound bonded thereto, the method comprising: a first step of introducing capture molecules for capturing a target substance onto the substrate; a second step of irradiating light from the back surface of the substrate on which the first step has been performed, In the second step, the photoreaction of the photoreactive compound is promoted by an enhanced electric field due to plasmons, and the capture molecules are bound to the plasmon generating region.

8. 8. The method for manufacturing a sensing chip according to claim 6, wherein the light irradiated in the second step has a wavelength of 300 nm or more and 550 nm or less, or 600 nm or more and 1100 nm or less.

9. The method for manufacturing a sensing chip according to any one of claims 6 to 8, wherein the photoreactive compound includes a compound represented by the following general formula (1) or general formula (2), or TFPA-PEG3-Biotin: 【Transformation 3】 In the general formula (1) and the general formula (2), R is any one of the following compounds A1 to A11. 【Chemistry 4】

10. The method for manufacturing a sensing chip according to claim 9 , wherein the light irradiated in the second step has a wavelength of 450 nm or more and 490 nm or less.

11. A substrate having a plasmon generating region including a metal layer with a periodic structure that generates propagating plasmons; a photoreactive compound; A sensing kit in which the photoreactive compound and a plurality of capture molecules for capturing a target substance are placed on the substrate, and light is irradiated from the back of the substrate, whereby the photoreaction of the photoreactive compound is promoted by an enhanced electric field caused by plasmons, and the photoreactive compound binds the plurality of capture molecules to the plasmon generating region at a higher density than in the region surrounding the plasmon generating region.

12. a first step of introducing the target substance bound to a fluorescent substance into the sensing chip according to any one of claims 1 to 5; a second step of irradiating light from the back of the sensing chip on which the first step has been performed and measuring the fluorescence emitted from the fluorescent material due to the enhanced electric field caused by plasmons from the front of the sensing chip.

13. A light source and a lens for focusing light from the light source; In a state where a photoreactive compound to which a capture molecule for capturing a target substance is bound is placed on a substrate having a plasmon generating region including a metal layer with a periodic structure that generates propagating plasmons, the light focused by the lens is irradiated from the back surface of the substrate, thereby promoting the photoreaction of the photoreactive compound by an enhanced electric field caused by the plasmons, and binding the capture molecule to the plasmon generating region, a measuring unit that measures fluorescence emitted from the fluorescent substance due to an enhanced electric field caused by plasmons by irradiating the back surface of the substrate with the light focused by the lens in a state in which a target substance containing a fluorescent substance is introduced into the substrate having the capture molecules bound to the plasmon generating region.

Images