Method for performing plasmon-enhanced fluorescence analysis to detect antigens in a sample using nanoparticles

Abstract

To provide a nanoparticle body having improved detection sensitivity capable of contributing to detection of a relatively low-concentration biomatter.SOLUTION: A nanoparticle body 1 is used for a plasmon excitation fluorometric analysis. It has a metal nanoparticle 2, and a nanoantibody 4 coupled to a surface of the metal nanoparticle 2, and molecular mass of the nanoantibody is equal to or less than 60,000 Da.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a nanoparticle, and in particular to a nanoparticle for use in plasmon excited fluorescence analysis. [Background technology]

[0002] A biosensor specifically reacts a specific antigen, which is the detection target, with a specific antibody to form a complex, and detects the antigen based on a signal derived from the specific binding in the complex. In plasmon excitation fluorescence analysis, the complex contains a fluorescent substance and a metal in addition to the antibody and antigen. When excitation light is irradiated onto the complex, surface plasmon resonance occurs in the metal in the complex, forming a near-field near the surface of the metal. This near-field increases the fluorescence intensity of the fluorescent substance.

[0003] For example, in the localized plasmon enhanced sensor described in Patent Document 1, a large number of convex portions each having a height of 100 to 1000 nm, a width of 20 to 1000 nm, and an aspect ratio of 2 to 10 are formed on one surface of a substrate, a metal film having a thickness of 40 to 120 nm is laminated on the surface of the convex portions, a dielectric layer is laminated on the metal surface, and a specific binding member capable of specifically binding to a substance to be measured in a specimen to form a specific binding complex is fixed to the surface of the dielectric layer. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2007-240361 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, as a result of extensive investigations by the present inventors, it has been found that there is room for further improvement in the detection sensitivity of the above-mentioned sensor.

[0006] The present invention has been made in view of the above problems. That is, a main object of the present invention is to provide a nanoparticle having excellent detection sensitivity, which is useful for detecting a biological material at a relatively low concentration. [Means for solving the problem]

[0007] The nanoparticle body according to one embodiment of the present invention comprises: A nanoparticle and a nanobody bound to a surface of the nanoparticle, The nanobody has a molecular mass of less than 60,000 Da. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a cross-sectional view showing a schematic diagram of a nanoparticle. [Diagram 2] FIG. 2 is a cross-sectional view that illustrates a schematic diagram of the composite body. [Diagram 3] FIG. 3 is a schematic diagram of a measuring device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, nanoparticles according to embodiments of the present invention will be described in detail with reference to the drawings. Note that the drawings include schematic drawings and may not reflect actual dimensions or proportions.

[0010] <First embodiment: nanoparticle body> The nanoparticle according to the first embodiment is A nanoparticle and a nanobody bound to a surface of the nanoparticle, The nanobody has a molecular mass of 60,000 Da or less.

[0011] The nanoparticle body according to this embodiment has excellent detection sensitivity. The reason is presumed to be as follows. The nanoparticle body according to this embodiment has a metal nanoparticle and a nanoantibody bound to the surface of the metal nanoparticle. Therefore, the nanoantibody of the nanoparticle body undergoes an antigen-antibody reaction with an antigen in a sample to form a complex. The formed complex is composed of two nanoparticle bodies bound via the antigen. That is, the two metal nanoparticle bodies are arranged at a certain distance apart by each nanoantibody binding to the same antigen in the complex. Here, since the molecular mass of the nanoantibody is 60,000 Da or less, the volume of the nanoantibody is relatively small. Therefore, the distance (separation distance) between the two nanoparticle bodies can be made small. Therefore, in surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), when excitation light is irradiated to the complex, a localized surface plasmon resonance (LSPR) occurs, and a near field is efficiently formed near the surface of the metal nanoparticle (particularly, near the surface between the two metal particles). It is believed that the fluorescent substance of the complex is efficiently excited by this near field, and the fluorescence intensity increases. Therefore, the nanoparticle according to this embodiment has excellent detection sensitivity. In particular, even if the concentration of the antigen in the sample is low, it can be detected.

[0012] The present inventors have investigated the reason why the detection sensitivity of conventional nanoparticles is low, and have focused on the fact that a complex capturing an antigen cannot effectively induce localized surface plasmon resonance. The inventors have found that the reason is that the distance between metal nanoparticles is relatively large. Based on this finding, the present inventors have further investigated and have come up with the idea of ​​forming a complex in which the distance between nanoparticles is sufficiently narrowed by adopting a nanoantibody having a relatively small volume, thereby increasing the fluorescence intensity and improving the detection sensitivity. In other words, the present inventors have come up with the idea that "the molecular mass of the nanoantibody is 60,000 Da or less."

[0013] The nanoparticle body can capture an antigen through an antibody-antigen reaction and form a complex consisting of two nanoparticle bodies and the antigen. The nanoparticle body will be described with reference to FIG. 1. FIG. 1 is a cross-sectional view showing a schematic diagram of a nanoparticle body. The nanoparticle body 1 according to this embodiment has a metal nanoparticle 2 and a nanoantibody 4 bound to the surface of the metal nanoparticle 2.

[0014] The nanoparticle 1 can be used for plasmon excitation fluorescence analysis. The nanoparticle 1 captures an antigen in a sample through an antigen-antibody reaction, forming a complex, which will be described later. When the complex is irradiated with excitation light, localized surface plasmon resonance occurs, forming a near field. This near field increases the fluorescence intensity.

[0015] The nanoparticle body 1 may also have non-specific binding sites blocked by a blocking agent. The blocked nanoparticle body 1 suppresses the formation of non-specific binding to substances other than the detection target of the nanobody 4, thereby reducing background and false positive signals. Examples of blocking agents include proteins such as bovine serum albumin (BSA), skim milk, and casein, as well as chemically synthesized polymers.

[0016] (Metal Nanoparticles) The metal nanoparticles 2 interact with light having a specific wavelength, which varies depending on the type of metal, and cause localized surface plasmon resonance. The plasmon resonance peak is between 400 nm and 530 nm for silver nanoparticles and between 510 nm and 580 nm for gold nanoparticles. This varies depending on the particle diameter. For example, silver nanoparticles with a particle diameter of 20 nm resonate with light with a wavelength of 405 nm, and gold nanoparticles with a particle diameter of 20 nm resonate with light with a wavelength of 524 nm. The particle diameter (average primary particle diameter) of the metal nanoparticles 2 is, for example, 5 nm to 100 nm. The particle diameter of the metal nanoparticles 2 can be obtained by capturing an image of the metal nanoparticles 2 using a scanning electron microscope (SEM) or a transmission electron microscope (TEM), measuring the particle diameter of the metal nanoparticles 2 in the image, and calculating the average value of multiple particle diameters (number of measurements: at least 10 or more). The metal nanoparticles 2 preferably comprise gold or silver, more preferably comprise silver.

[0017] (Nanoantibody) The nanoantibody 4 specifically binds to a specific antigen at its tip (antigen binding site) through an antigen-antibody reaction. This specific binding is a non-covalent bond, for example, a bond resulting from hydrogen bonds, as well as intermolecular forces, hydrophobic interactions, and charge interactions. The nanoantibody 4 is, for example, a variable domain heavy chain antibody (VHH) antibody and a variant thereof. The VHH antibody is a single domain antibody. The variant is an antibody in which a part of the amino acid sequence has been modified or a substituent has been introduced within a range that has specific binding ability to the antigen. The nanoantibody 4 is preferably a VHH antibody. When the nanoantibody 4 is a VHH antibody, the VHH antibody has a relatively small volume, so that the separation distance in the complex can be narrowed, the near field can be formed more efficiently, and the fluorescence intensity can be further increased.

[0018] The molecular mass of the nanobody 4 is 60,000 Da or less, preferably 30,000 Da or less, more preferably 20,000 Da or less. When the molecular mass is 60,000 Da or less (particularly 30,000 Da or less or 20,000 Da or less), the volume of the nanobody 4 is relatively small, so that the separation distance in the complex can be narrowed, the near field can be formed more efficiently, and the fluorescence intensity can be further increased. Methods for measuring the molecular mass include electrophoresis (SDS-PAGE), gel filtration chromatography, and static light scattering.

[0019] The nanoantibody 4 is directly bonded to the metal nanoparticle 2. Here, the term "direct bond" includes not only chemical bonds such as single covalent bonds, but also non-chemical bonds such as physical adsorption. When the nanoantibody 4 is directly bonded to the surface of the metal nanoparticle 2, the distance between the two metal nanoparticles in the complex can be further narrowed, which is preferable.

[0020] <Second embodiment: composite> The complex will be described with reference to FIG. 2. FIG. 2 is a cross-sectional view that shows a schematic diagram of the complex. The complex 40 comprises an antigen 30 to be detected, and two nanoparticles 10, 20. In the complex 40, the two nanoparticles 10, 20 are bound via the antigen 30. One of the two nanoparticles 10, 20 will be referred to as the first nanoparticle 10, and the other nanoparticle will be referred to as the second nanoparticle 20. In this way, the complex 40 includes the first nanoparticle 10 and the second nanoparticle 20 as the nanoparticle 1.

[0021] In the complex 40, the first nanoparticle body 10 has a first metal nanoparticle 12, a first nanoantibody 14 bound to the surface of the first metal nanoparticle 12, and a fluorescent substance 16 labeled to the first nanoantibody 14. In this manner, the first nanoparticle body 10 can further have the fluorescent substance 16. The second nanoparticle body 20 has a second metal nanoparticle 22, and a second nanoantibody 24 bound to the surface of the second metal nanoparticle 22.

[0022] Distance between the first nanoparticle body 10 and the second nanoparticle body 20 between The distance L is 10 nm to 50 nm, and preferably 10 nm to 25 nm. between The distance L is the distance between the first metal nanoparticle 12 and the second metal nanoparticle 22, and is the distance at which the line segment connecting the first point P1 on the surface of the first nanoparticle 10 and the second point P2 on the surface of the second nanoparticle 20 is the shortest. between When the distance L is 50 nm or less, when the complex 40 is irradiated with excitation light, a near-field is generated more efficiently in the space near the surface between the first and second metal nanoparticles 12, 22, and the fluorescence intensity can be increased further.

[0023] (Nanoparticles) Hereinafter, the first metal nanoparticle 12 will be described as an example, but the second metal nanoparticle 22 is substantially the same as the first metal nanoparticle 12. The first metal nanoparticle 12 has a first core 12a and a first coating layer 12b that covers the surface of the first core 12a. In other words, the first metal nanoparticle 12 has a core-shell structure. The first coating layer 12b can function as a metal quenching prevention film. The first coating layer 12b completely covers the first core 12a. When the first coating layer 12b completely covers the first core 12a, the fluorescent substance 16 does not contact the first core 12a, and the fluorescent substance 16 is reliably arranged at a predetermined distance (at least a distance equivalent to the thickness of the coating layer 12b) from the surface of the first core 12a. In this case, the excited fluorescent substance 16 is less likely to be quenched by the material (for example, a metal (more specifically, gold or silver, etc.)) that constitutes the first core 12a, so that the decrease in the detected fluorescence intensity is suppressed and the detection sensitivity is improved. This quenching is believed to be a process of energy transfer from excited fluorescent material 16 to the material of first metal nanoparticles 12 and / or a non-radiative (thermal emission) process induced by the material of first metal nanoparticles 12 .

[0024] The first core 12a preferably comprises gold or silver, more preferably silver. The first coating layer 12b comprises, for example, an inorganic material (more specifically, SiO2). When the first coating layer 12b comprises an inorganic material, the excited fluorescent material 16 is less likely to be quenched in the plasmon excitation fluorescence analysis, and a decrease in the detected fluorescence intensity is suppressed.

[0025] The thickness of the first coating layer 12b is preferably 5 nm to 50 nm. When the thickness of the first coating layer 12b is 50 nm or less, the distance between the two metal nanoparticles 12, 22 is such that a near field is efficiently formed in the space between them, further improving the detection sensitivity. When the thickness of the first coating layer 12b is 5 nm or more, the first core 12a and the fluorescent substance 16 are disposed at a predetermined distance, so that quenching of the excited fluorescent substance 16 during measurement is suppressed, further improving the detection sensitivity.

[0026] (Fluorescent material) The fluorescent substance 16 is labeled on the first nanoparticle 10, but is not labeled on the second nanoparticle 20. The fluorescent substance 16 is excited by a near field formed by localized surface plasmon resonance and emits fluorescence. The fluorescent substance 16 is, for example, a complex (metal complex) of a metal such as europium or ruthenium. As shown in FIG. 2, the fluorescent substance 16 is preferably positioned between the first metal nanoparticle 12 and the second metal nanoparticle 22. This is because the fluorescent substance 16 is positioned in the space between the metal nanoparticles 12 and 22, which is a space in which a near field is efficiently generated, and therefore, by positioning the fluorescent substance 16 in the space between the metal nanoparticles 12 and 22, the fluorescence intensity is likely to be increased.

[0027] It is preferable that the fluorescent substance 16 has a large Stokes shift. Here, the Stokes shift is the difference between the absorption peak wavelength (maximum excitation wavelength) in the absorption spectrum of the fluorescent substance 16 and the fluorescence peak wavelength (maximum fluorescence wavelength) in the fluorescence spectrum. When the Stokes shift of the fluorescent substance 16 is large, the absorption spectrum and the fluorescence spectrum are less likely to overlap, and excitation light (scattered light) is less likely to enter the fluorescence to be detected, allowing for more accurate measurement of the fluorescence intensity.

[0028] It is preferable that the fluorescent spectrum of the fluorescent substance 16 is sharp. If the fluorescent spectrum is sharp, it is less likely to overlap with the absorption spectrum, so that (scattered) excitation light is less likely to enter the fluorescence to be detected, and the fluorescence intensity can be measured more accurately.

[0029] (antigen) The antigen 30 has at least two epitopes, and forms a specific bond with the first and second nanobodies 14 and 24 at the epitopes. The antigen 30 is, for example, a protein such as TnT (registered trademark) and a virus. The antigen 30 is, for example, an antigen derived from blood, plasma, serum, urine, and saliva. That is, examples of samples containing the antigen 30 include blood, plasma, serum, urine, and saliva. The sample may further contain a solvent and a buffer solution (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, MES buffer, etc.).

[0030] <Third embodiment: Measuring device> The measurement device will be described with reference to Fig. 3. Fig. 3 is a diagram showing the measurement device. As shown in Fig. 3, the measurement device 100 includes an excitation light source 110, an excitation light irradiation optical system 120, a reagent container 130, a light receiving optical system 140, and a light receiving element 150. The excitation light source 110 irradiates excitation light 112. The excitation light source 110 is, for example, a laser. The excitation light irradiation optical system 120 adjusts the cross-sectional diameter, such as by focusing the excitation light 112, and outputs the incident excitation light 122. The excitation light irradiation optical system 120 is a lens 124 and a polarizing element (λ / 2 plate) 126. The incident excitation light 122 output from the excitation light irradiation optical system 120 enters a reagent container 130 and is irradiated onto the measurement sample in the reagent container 130. The reagent container 130 is, for example, a removable container (more specifically, a cell, a slide, etc.) and a microchannel chip. The microchannel chip is a chip having a minute flow channel. When the reagent container 130 is a microchannel chip, for example, the nanoparticle body (reagent) according to the first embodiment and a specimen can be mixed and continuously supplied. Therefore, it is not necessary to mix and prepare the measurement sample in advance, and it is possible to perform continuous measurement.

[0031] The measurement sample irradiated with the incident excitation light 122 emits fluorescence (detection light 132). The light receiving optical system 140 is disposed in a direction perpendicular to the traveling direction of the incident excitation light 122 into the reagent container 130. The light receiving optical system 140 can adjust the cross-sectional diameter, etc., of the detection light 132 emitted from the measurement sample, and can remove scattered light of the incident excitation light 122 or adjust the amount of light. The light receiving optical system 140 includes a lens 144 and an optical filter 146. The optical filter 146 is, for example, a band-pass filter and a dichroic mirror.

[0032] The fluorescence 142 that passes through the light receiving optical system 140 is detected by the light receiving element 150. The light receiving element 150 is, for example, a PD, an APD, a PMT, a CCD camera, or a spectrometer. The light receiving element 150 is capable of measuring the amount of fluorescence at a single wavelength, measuring the fluorescence spectrum, and creating a two-dimensional fluorescent image.

[0033] The present invention is not limited to the above-described embodiment, and the design can be modified without departing from the gist of the present invention.

[0034] In the above-described embodiments, the nanoparticles 1, 10, 20 each have one nanobody 4, 14, 24, but are not limited to this. The nanoparticles 1, 10, 20 may each have two or more nanobodies 4, 14, 24.

[0035] In the above embodiment, the fluorescent substance 16 is labeled on the first nanoparticle body 10, but not on the second nanoparticle body 20, but this is not limiting. The fluorescent substance 16 may be labeled on at least one of the first nanoparticle body 10 and the second nanoparticle body 20. More specifically, the fluorescent substance 16 may be labeled on only the second nanoparticle body 20, or on both the first nanoparticle body 10 and the second nanoparticle body 20.

[0036] In the above embodiment, the nanoantibody 4, 14, 24 is directly bound to the surface of the metal nanoparticle 2, 12, 22, but is not limited thereto. The nanoantibody 4, 14, 24 may be indirectly bound to the surface of the metal nanoparticle 2, 12, 22 by, for example, interposing any compound that forms a biotin-avidin bond between the surface of the metal nanoparticle 2, 12, 22 and the nanoantibody 4, 14, 24.

[0037] In the above embodiment, the metal nanoparticles 12, 22 have a core-shell structure, but may not have such a structure. Also, in the above embodiment, the coating layer 12b, 22b completely (continuously) covers the core 12a, 22a, but may partially cover the core 12a, 22a.

[0038] In the above embodiment, the fluorescent substance 16 is labeled to the first nano antibody 14, but this is not limiting. The fluorescent substance 16 may be labeled to the first metal nanoparticle 12.

[0039] In the embodiment, the light receiving optical system 140 in the measuring device 100 is disposed in a direction perpendicular to the traveling direction of the incident excitation light 122 to the reagent container 130, but is not limited to this. The light receiving optical system 140 may be disposed, for example, in a direction parallel to the traveling direction of the incident excitation light 122, or in a direction forming an acute angle or an obtuse angle with respect to the traveling direction of the incident excitation light 122. [Explanation of symbols]

[0040] 1. Nanoparticles 2. Metal Nanoparticles 4. Nano antibodies 10. First nanoparticle body 12. First metal nanoparticles 12a 1st core 12b...First coating layer 14. The first nano antibody 16. Fluorescent materials 20 Second nanoparticle body 22 Second metal nanoparticles 22a 2nd core 22b...Second coating layer 24 Second Nanobody 30...antigen 40 Complex L...Separation distance

Claims

1. 1. A method for performing plasmon enhanced fluorescence analysis to detect an antigen in a sample using a nanoparticle, comprising: capturing an antigen in a specimen using a first nanoparticle and a second nanoparticle contained in the nanoparticle, and forming a complex in which the first nanoparticle and the second nanoparticle are bound to each other via the antigen; irradiating the complex with excitation light; Consisting of In the composite, the distance between the first nanoparticle and the second nanoparticle is 10 nm to 25 nm; The first nanoparticle body has a first metal nanoparticle and a first nanobody bound to a surface of the first metal nanoparticle, the second nanoparticle body has a second metal nanoparticle and a second nanobody bound to a surface of the second metal nanoparticle; At least one of the first nanoparticle and the second nanoparticle is labeled with a fluorescent substance; A method wherein said first and second Nanobodies have a molecular mass of 20,000 Da or less.

2. The method of claim 1 , wherein in the composite, the fluorescent material is located between the first metal nanoparticle of the first nanoparticle body and the second metal nanoparticle of the second nanoparticle body.

3. The method of claim 1 or 2, wherein the first and second nanobodies are VHH antibodies.

4. The method of any one of claims 1 to 3, wherein the first and second metal nanoparticles comprise gold or silver.

5. The method according to any one of claims 1 to 4, wherein the first and second metal nanoparticles have a core and a coating layer coating the surface of the core.

6. The coating layer is made of SiO ２ The method of claim 5, comprising:

7. The method of any one of claims 1 to 6, wherein the first nanoantibody is directly bound to the first metallic nanoparticle and the second nanoantibody is directly bound to the second metallic nanoparticle.

8. The method according to any one of claims 1 to 7, wherein the first nanoparticles are labeled with the fluorescent substance, whereas the second nanoparticles are not labeled with the fluorescent substance.

9. The method according to any one of claims 1 to 8, wherein the antigen is derived from blood, plasma, urine, or saliva.

10. A nanoparticle body having a metal nanoparticle and a nanobody bound to a surface of the metal nanoparticle, the nanobody has a molecular mass of 20,000 Da or less; The nanoparticles include a first nanoparticle and a second nanoparticle, At least one of the first nanoparticle and the second nanoparticle is labeled with a fluorescent substance; A nanoparticle body, in which in a complex in which the first nanoparticle body and the second nanoparticle body are bound via an antigen, the distance between the first nanoparticle body and the second nanoparticle body is 10 nm to 25 nm.

11. The nanoparticle according to claim 10, wherein the nanoparticle is a nanoparticle used for plasmon excited fluorescence analysis.

12. The nanoparticle according to claim 10 or 11, wherein the nanobody is a VHH antibody.

13. The nanoparticle of any one of claims 10 to 12, wherein the metal nanoparticles comprise gold or silver.

14. The nanoparticle according to any one of claims 10 to 13, wherein the metal nanoparticle has a core and a coating layer that coats the surface of the core.

15. The coating layer is made of SiO ２ The nanoparticle of claim 14, comprising:

16. The nanoparticle according to any one of claims 10 to 15, wherein the nanobody is directly bound to the metal nanoparticle.

17. The nanoparticle body according to any one of claims 10 to 16, wherein the first nanoparticle body is labeled with the fluorescent substance, whereas the second nanoparticle body is not labeled with the fluorescent substance.

18. The nanoparticle body according to any one of claims 10 to 17, wherein in the complex, the fluorescent substance is positioned between the metal nanoparticles of the first nanoparticle body and the metal nanoparticles of the second nanoparticle body.

19. The nanoparticle body according to any one of claims 10 to 18, wherein the antigen is an antigen derived from blood, plasma, urine or saliva.

Images