Method for detecting a substance to be detected and system for detecting a substance to be detected

Abstract

This detection method for a substance to be detected includes a step in which a sample is prepared that includes: a plurality of metal nanoparticles (11) (probe particles (1)), each of which is modified by a host molecule (12) that bonds specifically to the substance to be detected; and a plurality of microparticles (21) (probe particles (2)), each of which is modified by a host molecule (22) that bonds specifically to the substance to be detected. The preparation step includes combining the plurality of probe particles (1) and the plurality of probe particles (2), which mutually differ in size and material, so that: each of the plurality of probe particles (1) is of a size that allows diffusion by Brownian motion within the sample; each of the plurality of probe particles (2) is of a size that allows being subject to dissipative force caused by Mie scattering of nonresonant light; and each of the plurality of probe particles (1) includes a material that causes thermal convection to be produced within the sample by irradiation with nonresonant light.

Description

Technical Field

[0001] The present disclosure relates to a method for detecting a target substance and a detection system for a target substance, and more particularly to a technique for detecting a target substance that may be contained in a sample by light irradiation.

Background Art

[0002] International Publication No. 2014 / 192937 (Patent Document 1) and International Publication No. 2021 / 040021 (Patent Document 2) disclose techniques for detecting a target substance that may be contained in a sample by light irradiation. In addition, International Publication No. 2017 / 213107 (Patent Document 3) discloses a technique for integrating nanocapsules encapsulating metal nanoparticles.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] There is always a need for a technique for detecting a trace amount of a target substance or shortening the detection time of a target substance, that is, a technique capable of detecting a target substance with high sensitivity and rapidly.

[0005] The present disclosure has been made to solve the above problems, and one of the objects of the present disclosure is to detect a target substance with high sensitivity and rapidly.

Means for Solving the Problems

[0006] (1) A method for detecting a substance to be detected according to the first aspect of this disclosure comprises first to third steps. The first step is to prepare a liquid sample comprising a plurality of nanoparticles, each modified by a first host molecule that specifically binds to the substance to be detected, and a plurality of microparticles, each modified by a second host molecule that specifically binds to the substance to be detected. The second step is to irradiate the liquid sample with non-resonant light, which is light outside the wavelength range of the electronic resonance of the plurality of nanoparticles and outside the wavelength range of the electronic resonance of the plurality of microparticles. The third step is to detect the substance to be detected based on the output signal from a photodetector that has received light from the liquid sample. The preparation step (first step) includes combining a plurality of nanoparticles and a plurality of microparticles of different sizes and materials such that each of the plurality of nanoparticles has a size that diffuses in the liquid sample by Brownian motion, and each of the plurality of microparticles has a size that is subject to dissipative force by Mie scattering of non-resonant light, and each of the plurality of nanoparticles contains a material that generates thermal convection in the liquid sample upon irradiation with non-resonant light.

[0007] (2) The preparation step (first step) further includes the step of combining a plurality of nanoparticles and a plurality of microparticles having different concentrations from each other such that the average interparticle distance of the plurality of nanoparticles is shorter than the average interparticle distance of the plurality of microparticles.

[0008] (3) The preparation step further includes the step of uniformly mixing multiple nanoparticles and multiple microparticles.

[0009] (4) A method for detecting a substance to be detected further includes the steps of holding a liquid sample on a substrate and, after the holding step, adjusting the irradiation position of the non-resonant light so that the focal point of the non-resonant light is located behind the substrate in the direction of propagation of the non-resonant light.

[0010] (5) A method for detecting a substance to be detected further includes the steps of adjusting the irradiation position of non-resonant light such that the focal point of the non-resonant light is located behind the substrate in the direction of propagation of the non-resonant light, and after the adjustment step, holding the liquid sample on the substrate.

[0011] (6) A method for detecting a substance to be detected further includes the step of holding a liquid sample in a well provided in a substrate. The irradiation step (second step) includes the step of focusing non-resonant light using a focusing lens including a correction ring. A method for detecting a substance to be detected further includes the step of adjusting the correction ring so that spherical aberration correction is performed according to the thickness of the substrate and the depth of the well.

[0012] (7) The substance to be detected is labeled with a fluorescent dye. The detection step (third step) includes measuring the fluorescence intensity of a liquid sample and detecting the substance to be detected based on the change in fluorescence intensity upon irradiation with non-resonant light.

[0013] (8) The substance to be detected is DNA containing multiple bases. The detection step includes detecting single nucleotide polymorphisms in DNA.

[0014] (9) The detection step includes measuring the absorbance spectrum of a liquid sample and detecting the substance to be detected based on the amount of change in the absorbance spectrum due to irradiation with non-resonant light.

[0015] (10) The detection step includes taking an image of an accumulation region in which multiple nanoparticles and multiple microparticles are accumulated, and detecting the substance to be detected based on the area of ​​the accumulation region.

[0016] (11) The liquid sample is a highly viscous liquid sample containing impurities. The detection step includes the step of specifically detecting the substance to be detected from the highly viscous liquid sample.

[0017] (12) The detection system for the analyte according to the second aspect of the present disclosure includes a plurality of nanoparticles each modified with a first host molecule that specifically binds to the analyte, a plurality of microparticles each modified with a second host molecule that specifically binds to the analyte, a light source that emits non-resonant light, which is light outside the wavelength range of the electronic resonance of the plurality of nanoparticles and outside the wavelength range of the electronic resonance of the plurality of microparticles, a light receiver that receives light from a liquid sample containing the plurality of nanoparticles and the plurality of microparticles irradiated with the non-resonant light, and a processor that executes a detection process for detecting the analyte based on an output signal from the light receiver. The plurality of nanoparticles and the plurality of microparticles are sized such that each of the plurality of nanoparticles undergoes Brownian motion due to collisions with the dispersion medium molecules of the liquid sample, and each of the plurality of microparticles is sized to receive a dissipative force due to Mie scattering of the non-resonant light, and the sizes and materials are combined such that each of the plurality of nanoparticles contains a material that generates thermal convection in the liquid sample upon irradiation with the non-resonant light, and the sizes and materials are different from each other.

Advantages of the Invention

[0018] According to the present disclosure, the analyte can be detected with high sensitivity and quickly.

Brief Description of the Drawings

[0019] [Figure 1] It is a diagram showing the first probe particles in the present embodiment. [Figure 2] It is a diagram showing the second probe particles in the present embodiment. [Figure 3] It is a diagram for explaining the detection principle of target DNA using the first and second probe particles. [Figure 4] It is a diagram showing an example of the overall configuration of the detection system for the analyte according to the present embodiment. [Figure 5] It is a diagram showing another example of the overall configuration in the upright arrangement of the detection system for the analyte according to the present embodiment. [Figure 6] It is a perspective view schematically showing the configuration of the detection kit. [Figure 7]This figure shows an image of the detection kit that was actually manufactured. [Figure 8] This diagram illustrates the method of irradiating the detection kit with laser light. [Figure 9] This flowchart shows the processing procedure for detecting the substance to be detected. [Figure 10] This diagram illustrates the dissipative force acting on the probe particle. [Figure 11] This figure shows the simulation results regarding the Brownian motion of the probe particle. [Figure 12] This is a conceptual diagram to explain the average interparticle distance of probe particles. [Figure 13] This diagram illustrates the evaluation results regarding the combination of probe particles. [Figure 14] This figure shows an example of an image captured by the camera in Example 2. [Figure 15] This figure shows an example of an absorbance spectrum measured by a spectrometer in Example 2. [Figure 16] This figure shows the dependence of the absorbance difference on the target concentration. [Figure 17] This figure shows the dependence of the probe particle accumulation area on the target concentration. [Figure 18] This diagram summarizes the measurement conditions in Example 3. [Figure 19] This figure shows the transmission and fluorescence images of Sample 1. [Figure 20] This figure shows the transmission and fluorescence images of Sample 2. [Figure 21] This figure shows the transmission and fluorescence images of Sample 3. [Figure 22] This figure shows the transmission and fluorescence images of sample 4. [Figure 23] This figure shows the transmission and fluorescence images of sample 5. [Figure 24] This figure shows the transmission and fluorescence images of sample 6. [Figure 25] This figure shows the transmission and fluorescence images of sample 7. [Figure 26] This figure shows an example of measurement results for the positional dependence of fluorescence intensity. [Figure 27] This figure shows an example of a calibration curve obtained from the fluorescence intensity shown in Figure 26. [Figure 28] This figure shows an image capturing the enhancement of fluorescence on the surface of metal nanoparticles contained in probe particles. [Figure 29] Figure 1 shows the sequence dependence of fluorescence intensity. [Figure 30] The second figure shows the sequence dependence of fluorescence intensity. [Figure 31] This figure shows the transmission and fluorescence images of a low-concentration sample. [Figure 32] This figure summarizes the fluorescence intensity measurements at various concentrations of target DNA9. [Figure 33] This is a diagram to explain the third improvement. [Figure 34] This is a diagram illustrating the eight types of target DNA. [Figure 35] This figure summarizes the fluorescence intensity measurement results for eight different target DNAs. [Figure 36] This figure shows the detection results of target DNA in highly viscous samples. [Modes for carrying out the invention]

[0020] The embodiments of this disclosure will be described in detail below with reference to the drawings. In the drawings, the same or corresponding parts are denoted by the same reference numerals, and their descriptions will not be repeated.

[0021] <Explanation of Terms> In this disclosure and its embodiments, "nanometer order" includes the range from 1 nm to 1000 nm (= 1 μm). "Micrometer order" includes the range from 1 μm to 1000 μm (= 1 mm). Therefore, "from nanometer order to micrometer order" includes the range from 1 nm to 1000 μm. The term "from nanometer order to micrometer order" typically means a range of a few nm to several hundred μm, preferably a range of 100 nm to 100 μm, and more preferably a range of 1 μm to several tens of μm.

[0022] In this disclosure and its embodiments, “sample” means a substance containing or potentially containing the substance to be detected. A sample may be a biological sample from an animal (such as a human, cattle, horse, pig, goat, chicken, rat, or mouse). A biological sample may include, for example, blood, tissue, cells, secretions, body fluids, etc. A “sample” may also include dilutions or isolates thereof (such as serum or plasma). A “liquid sample” is a liquid containing a sample.

[0023] In this disclosure and its embodiments, "analyte" means a substance to be detected having a size ranging from the nanometer order to the micrometer order. The shape of the analyte is not particularly limited and may be spherical, ellipsoidal, or rod-shaped. If the analyte is ellipsoidal, at least one of the lengths in the short axis and long axis directions of the ellipsoid may be in the range of nanometer to micrometer order. If the analyte is rod-shaped, at least one of the width and length of the rod may be in the range of nanometer to micrometer order.

[0024] The substances to be detected include, for example, cells, microorganisms (bacteria, fungi, etc.), vesicles (exosomes, microvexyl, apoptotic bodies, etc.), biomacromolecules (proteins, nucleic acids, lipids, polysaccharides, etc.), antigens (allergens, etc.), and viruses. Specific examples of proteins include CD (Cluster of Differentiation) classified antibodies such as CD9, CD63, CD80, and CD81, cytokines such as IL-6, and albumin. Nucleic acids include DNA or RNA. Specifically, nucleic acids may include cell-free DNA (cfDNA), circulating tumor DNA (ctDNA) derived from cancer cells, messenger RNA (mRNA), and microRNA (miRNA). However, the substances to be detected are not limited to biologically derived substances (biomaterials), but may also include resin beads, metal nanoparticles, metal nanoparticle aggregates, metal nanoparticle aggregate structures, semiconductor nanoparticles, and organic nanoparticles.

[0025] In this disclosure and its embodiments, "fine particle" means a substance having a size ranging from the nanometer order to the micrometer order. Fine particles include nanoparticles and microparticles.

[0026] In this disclosure and its embodiments, "nanoparticles" means particles having a size on the order of nanometers. The nanoparticles may be metal nanoparticles, semiconductor nanoparticles, or organic nanoparticles.

[0027] In this disclosure and its embodiments, "metal nanoparticles" means metal particles having a size on the order of nanometers. Metal nanoparticles may include metal nanoparticle aggregates and metal nanoparticle aggregate structures. A "metal nanoparticle aggregate" is an aggregate formed by the aggregation of multiple metal nanoparticles. A "metal nanoparticle aggregate structure" is, for example, a structure in which multiple metal nanoparticles are fixed to the surface of a bead via interaction sites, with gaps between them, and arranged at intervals smaller than or equal to the diameter of the metal nanoparticles.

[0028] The shape of the metal nanoparticles is not limited to a spherical shape; they may also be ellipsoidal or rod-shaped. If the metal nanoparticles are ellipsoidal, at least one of the length along the major axis and the length along the minor axis of the ellipsoid may be on the order of nanometers. If the metal nanoparticles are rod-shaped, at least one of the width and length of the rod may be on the order of nanometers.

[0029] In this disclosure and its embodiments, "semiconductor nanoparticles" means semiconductor particles having a size on the order of nanometers. "Organic nanoparticles" means particles made of organic compounds having a size on the order of nanometers. Similar to the shape of metal nanoparticles, the shape of semiconductor nanoparticles and organic nanoparticles is not particularly limited and may be spherical, ellipsoidal, rod-shaped, etc.

[0030] In this disclosure and its embodiments, "microparticles" means particles having a size on the order of micrometers. Microparticles may be resin beads, magnetic beads, or PM (particulate matter). "Resin beads" are particles made of resin having a size on the order of micrometers. "Magnetic beads" are magnetic particles (polymer particles with magnetic material dispersed or embedded inside) having a size on the order of micrometers. "PM" refers to particulate matter having a size on the order of micrometers. The material of the microparticles is not limited to resins or organic compounds, but may also be metals, semiconductors, etc.

[0031] In this disclosure and its embodiments, "host molecule" means a substance that can specifically bind to (or specifically adhere to) a substance to be detected. Examples of combinations of host molecules and substances to be detected include antigens and antibodies, glycans and proteins, lipids and proteins, small molecule compounds (ligands) and proteins, proteins and proteins, single-stranded DNA and single-stranded DNA. When either of these two substances with specific affinity is the substance to be detected, the other can be used as the host molecule.

[0032] For example, if the substance to be detected is single-stranded DNA (target DNA), the host molecule is another single-stranded DNA (probe DNA). Anti-DNA antibodies that specifically bind to DNA (e.g., anti-dsDNA that specifically binds to double-stranded DNA, anti-ssDNA that specifically binds to single-stranded DNA, etc.) can also be used as host molecules.

[0033] When the antigen is the substance to be detected, an antibody can be used as the host molecule. Conversely, when the antibody is the substance to be detected, the antigen can be used as the host molecule. The antigen may include allergens, microorganisms (bacteria, fungi, etc.), viruses, vesicles, etc. Furthermore, by changing the type of antibody, the types of allergens, microorganisms, or viruses that can be detected can also be changed. Therefore, the types of allergens, microorganisms, or viruses that can be detected by this disclosure are not particularly limited. In addition, when the substance to be detected is a heavy metal, a substance capable of capturing heavy metal ions can be used as the host molecule.

[0034] Host molecules are fixed to the surface of microparticles through interactions between the host molecules and the microparticles. The type of interaction used to fix the host molecules to the microparticle surface depends on the type of microparticle. Interactions include covalent bonds, ionic bonds, metallic bonds, van der Waals forces, electrostatic interactions, hydrophobic interactions, intermolecular forces (e.g., hydrogen bonds), and adsorption forces.

[0035] In this disclosure and its embodiments, the term "photo-induced force" is used as a general term for dissipative force, gradient force, and intermaterial photo-induced force. Dissipative force is a force that arises when the momentum of light is transferred to a substance in a dissipative process such as light scattering or light absorption. Gradient force is a force that moves a substance to a stable point in its electromagnetic potential when a substance that has undergone photo-induced polarization is placed in a non-uniform electromagnetic field. Intermaterial photo-induced force is the sum of the force due to the longitudinal electric field and the force due to the transverse electric field (radiation field) arising from the induced polarization in multiple photo-excited substances. Photo-induced polarization is electric polarization that arises when electrons inside a substance are excited by light.

[0036] In this disclosure and its embodiments, "visible light" means light in the wavelength range of 400 nm to 700 nm. "Infrared light" means light in the wavelength range of 700 nm to 700 nm. 1,000 "μm" refers to light in the wavelength range of μm (=1 mm), preferably light in the wavelength range of 700 nm to 2,500 nm, and more preferably light in the wavelength range of 700 nm to 1,400 nm. "White light" refers to light in the wavelength range spanning from the ultraviolet region to the near-infrared region (for example, the wavelength range of 200 nm to 1100 nm).

[0037] In this disclosure and its embodiments, "resonant light" means light that, upon incidence to a microparticle, causes a large photo-induced polarization in the microparticle. Resonant light has a wavelength within the wavelength range of the electronic resonance of the microparticle. On the other hand, "non-resonant light" means light that, upon incidence to a microparticle, causes a small photo-induced polarization in the microparticle. Non-resonant light has a wavelength outside the wavelength range of the electronic resonance of the microparticle.

[0038] When the microparticles are metal microparticles, the "wavelength range of the electronic resonance of the microparticles" is the wavelength range corresponding to the full width at half maximum of the peak of the localized surface plasmon resonance. The wavelength range in which localized surface plasmon resonance occurs in metal microparticles is determined by the size of the metal microparticles. When the metal microparticles are metal nanoparticles, this wavelength range is typically the visible light wavelength range of 400 nm to 700 nm. Therefore, light "outside the wavelength range of the electronic resonance of the microparticles" (non-resonant light) is typically infrared light with wavelengths longer than 700 nm.

[0039] When the nanoparticles are semiconductor nanoparticles or organic nanoparticles, the "wavelength range of the electronic resonance of the nanoparticles" refers to the wavelength range in which interband transitions or exciton resonances (electron-hole pair resonances) occur. When the nanoparticles are organic nanoparticles (such as microparticles made of organic materials like polystyrene), this wavelength range is typically shorter than 400 nm. Therefore, light "outside the wavelength range of the electronic resonance of the nanoparticles" (non-resonant light) is typically visible light with wavelengths longer than 400 nm.

[0040] [Embodiment] Embodiments of this disclosure will be described in detail below with reference to the drawings. In the drawings, the same or corresponding parts are denoted by the same reference numerals, and their descriptions will not be repeated. In the following description, the x and y directions represent the horizontal directions. The x and y directions are orthogonal to each other. The z direction represents the vertical direction. The direction of gravity is downward in the z direction. Upward in the z direction is abbreviated as "up," and downward in the z direction is abbreviated as "down."

[0041] <Detection principle of the substance to be detected> Let's describe an example where the substance to be detected is DNA. This DNA will be referred to as "target DNA." Target DNA is single-stranded DNA having a predetermined base sequence. In this embodiment, two types of probe particles are used to detect the target DNA.

[0042] Figure 1 shows a probe particle 1 in this embodiment. The probe particle 1 includes, for example, a metal nanoparticle 11, a host molecule 12, and an interaction site 13.

[0043] The metal nanoparticles 11 are, for example, gold nanoparticles with a diameter of 30 nm. As mentioned above, generally, the wavelength of localized surface plasmon resonance of gold nanoparticles in a liquid (e.g., water) is within the visible light wavelength range (typically 400 nm to 700 nm). However, depending on the application, it may be desirable to use light outside the wavelength range of localized surface plasmon resonance (e.g., infrared light). In this embodiment, such non-resonant light is irradiated onto the metal nanoparticles 11. In this case as well, localized surface plasmon resonance is induced on the surface of the gold nanoparticles. The metal nanoparticles 11 may be metal nanoparticles other than gold nanoparticles, as long as they can induce localized surface plasmon resonance; for example, they may be silver nanoparticles.

[0044] The size (diameter) of the metal nanoparticles 11 is not particularly limited as long as it is on the order of nanometers, but is preferably several tens of nanometers or larger, as in this example. This is because the size of metal nanoparticles that can be captured by the photo-induced force generated by non-resonant light irradiation at a typical light intensity (around several hundred mW) is several tens of nanometers or larger.

[0045] Metal nanoparticles 11 are an example of the "multiple nanoparticles" relating to this disclosure. The "multiple nanoparticles" relating to this disclosure may be semiconductor nanoparticles or organic nanoparticles. The "multiple nanoparticles" relating to this disclosure may include two or three types from among metal nanoparticles, semiconductor nanoparticles, and organic nanoparticles.

[0046] The host molecule 12 is a substance that specifically binds to the target DNA 9. In this embodiment, the host molecule 12 is a probe DNA having a base sequence between its 3' and 5' ends that is complementary to the base sequence at the 5' end of the target DNA 9. The host molecule 12 corresponds to the "first host molecule" in this disclosure.

[0047] The interaction site 13 is a site where the metal nanoparticle 11 and the host molecule 12 can interact. The interaction site 13 fixes the host molecule 12 to the surface of the metal nanoparticle 11. As a result, the host molecule 12 is modified on the surface of the metal nanoparticle 11. When the metal nanoparticle 11 is a gold nanoparticle and the host molecule 12 is probe DNA, the interaction site 13 is, for example, a thiol group (indicated by SH) located at the 3' end of the probe DNA.

[0048] Figure 2 shows the probe particle 2 in this embodiment. The probe particle 2 includes a microparticle 21, a host molecule 22, and an interaction site 23.

[0049] The microparticles 21 are, for example, resin beads made of polystyrene with a diameter of 2 μm. The material of the microparticles 21 is ,a Other resins such as krill, polyolefin, polyethylene, and polypropylene may also be used. The size (diameter) of the microparticles 21 is not particularly limited as long as it is on the order of micrometers, but it may be similar in size to that of general resin beads (so-called latex beads) (approximately 1 μm to 5 μm).

[0050] The host molecule 22 is a substance that specifically binds to the target DNA 9. In this embodiment, the host molecule 22 is a probe DNA having a base sequence between its 3' and 5' ends that is complementary to the base sequence at the 3' end of the target DNA 9. The host molecule 22 corresponds to the "second host molecule" in this disclosure.

[0051] The interaction site 23 is a site where the microparticle 21 and the host molecule 22 can interact. The interaction site 23 immobilizes the host molecule 22 on the surface of the microparticle 21. As a result, the host molecule 22 is modified on the surface of the microparticle 21. When the microparticle 21 is a resin bead and the host molecule 22 is probe DNA, the interaction site 23 includes, for example, avidin (indicated as A) 231 and biotin (indicated as B) 232. Avidin 231 is immobilized on the surface of the microparticle 21 by interaction between avidin 231 and the microparticle 21. Biotin 232 binds to the host molecule 22 (the 5' end of the probe DNA) (biotinization). The host molecule 22 is modified on the surface of the microparticle 21 due to the strong affinity between avidin 231 and biotin 232.

[0052] Note that Figure 1 shows an example where two host molecules 12 are modified on the surface of the metal nanoparticles 11, in order to avoid cluttering the paper. However, in reality, more host molecules 12 are modified on the surface of the metal nanoparticles 11. The same applies to the host molecules 22 modified on the surface of the microparticles 21.

[0053] Figure 3 illustrates the detection principle of target DNA 9 using probe particles 1 and 2. In this example, target DNA 9 is labeled with a fluorescent dye 90 (specifically, Alexa Flour® 488 from Thermo Fisher Scientific).

[0054] When probe particles 1 and 2 are introduced into a sample containing target DNA 9, hybridization occurs between the host molecule 12 of probe particle 1 and the target DNA 9, and between the host molecule 22 of probe particle 2 and the target DNA 9. As a result, multiple probe particles 1 and multiple probe particles 2 aggregate to form aggregates 3. When aggregates 3 grow to a certain size, they become optically detectable (for example, they can be photographed by camera 57 in the example described later). Therefore, if aggregates 3 are optically detected, it can be determined that target DNA 9 is present in the sample.

[0055] "Hybridization" refers to the reassociation reaction between two single-stranded nucleic acids. In this embodiment, a double helix is ​​formed between two single-stranded DNA molecules whose base sequences are complementary. However, hybridization is not limited to this and includes double helix formation between one single-stranded DNA molecule and one RNA molecule, or between two RNA molecules.

[0056] <Overall System Configuration> Figure 4 shows an example of the overall configuration of the target DNA9 detection system according to this embodiment. The detection system 100 comprises a detection kit 4, a sample stage 51, an adjustment mechanism 52, a laser light source 53, a focusing lens 54, an illumination light source 55, an objective lens 56, a camera 57, a spectrometer 58, optical components 59, and a controller 6. The optical components 59 include, for example, a mirror 591, a half mirror 592, and a lens 593.

[0057] Detection kit 4 holds the sample. The sample is a liquid sample that may contain target DNA9. The type of liquid (dispersion medium) is not particularly limited, but in this example it is water. Probe particle 1 and probe particle 2 have been introduced into the sample. An example of the configuration of detection kit 4 is shown in Figures 6 and 7.

[0058] The sample stage 51 is configured to hold the detection kit 4. Although not shown in the diagram, multiple detection kits 4 may be prepared. In that case, the multiple detection kits 4 are placed sequentially on the sample stage 51, and the detection process described later (see Figure 9) is performed.

[0059] The adjustment mechanism 52 is, for example, an adjustment mechanism for a 3-axis (XYZ axis) stage. The adjustment mechanism 52 adjusts the horizontal position and vertical height of the sample stage 51 according to commands from the controller 6. This allows for adjustment of the relative positional relationship between the detection kit 4 and the focusing lens 54, or between the detection kit 4 and the objective lens 56. The adjustment mechanism 52 may be provided on the focusing lens 54 and / or the objective lens 56 instead of or in addition to the sample stage 51.

[0060] The laser light source 53 emits continuous wave (CW) laser light (indicated as L1) according to a command from the controller 6. The laser light emitted from the laser light source 53 is reflected by the mirror 591 and directed towards the focusing lens 54. The wavelength of the laser light is outside the wavelength range of the electronic resonance of probe particle 1 (localized surface plasmon resonance of metal nanoparticle 11) and outside the wavelength range of the electronic resonance of probe particle 2 (exciton resonance of microparticle 21). In this embodiment, the wavelength of the laser light is in the near-infrared region (for example, 1064 nm). The laser light corresponds to the "non-resonant light" as described in this disclosure. The laser light source 53 corresponds to the "light source" as described in this disclosure.

[0061] The focusing lens 54 concentrates the laser light reflected by the mirror 591. The focusing lens 54 is typically an objective lens provided in a microscope. In particular, it is preferable that the focusing lens 54 is an objective lens with a correction ring. The laser light focused by the focusing lens 54 is irradiated onto the detection kit 4.

[0062] The illumination light source 55 emits white light (indicated as L2) to illuminate the sample on the detection kit 4. For example, a halogen lamp or a mercury lamp can be used as the illumination light source 55. In this embodiment, a mercury lamp is used in combination with a fluorescent filter (Nikon's standard series FITC Basic C-FL). The white light emitted from the illumination light source 55 passes through the detection kit 4.

[0063] The objective lens 56 captures the white light that has passed through the detection kit 4. A portion of the white light captured by the objective lens 56 passes through the half mirror 592. The remaining white light is reflected by the half mirror 592. The transmitted white light is focused by the lens 593 and directed to the camera 57. Meanwhile, the reflected white light is directed to the spectrometer 58.

[0064] Camera 57 is a photodetector that includes, for example, a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor. Camera 57 takes a picture of the sample on the detection kit 4 according to a command from the controller 6 and outputs a signal to the controller 6 indicating the captured image. The image captured by camera 57 may be a still image or a video.

[0065] The spectrometer 58 is a photodetector capable of spectroscopy in the ultraviolet to near-infrared region, for example. The spectrometer 58 measures the spectral characteristics of the sample on the detection kit 4 and outputs a signal indicating the measured spectral characteristics to the controller 6. More specifically, the spectrometer 58 measures the absorbance spectrum or fluorescence intensity of the aggregates 3 formed in the sample on the detection kit 4 and outputs a signal indicating the measurement result to the controller 6. The spectrometer 58 used for fluorescence observation may also be a fluorescence spectrum detector.

[0066] The controller 6 includes a processor 61 such as a CPU (Central Processing Unit), memory 62 such as ROM (Read Only Memory) and RAM (Random Access Memory), and input / output ports 63 to which various signals are input and output. The controller 6 controls each instrument in the detection system 100 (adjustment mechanism 52, laser light source 53, illumination light source 55, camera 57, spectrometer 58). Furthermore, the controller 6 detects target DNA 9 in the sample based on the image captured by the camera 57. The controller 6 also detects target DNA 9 in the sample based on the absorbance spectrum or fluorescence intensity measured by the spectrometer 58. The results of these detection processes will be described later.

[0067] Note that the optical system for imaging or measurement shown in Figure 4 (illumination light source 55, objective lens 56, half mirror 592, lens 593) is just one example. For example, the optical system of the detection system 100 shown in Figure 4 is configured such that white light from the illumination light source 55 is irradiated onto the sample from above downwards (see downward illumination in Figure 8), and the camera 57 is positioned below to photograph the sample above (transmission mode of an inverted microscope). However, the optical system of the detection system 100 may also be configured such that white light from the illumination light source 55 is irradiated onto the sample from below upwards (see upward illumination in Figure 8), and the camera 57 is positioned above to photograph the sample below (transmission mode of an upright microscope). The same applies to the spectrometer 58. The optical system of the detection system 100 may include other optical components (mirrors, dichroic mirrors, beam splitters, filters, optical fibers, etc.) in place of or in addition to the optical component 59 shown in Figure 4.

[0068] The optical system shown in Figure 4 can also be realized using the optical system of an upright microscope (upright configuration). A more detailed description of the upright configuration follows.

[0069] Figure 5 shows another example of the overall configuration of the detection system for a substance to be detected according to this embodiment in an upright configuration. The detection system 100A comprises a detection kit 4, a sample stage 51, an adjustment mechanism 52, a laser light source 53, an illumination light source 55, an excitation light source 55A, an objective lens 56A, a camera 57, a spectrometer 58, optical components 59, and a controller 6. The optical components 59 include, for example, a shutter 594, mirrors 595A, 595B, a dichroic mirror 596, beam splitters 597A, 597B, and a lens 598.

[0070] The laser light from the laser light source 53 (indicated as L1) passes through the shutter 594, is reflected by mirrors 595A, 595B and the dichroic mirror 596, and is directed towards the objective lens 56A. In the optical system shown in Figure 5, the objective lens 56A combines the functions of the focusing lens 54 and the objective lens 56 in Figure 4. The laser light is focused by the objective lens 56A and irradiated onto the detection kit 4 from above downwards.

[0071] Figure 5 also shows an excitation light source 55A (for example, a mercury lamp) that emits excitation light (indicated as L3) for fluorescence observation. The excitation light is reflected by the beam splitter 597A and transmitted through the dichroic mirror 596. The excitation light is then focused by the objective lens 56A and irradiated onto the detection kit 4 from above downwards. Although not shown in the figure, a filter cube matched to the color (wavelength) of the fluorescence may be placed above the objective lens 56A during fluorescence observation.

[0072] White light (indicated as L2) from the illumination source 55 is directed onto the detection kit 4 from below upward. The white light that has passed through the detection kit 4 passes through the dichroic mirror 596 and the beam splitter 597A. A portion of the white light that has passed through the beam splitter 597A passes further through the beam splitter 597B, is focused by the lens 598, and is directed to the camera 57. The remaining white light that has passed through the beam splitter 597A is reflected by the beam splitter 597B and directed to the spectrometer 58. The other configurations shown in Figure 5 are the same as the corresponding configurations shown in Figure 4, so a detailed explanation will not be repeated.

[0073] Figure 6 is a schematic perspective view showing the configuration of the detection kit 4. Figure 7 is a diagram showing an image of the actual fabricated detection kit 4. Referring to Figures 6 and 7, the detection kit 4 includes a substrate 41, a cover 42, and a spacer 43.

[0074] The materials of the substrate 41, cover 42, and spacer 43 are transparent to both laser light and white light. Such materials include glass, quartz, and silicone. In this example, both the substrate 41 and cover 42 are cover glass. The cover glass measures 32 mm in length, 24 mm in width, and 0.17 mm in height. The spacer 43 is placed between the substrate 41 and cover 42. In this example, the spacer 43 is double-sided tape. The size of the double-sided tape is smaller than the size of the substrate 41 and cover 42 (both cover glass). A cylindrical well 44 (6 mm in diameter) is provided in the center of the double-sided tape as an internal space for holding the sample.

[0075] <Upward and downward irradiation> Figure 8 illustrates the irradiation method of the detection kit 4 with laser light. Figure 8 shows how the laser light focused by the focusing lens 54 is irradiated onto the sample (indicated as SP) held in the well 44. A beam waist of the laser light is formed at the focal point of the focusing lens 54. The beam diameter (minimum spot diameter φ0) at the beam waist is, for example, several μm to several tens of μm.

[0076] In this embodiment, the laser light is irradiated onto the sample in one of the following two irradiation modes. The irradiation mode in which the focusing lens 54 is placed above the detection kit 4 and the laser light is irradiated from above the detection kit 4 downwards is called "downward irradiation". Conversely, the irradiation mode in which the focusing lens 54 is placed below the detection kit 4 and the laser light is irradiated from below the detection kit 4 upwards is called "upward irradiation". Downward irradiation can be achieved by an upright arrangement as shown in Figure 5. Upward irradiation is achieved by inverting the optical system as shown in Figure 5 and irradiating the sample from the laser light source 53 through the objective lens 56A below the sample stage 51. light This can be achieved by the optical system (inverted configuration) of an inverted microscope that irradiates the detection kit 4.

[0077] In downward irradiation, the vertical position of the beam waist is preferably below the bottom surface BS of well 44. In upward irradiation, the vertical position of the beam waist is preferably above the top surface TS of well 44. In other words, it is preferable that the condition is met where the beam waist is off the top surface TS or bottom surface BS of well 44, or in other words, the condition is met where the laser light is located behind the detection kit 4 in its propagation direction. Hereafter, this condition will be referred to as the "defocus condition". The defocus condition will be explained in Figure 10.

[0078] <Processing Flow> Figure 9 is a flowchart showing the processing steps for detecting target DNA9. This flowchart is executed when predetermined conditions are met (for example, when the user operates the start button (not shown)). Each step is basically implemented by software processing by the controller 6, but some or all of it may be implemented by hardware (electrical circuits) manufactured within the controller 6. Hereinafter, steps will be abbreviated as "S".

[0079] In S1, the controller 6 prepares the sample. More specifically, the controller 6 introduces probe particles 1 and 2 into the sample, which may contain target DNA 9. For example, the controller 6 controls a dispenser (not shown) to dropwise dispense dispersions of probe particle 1 and probe particle 2, each prepared to a predetermined concentration, onto the sample. The user may also manually introduce probe particles 1 and 2 into the sample.

[0080] In S2, the controller 6 uses the adjustment mechanism 52 to adjust the height so that the position of the laser beam waist is at a desired position above the top surface TS of the well 44 (in the case of upward irradiation) or at a desired position below the bottom surface BS of the well 44 (in the case of downward irradiation).

[0081] In general, both the substrate 41 and the cover 42 (cover glass), as well as the spacer 43 (double-sided tape), may have a certain degree of thickness variation (for example, several μm to several tens of μm). This thickness variation can be absorbed by adjusting the height after sample preparation. However, the order of sample preparation and height adjustment is not limited to this. For example, if the detection kit 4 is manufactured with high precision and the thickness variation is negligibly small, the order of processing S1 and processing S2 may be reversed.

[0082] In S3, the controller 6 places the detection kit 4 on the sample stage 51. This process can be achieved, for example, by a feeding mechanism for the detection kit 4 (not shown). The user may also manually place the detection kit 4.

[0083] In S4, the controller 6 controls the laser light source 53 to start (or continue) irradiating the detection kit 4 with laser light. If the target DNA 9 is present in the sample, this causes the probe particles 1 and 2 to bind to the target DNA 9 (hybridization occurs), forming aggregates 3 of the probe particles 1 and 2 (see Figure 3). The formed aggregates 3 grow as the laser light irradiation time increases. During laser light irradiation, the formation and growth of aggregates 3 are promoted according to the mechanism described later in Figures 10 to 13.

[0084] In S5, the controller 6 controls the illumination light source 55 to start irradiating the detection kit 4 with white light.

[0085] In S6, the controller 6 controls the camera 57 to capture an image of the detection kit 4 at the laser spot (the position where the laser light is irradiated). Alternatively, the controller 6 controls the spectrometer 58 to measure the absorbance spectrum or fluorescence intensity of the detection kit 4 at the laser spot. The controller 6 may perform both of the above two types of measurements simultaneously.

[0086] In S7, the controller 6 determines whether the elapsed time since the start of laser light irradiation has reached a specified time (e.g., 180 seconds, 240 seconds, etc., in the embodiment described later). If the light irradiation time has not reached the specified time (NO in S7), the controller 6 returns to S4. As a result, irradiation of laser light and white light continues, and image acquisition, spectral measurement, etc., continue. When the light irradiation time reaches the specified time (YES in S7), the controller 6 proceeds to S8.

[0087] In S8, the controller 6 determines whether aggregates 3 of probe particles 1 and 2 are observed in the image by applying predetermined image processing to the captured image. For example, the controller 6 can determine that aggregates 3 are observed if the size of aggregates 3 (the accumulation area of ​​probe particles 1 and 2, described later) exceeds a reference area (see Figures 14 and 17).

[0088] Alternatively, the controller 6 may determine the presence of aggregates 3 by analyzing the measured absorbance spectrum or fluorescence intensity. For example, the controller 6 may determine that aggregates 3 have been observed if the difference in absorbance or fluorescence intensity before and after light irradiation exceeds a predetermined amount (see Figures 15, 16, and 26). For example, the controller 6 may determine that aggregates 3 have been observed if, in the image or fluorescence image after light irradiation, the difference in absorbance or fluorescence intensity between the irradiated area and the background area (an area sufficiently far from the light irradiation position) exceeds a predetermined amount (see Figures 19-25, 28, 31, 36, etc.). In this way, the controller 6 detects aggregates 3 based on the amount of change in absorbance or fluorescence intensity associated with light irradiation. This amount of change is typically a difference, but may also be a ratio, for example.

[0089] Many fluorescent probes undergo bleaching, so the difference in fluorescence intensity before and after light irradiation may include changes in intensity due to the bleaching of the fluorescent probe. Therefore, especially in fluorescence measurements, it is desirable to use the difference in fluorescence intensity between the irradiated area and the background area in the fluorescence image after light irradiation for accurate comparison. On the other hand, there are also fluorescent probes that are less prone to bleaching (such as nanodiamonds). When using a fluorescent probe that is less prone to bleaching, the difference in fluorescence intensity before and after light irradiation of the same area may be used.

[0090] If aggregates 3 of probe particles 1 and 2 are observed (YES in S8), controller 6 determines that target DNA 9 is present in the sample (S9). On the other hand, if aggregates 3 are not observed (NO in S8), controller 6 determines that target DNA 9 is not detected (not present in the sample) (S10).

[0091] In S11, the controller 6 controls the laser light source 53 to stop emitting laser light and the illumination light source 55 to stop emitting white light. This completes the series of processes.

[0092] <Mechanism> As explained in Figure 3, probe particles 1 and 2 repeatedly encounter and hybridize with target DNA 9, forming aggregates 3 of probe particles 1 and 2 via target DNA 9. As aggregates 3 grow, the probability of probe particles 1 and 2 and target DNA 9 surrounding aggregates 3 encountering aggregates 3 increases. As a result, the frequency of hybridization increases exponentially, promoting the growth of aggregates 3. Thus, according to this embodiment, "photo-induced acceleration" can be achieved by irradiating probe particles 1 and 2 with laser light. Photo-induced acceleration is achieved through a mechanism involving dissipative force, thermal convection, and Brownian motion.

[0093] Dissipative force Figure 10 is a diagram illustrating the dissipative forces acting on probe particles 1 and 2. Downward irradiation is illustrated in Figure 10.

[0094] When laser light is shone onto the detection kit 4, photo-induced forces act on the probe particles 1 and 2 in the sample. More specifically, in addition to intermaterial photo-induced forces and gradient forces acting on the probe particles 1 and 2, a dissipative force acts in the same direction as the laser light irradiation direction. In the case of downward irradiation, a dissipative force acting from above to below presses the probe particles 1 and 2 against the bottom surface BS of the well 44. Although not shown in the diagram, in the case of upward irradiation, a dissipative force acting from below to above presses the probe particles 1 and 2 against the top surface TS of the well 44.

[0095] Generally, under non-resonant conditions, when the size (diameter) of the irradiated material is smaller than the wavelength of light (in this embodiment, the material is a nanoparticle), the gradient force among the components of the photoinduced force is proportional to the volume of the material, and the dissipative force is proportional to the square of the volume of the material. On the other hand, when the size of the irradiated material is about the same as the wavelength of light (in this embodiment, the material is a microparticle), the light undergoes Mie scattering. The dissipative force resulting from Mie scattering is proportional to the square of the diameter of the material.

[0096] In this embodiment, the diameter of probe particle 2 (microparticle 21) is 2 μm, and the wavelength of the laser light is 1064 nm. That is, the diameter of probe particle 2 and the wavelength of the laser light are approximately the same. Therefore, the laser light irradiated onto the detection kit 4 generates a dissipative force on probe particle 2 due to Mie scattering. The diameter of probe particle 2 (2 μm) is about two orders of magnitude larger than the diameter of probe particle 1 (30 nm). Therefore, the dissipative force acting on probe particle 2 is significantly stronger than the dissipative force acting on probe particle 1. Specifically, when irradiated with laser light of an intensity of about 100 mW, the dissipative force experienced by gold nanoparticles is about several tens of fN, while the dissipative force experienced by microparticles is about several tens of pN, which is three orders of magnitude larger.

[0097] In addition, in the case of downward irradiation, the dissipative force due to Mie scattering can strongly press the probe particles 2 against the bottom surface BS of well 44. As a result, the density of probe particles 2 at the bottom surface BS of well 44 becomes locally higher than the density of probe particles 2 at other locations (positions sufficiently far from the beam waist). Consequently, the probability of encounter between probe particles 2 and target DNA 9 increases near the bottom surface BS of well 44. When probe particles 2 and target DNA 9 encounter each other, hybridization occurs between the host molecule 22 (probe DNA) modified on the surface of probe particles 2 and the target DNA 9, as explained in Figure 3.

[0098] Furthermore, in this embodiment, when irradiating downwards, the beam waist is positioned below the bottom surface BS of the well 44. By intentionally defocusing the beam waist from the bottom surface BS of the well 44, the irradiation area of ​​the laser light on the bottom surface BS of the well 44 becomes larger compared to when the beam waist coincides with the bottom surface BS of the well 44 (when focused). By securing a certain amount of irradiation area, the probe particles 2 are pressed against the bottom surface BS over a wide area. Therefore, the probability of the probe particles 2 encountering the target DNA 9 can be further increased.

[0099] In this way, by irradiating probe particles 2, which are on the order of micrometers, with laser light of a similar wavelength, and locally increasing the density at the bottom surface BS of probe particles 2, hybridization between probe particles 2 and target DNA 9 can be brought about with high efficiency. While a detailed explanation will not be repeated, the same principle applies to upward irradiation.

[0100] ≪Thermal Convection≫ Probe particle 1 contains metal nanoparticles 11. When laser light is shone on probe particle 1, the free electrons of the metal nanoparticles 11 form localized surface plasmons and vibrate in response to the laser light. This causes polarization. The energy of this polarization is converted into lattice vibration energy through Coulomb interaction between the free electrons and atomic nuclei. As a result, the metal nanoparticles 11 generate heat (photothermal effect). The photothermal effect creates a temperature gradient in the sample. This causes regular thermal convection (buoyant convection and / or Marangoni convection) toward the beam west to occur steadily. New probe particles 1, 2 and target DNA 9 are carried toward the beam west by this thermal convection. This increases the probability of encounter between probe particle 1 and target DNA 9, as well as between probe particle 2 and target DNA 9, compared to a stationary liquid.

[0101] In this way, by irradiating the probe particle 1 containing metal nanoparticles 11 with laser light and generating thermal convection toward the beam west, hybridization between the probe particles 1 and 2 and the target DNA 9 can be brought about with even greater efficiency.

[0102] Brownian motion Probe particle 1 is more significantly affected by Brownian motion (random diffusive motion due to thermal fluctuations in water) than probe particle 2. The results of estimating the Brownian motion of each particle from the diffusion distance and the average inter-particle distance are explained below.

[0103] Figure 11 shows the simulation results regarding the Brownian motion of probe particle 1. Figure 11 shows the trajectory of each probe particle 1 over 0.1 seconds when N probe particles 1 are placed in water. Simulations were performed for three different values ​​of N: 4, 8, and 32. The diameter of probe particle 1 was set to 40 nm, and the water temperature to 25°C. The initial position of probe particle 1 was at the center of the image, indicated by START. The final position of probe particle 1 was at the position indicated by END.

[0104] The average diffusion radius σ of the particles after t seconds is calculated according to the following equation (1). Note that the average diffusion radius σ is located approximately midway between the two circles C1 and C2 in the figure.

[0105]

number

[0106] k is Boltzmann's constant, and T is the absolute temperature of water. ξ is the coefficient of friction, expressed as ξ = 6πηa, where η is the viscosity coefficient of water and a is the particle radius. From equation (1), it can be seen that the diffusion distance per unit time for each particle is inversely proportional to the square root of the particle size (radius or diameter). For example, probe particle 1 with a diameter of 30 nm diffuses approximately 8 times further than probe particle 2 with a diameter of 2 μm.

[0107] Figure 12 is a conceptual diagram illustrating the average interparticle distance of probe particles 1 and 2. Probe particle 1 is assumed to be a gold nanoparticle with a diameter of 30 nm, and probe particle 2 is assumed to be a polystyrene particle with a diameter of 2 μm. The concentration of probe particle 1 was set to 1.73 × 10⁻⁶. 11 [particles / mL], and the concentration of probe particle 2 is 1.58 × 10⁻⁶. 8 [particles / mL] was used.

[0108] The average interparticle distances of probe particle 1 and probe particle 2 are calculated to be 1.79 μm and 18.5 μm, respectively. In this case, as shown in Figure 12, when observed in a planar manner, there are approximately 100 probe particles 1 within a square, with probe particle 2 positioned at each of the four vertices. In the figure, the circle surrounding all of probe particles 1 and 2 represents a laser spot with a diameter of 30 μm. When observed in three dimensions, there are approximately 1000 probe particles 1 within a cube, with probe particle 2 positioned at each of the eight vertices.

[0109] Probe particle 1 diffuses at a faster rate, meaning it diffuses a longer distance per unit time compared to probe particle 2. Therefore, the probability of probe particle 1 encountering target DNA 9 is higher as probe particle 1 repeatedly performs a rapid random walk. Consequently, statistically speaking, target DNA 9 is likely to encounter probe particle 1 first, followed by probe particle 2 surrounding probe particle 1. This means that using both probe particles 1 and 2 can accelerate the first hybridization of target DNA 9, in particular, compared to using only probe particle 2.

[0110] ≪Summary≫ Even when only probe particle 2 is used, the density of probe particle 2 increases locally due to the strong dissipative force caused by Mie scattering. This increases the probability of encounter between probe particle 2 and target DNA 9, thereby promoting hybridization. However, this promotional effect is limited to a certain extent.

[0111] In contrast, when both probe particles 1 and 2 are used, the active Brownian motion of probe particle 1 increases the probability of encounter between probe particle 1 and target DNA 9. In addition, the photothermal effect of probe particle 1 induces thermal convection, further increasing the probability of encounter between probe particles 1 and 2 and target DNA 9. In Figure 12, it is assumed that the distribution of probe particle 2 is uniform throughout the sample. However, because probe particle 2 is concentrated due to a strong dissipative force, the distance between probe particle 2 within the laser spot is shorter. Therefore, the probability of probe particle 1 and the hybridized target DNA 9 encountering a nearby probe particle 2 before diffusing far away due to the Brownian motion of probe particle 1 becomes even higher. Due to this synergistic effect, hybridization between probe particles 1 and 2 and target DNA 9 can be achieved with extremely high efficiency according to this embodiment.

[0112] As described above, this embodiment employs a "heteroprobe method" using two different types of probe particles, 1 and 2, which differ in size and material from one another. This increases the probability of encounter between probe particles 1 and 2 and target DNA 9, compared to the case where only one of the probe particles 1 or 2 is used, enabling highly efficient hybridization and further enhancing photo-induced acceleration. As a result of the enhanced photo-induced acceleration, even if the amount of target DNA 9 is minute, aggregates 3, in which probe particles 1 and 2 are densely aggregated, are formed and grow in a short time. Therefore, according to this embodiment, target DNA 9 can be detected with high sensitivity and speed.

[0113] [Example 1] This section describes the evaluation results regarding the effect of probe particle combinations on the detection sensitivity of target DNA.

[0114] Figure 13 illustrates the evaluation results regarding probe particle combinations. Target DNA 9, labeled with a fluorescent dye and having 24 randomly arranged bases, was used. Probe particle 1, having a base sequence that is perfectly complementary to the 12 bases at the 5' end of target DNA 9, is referred to as "Probe particle 1A". On the other hand, probe particle 1, having a base sequence that is perfectly complementary to the 12 bases at the 3' end of target DNA 9, is referred to as "Probe particle 1B". The same applies to probe particle 2.

[0115] In this example, three samples were prepared. Sample 1 contained probe particle 2A and probe particle 1B. Sample 2 contained probe particle 2A and probe particle 2B. Sample 3 contained probe particle 1A and probe particle 1B. For each sample, the fluorescence intensity with and without target DNA9 was measured, and the difference between the two measured fluorescence intensities was calculated.

[0116] The differences in fluorescence intensity between the first, second, and third samples were 19.57, 9.07, and 1.06, respectively. Specifically, the difference in fluorescence intensity in the first sample was significantly larger than the differences in fluorescence intensity between the second and third samples. This indicates that introducing different types of probe particles (both probe particles 1 and 2) into the sample can improve the detection sensitivity of target DNA9 compared to introducing only one type of probe (either probe particle 1 or 2).

[0117] [Example 2] This section describes the results of transmission images and absorbance spectra measurements when target DNA, which is not labeled with a fluorescent dye, is used as the substance to be detected.

[0118] For comparison (control experiment), two types of target DNA 9 were prepared. The first target DNA has a base sequence that is completely complementary to the host molecules (probe DNA) of probe particles 1 and 2, and therefore undergoes hybridization with probe particles 1 and 2. Hereafter, such DNA will be referred to as "matching DNA". The second target DNA has a base sequence that is not complementary to the host molecules (probe DNA) of probe particles 1 and 2, and therefore does not undergo hybridization. Hereafter, such DNA will be referred to as "mismatching DNA".

[0119] In Example 2, an AT sequence containing only adenine (A) and thymine (T) was used as target DNA 9. Specifically, the host molecule 12 of probe particle 1 (gold nanoparticle) was 5'-TTT TTT TTT TTT-3'-(CH2)6-SH. The host molecule 22 of probe particle 2 (microparticle) was Biotin-3'-TTT TTT TTT TTT-5'. The matched DNA was 5'-AAA AAA AAA AAA AAA AAA AAA AAA-3'. The mismatched DNA was Biotin-5'-TTT TTT TTT TTT TTT TTT TTT TTT-3'.

[0120] The concentration of target DNA 9 (matched DNA or mismatched DNA) was 10 nM. Laser light was shone upwards. The laser beam waist was adjusted to 45 μm above the top surface TS of well 44. The magnification of the focusing lens 54 was 40x. The laser light intensity (output after passing through detection kit 4) was 525 mW. The light irradiation time was 240 seconds.

[0121] Figure 14 shows an example of an image captured by camera 57 in Example 2. Figure 15 shows an example of an absorbance spectrum measured by spectrometer 58 in Example 2. The horizontal axis represents wavelength, and the vertical axis represents absorbance.

[0122] As shown in Figure 14, when target DNA 9 was matched DNA, the accumulation of probe particles 1 and 2 at the laser spot was greater compared to when target DNA 9 was mismatched DNA. Furthermore, as shown in Figure 15, the difference in absorbance spectra before and after irradiation was also confirmed to be larger in the case of matched DNA compared to the case of mismatched DNA.

[0123] Figure 16 shows the dependence of the absorbance difference on the target concentration. The horizontal axis represents the target concentration (concentration of target DNA9). The vertical axis represents the absorbance difference (difference in the peak value of the absorbance spectrum before and after irradiation).

[0124] The results showed that the absorbance difference increased linearly with increasing target concentration, regardless of whether target DNA9 was matched or mismatched. On the other hand, when target DNA9 was matched, the slope of the line was 0.0071. When target DNA9 was mismatched, the slope of the line was 0.0019. In other words, when target DNA9 was matched, the increase in absorbance difference with increasing target concentration was greater compared to when target DNA9 was mismatched.

[0125] Figure 17 shows the dependence of the accumulation area of ​​probe particles 1 and 2 on the target concentration. The horizontal axis represents the target concentration, and the vertical axis represents the accumulation area of ​​probe particles 1 and 2. The accumulation area of ​​probe particles 1 and 2 is calculated by image processing, specifically by determining the area of ​​the region where probe particles 1 and 2 have accumulated and become darker in color.

[0126] Regardless of whether target DNA9 was matched or mismatched, the accumulation area increased linearly with increasing target concentration. However, when target DNA9 was matched, the slope of the line was 25.16. When target DNA9 was mismatched, the slope was 6.36. In other words, when target DNA9 was matched, the increase in accumulation area with increasing target concentration was greater compared to when target DNA9 was mismatched.

[0127] [Example 3] This section describes the transmission image, fluorescence image, and fluorescence intensity measurement results when target DNA labeled with a fluorescent dye is used as the substance to be detected.

[0128] Figure 18 summarizes the measurement conditions in Example 3. Seven samples, 1 to 7, were prepared as shown in Figure 18. The following conditions were common to all samples 1 to 7. The volume of each sample was 39 μL. Laser light was shone downwards. The beam waist of the laser light was adjusted to be 45 μm below the bottom surface BS of well 44. The magnification of the focusing lens 54 was 40x. The intensity of the laser light was 640 mW. The light irradiation time was 240 seconds.

[0129] Figures 19 to 25 show transmission and fluorescence images of samples 1 to 7, respectively. In the transmission images on the left and in the center, no significant difference was observed depending on whether the target DNA 9 was matched or mismatched. However, in the fluorescence image on the right, clear fluorescence was observed only when the target DNA 9 was matched.

[0130] Figure 26 shows an example of the measurement results of the position dependence of fluorescence intensity. The horizontal axis represents the measurement position of fluorescence intensity along the principal plane direction (horizontal direction) of detection kit 4. The left edge of the transmission and fluorescence images corresponds to 0 μm, and the right edge corresponds to 240 μm. The vertical axis represents fluorescence intensity. Figure 26 shows the average values ​​of the fluorescence intensity measurement results for three samples for four different target concentrations. From Figure 26, it can be seen that when target DNA 9 is match DNA, the fluorescence intensity was higher with increasing target concentration. Thus, even at very low target concentrations of 10 nM or less, the fluorescence intensity clearly depends on the target concentration.

[0131] Figure 27 shows an example of a calibration curve obtained from the fluorescence intensity shown in Figure 26. In Figure 27, target DNA9 Miss Calibration curves for the concentration of target DNA9 are shown for both the case where match DNA is used (left figure) and the case where target DNA9 is match DNA (right figure). These were obtained by integrating the measured fluorescence intensity minus the background intensity over the position range of 105–135 μm in Figure 26. The error bars indicate the standard deviation for n=3 measurements.

[0132] As shown in Figure 27, a linear calibration curve was obtained. This indicates that even when the concentration of match DNA is less than 10 nM, the concentration of match DNA can be quantitatively evaluated from the fluorescence intensity using the calibration curve. When the concentration of match DNA is 10 nM, the mass concentration of match DNA calculated from the molecular weight of the base pairs of match DNA is approximately 79.2 [fg / μL]. Similarly, the detection limit calculated was approximately 7.92 to 39.6 [fg / μL] in mass concentrations.

[0133] Figure 28 shows images (transmission and fluorescence images) of the fluorescence enhancement on the surface of metal nanoparticles 11 (gold nanoparticles) contained in probe particle 1. Laser light was shone downward using a focusing lens 54 provided on an upright microscope (not shown). The laser beam waist was adjusted to 45 μm below the bottom surface BS of well 44. The magnification of the focusing lens 54 was 40x. The laser output was 640 mW. The light irradiation time was 240 seconds. The concentration of target DNA 9 was 10 nM. The concentration of probe particle 2 was 5.3 × 10⁻⁶. 8 The value was [particles / mL].

[0134] The base sequence of target DNA9 was a random sequence. A random sequence is a sequence that can contain all four types of bases: adenine (A), thymine (T), guanine (G), and cytosine (C). Specifically, the host molecule 12 of probe particle 1 (gold nanoparticle) was 5'-ATG CTC AAC TCT-3'-(CH2)6SH (3' end modification). The host molecule 22 of probe particle 2 (microparticle) was Biotin-3'-TCT CAA CTC GTA-5' (3' end modification). The matched DNA was 5'-AGA GTT GAG CAT X TAC GAG TTG AGA-3'. The mismatched DNA was 5'-TCT CAA CTC GTA X ATG CTC AAC TCT-3'. X is a fluorescent dye (Alexa Flour488 as mentioned above).

[0135] As shown in Figure 28, when target DNA 9 was a matched DNA, strong fluorescence was observed not only in the aggregate 3 of probe particles 1 and 2, but also around the aggregate 3. In contrast, when target DNA 9 was a mismatched DNA, strong fluorescence was not observed around the aggregate 3.

[0136] Since the target DNA9 is labeled with a fluorescent dye, when aggregate 3 is formed by hybridization of probe particles 1 and 2 with the target DNA9, metal nanoparticles 11 of probe particle 1 are present near the fluorescent dye. This increases the excitation probability of the fluorescent dye due to the electric field enhancement under light irradiation caused by the localized surface plasmon resonance of the metal nanoparticles 11, and enhances the green fluorescence from the fluorescent dye. As a result, the measured fluorescence intensity becomes stronger, improving the detection sensitivity of the target DNA9. Therefore, fluorescence imaging is a particularly suitable method for detecting trace amounts of substances to be detected.

[0137] In Example 3, the total amount of target DNA 9 present in well 44 is calculated to be approximately 900 zmol (zeptomoles). Since the thickness of aggregate 3 is approximately 6 μm, it is thought that aggregate 3 has a three-dimensional structure in which probe particles 1 and 2 are stacked on top of each other. It is estimated that the target DNA 9 that can undergo hybridization is approximately 1 / 100 of the total. In other words, the results of this example suggest that it is possible to detect a trace amount of target DNA 9 of approximately 9 zmol.

[0138] Figures 29 and 30 show the sequence dependence of fluorescence intensity. Figure 29 shows the observation results when the target DNA 9 sequence is an AT sequence, and Figure 30 shows the observation results when the target DNA 9 sequence is a random sequence. Specifically, the matched DNA was 5'-AAA AAA AAA AAA X AAA AAA AAA AAA-3'. The mismatched DNA was Biotin-5'-TTT TTT TTT TTT X TTT TTT TTT TTT-3'. Laser light was shone upward using a focusing lens 54 provided on an inverted microscope (not shown). The beam waist of the laser light was adjusted to 45 μm above the top surface TS of well 44. The magnification of the focusing lens 54 was 40x. The laser light intensity was 525 mW. The light irradiation time was 240 seconds. Target DNA The concentration of particle 9 was 10 nM. The concentration of probe particle 2 was 5.3 × 10⁻⁶. 8 The value was [particles / mL].

[0139] The random sequence (see Figure 30) showed a larger accumulation area for probe particles 1 and 2, and also exhibited stronger fluorescence intensity, compared to the AT sequence (see Figure 29). In the AT sequence, only adenine-thymine binding is formed, whereas in the random sequence, both adenine-thymine binding and guanine-cytosine binding can be formed. The adenine-thymine binding is a double hydrogen bond, while the guanine-cytosine binding is a triple hydrogen bond. Therefore, the binding force between guanine and cytosine is stronger than that between adenine and thymine. It is thought that the random sequence showed a larger accumulation area and stronger fluorescence intensity because of its stronger binding force.

[0140] [Example 4] This section describes attempts to further improve the sensitivity of target DNA detection (further improve the detection limit).

[0141] Figure 31 shows transmission and fluorescence images of low-concentration samples. The concentration of matched DNA or mismatched DNA as target DNA9 was set in the range of 0 nM to 1000 pM (= 1 nM). The measurement results shown in Figure 31 are for a sample with 1000 pM matched DNA (top), a sample with 1000 pM mismatched DNA (middle), and a sample without target DNA (bottom). From left to right, the images show the transmission image before laser irradiation, the transmission image after laser irradiation, and the fluorescence image after laser irradiation. The light irradiation time was 180 seconds.

[0142] Figure 32 summarizes the fluorescence intensity measurements at various concentrations of target DNA9. The horizontal axis represents the concentration of target DNA9, and the vertical axis represents the fluorescence intensity. As shown in Figure 32, a clear difference in fluorescence intensity was observed between matched DNA and mismatched DNA in the concentration range of 62.5 pM to 1000 pM (mass concentration of 514.8 [pg / μL] to 7.92 [pg / μL]). In particular, the coefficient of determination R for matched DNA was... 2A linear validation curve close to 1 was obtained. This indicates that while Example 3 targeted concentrations of 1 nM, 5 nM, or 10 nM (see Figures 18 and 27), Example 4 allows for the quantitative evaluation of extremely low concentrations of match DNA, such as 1 nM or less.

[0143] When σ is used as the standard deviation and the detection limit is defined as "the minimum amount at which the substance to be detected is not detected in the background (blank) signal distribution with a certainty of 3σ (≒99.7%)" (detection limit = mean value of the blank ± 3σ), the detection limit of match DNA in Example 4 was calculated to be 125~250 pM ≒ 1.0~2.0 [pg / μL]. This detection limit is comparable to the concentration (mass concentration 5~30 [pg / μL]) of free DNA (cfDNA) in patient-derived samples (plasma) diluted 100-fold, which is used as a biomarker in the fields of cancer and reproductive medicine.

[0144] Furthermore, the detection limit mentioned above is comparable to that of digital PCR (Polymerase Chain Reaction) (mass concentration 166 [fg / μL]). On the other hand, while digital PCR requires approximately 5 hours (=300 minutes), the light irradiation time in Example 4 was 180 seconds (=3 minutes). Therefore, it can be said that we have succeeded in significantly speeding up detection by reducing the detection time to 1 / 100th.

[0145] Such increased sensitivity (and speed) in target DNA detection can be achieved primarily through the following three improvements.

[0146] The first improvement is a change in the laser irradiation conditions. In Example 3, under defocus conditions for downward irradiation, the beam waist was set 45 μm below the bottom surface BS of well 44 (see Figure 8). In contrast, in Example 4, under defocus conditions for upward irradiation, the beam waist was set 30 μm above the top surface TS of well 44. In other words, in Example 4, the size of the laser spot (spot diameter) at the top surface TS of well 44 was reduced.

[0147] By increasing the laser irradiation area to a certain extent, it becomes possible to press the probe particles 2 against the top surface TS of well 44 over a wide area. On the other hand, if the laser irradiation area is excessively large, the power density of the laser light decreases, and the dissipative force acting on each probe particle 2 decreases. In Example 4, the balance between the laser irradiation area and power density was optimized, and conditions were achieved in which a strong dissipative force acted on the probe particles 2 over a wide area. As a result, it is thought that the probability of the probe particles 2 encountering the target DNA 9 was further increased.

[0148] The second improvement is the adjustment of the correction ring provided on the condensing lens 54, which is the objective lens. In Example 3, the correction ring was adjusted to a position corresponding to the thickness of the cover 42 (in this example, the thickness of the cover glass, approximately 0.17 mm). On the other hand, in Example 4, the correction ring was adjusted to a position equal to the thickness of the cover 42 plus the depth of the well 44 (0.17 mm + 1 mm ≈ 1.2 mm) so that spherical aberration correction is performed not only according to the thickness of the cover 42 but also according to the depth of the well 44. It is believed that this resulted in appropriate correction and an improvement in the signal-to-noise ratio.

[0149] Figure 33 illustrates the third improvement. The third improvement is a change in the sample preparation procedure. In Example 3, the dispersion of probe particle 1, the dispersion of probe particle 2, and the dispersion of target DNA 9 were introduced into well 44 separately. In contrast, in Example 4, the three dispersions were mixed using a tube before being introduced into well 44. Pre-mixing allows light irradiation to begin under conditions where probe particle 1, probe particle 2, and target DNA 9 are uniformly distributed. This ensures that probe particle 2 is located near probe particle 1 after it encounters target DNA 9 due to Brownian motion, and as a result, the probability of encounter between probe particle 1 and probe particle 2 is thought to have improved.

[0150] [Example 5] This section describes the results of evaluating the detection specificity of target DNA9 for its base sequence. In this example, two probe DNAs were used in common: 5'-ATG CTC AACTCT-3'-Biotin, which contains 12 bases, and SH-5'-TCT CAA CTC GTAT-3', which also contains 12 bases. Eight types of target DNA9, each containing 24 bases, were prepared.

[0151] Figure 34 is a diagram illustrating the eight types of target DNA9. Hereafter, bases that are complementary to the two probe DNAs mentioned above will be referred to as "complementary bases," and bases that are not complementary to the two probe DNAs will be referred to as "non-complementary bases." In Figure 34, non-complementary bases are shown in bold.

[0152] Target DNA 9a is a match DNA in which all 24 bases are complementary bases. On the other hand, target DNAs 9b to 9h contain at least one non-complementary base. Specifically, target DNA 9b contains 24 non-complementary bases. Target DNA 9c contains 6 non-complementary bases (and 18 complementary bases). Target DNA 9d contains 4 non-complementary bases (and 20 complementary bases). Target DNA 9e and target DNA 9f contain 2 non-complementary bases (and 22 complementary bases). Target DNA 9e and target DNA 9f differ in the arrangement of the 2 non-complementary bases. Target DNA 9g and target DNA 9h contain 1 non-complementary base (and 23 complementary bases). Target DNA 9g and target DNA 9h differ in the arrangement of the 1 non-complementary base.

[0153] Figure 35 summarizes the fluorescence intensity measurements for eight different target DNA9s. The horizontal axis represents the number of non-complementary base pairs, and the vertical axis represents fluorescence intensity. All eight target DNA9s were prepared at a concentration of 1 nM. The number of measurements was n=3. For comparison, the fluorescence intensity without target DNA9 is shown as a baseline by a dashed line.

[0154] The fluorescence intensity was significantly higher when all bases were complementary (see 9a). The fluorescence intensity was at the same level as the baseline when there were four or more non-complementary bases (see 9b-9d). When there were one or two non-complementary bases, the fluorescence intensity increased compared to the baseline, but the increase was very small (see 9e-9h). Specifically, the difference between the fluorescence intensity with one non-complementary base and the baseline was only about 25% of the difference between the fluorescence intensity with all bases being complementary and the baseline. This indicates that the target DNA9 detection method according to this embodiment exhibits high base sequence specificity, and in particular, that it can detect single nucleotide polymorphisms (SNPs) of target DNA9.

[0155] One reason why the detection method for target DNA9 according to this embodiment exhibits base sequence specificity is thought to be the photothermal effect of probe particle 1. Even if a small number of non-complementary bases are present, such as in target DNA9e~9h, hybridization can occur and a double helix can be formed. However, the more non-complementary bases present in target DNA9, the weaker the binding between probe DNA and target DNA9, resulting in a lower temperature at which the double helix dissociates into a single helix (for example, the melting temperature Tm at which 50% of the double helix dissociates). The photothermal effect of probe particle 1 raises the temperature of the double helix, causing the double helix containing many non-complementary bases to preferentially dissociate, leaving behind double helix that does not contain non-complementary bases. As a result, target DNA9a, in which all bases are complementary bases, is selectively detected.

[0156] [Example 6] This section describes the detection results of target DNA in highly viscous samples. High-viscosity and low-viscosity samples with different liquid crystal concentrations were prepared.

[0157] Figure 36 shows the detection results of target DNA in a highly viscous sample. The liquid crystal concentration in the highly viscous sample was 3.0 wt%, and in the low-viscosity sample it was 0.3 wt%. The concentration of target DNA 9 (match DNA) was set to 1 nM. The concentration of probe particle 2 was 1.58 × 10⁻⁶. 8 [particles / mL] was used. Upward irradiation was performed, and the beam waist position was set 45 μm above the top surface TS of well 44. The laser light intensity was set to 525 m W The settings were changed, and the irradiation time was set to 240 seconds.

[0158] As shown in Figure 36, fluorescence was observed even in highly viscous samples. Therefore, it can be seen that photo-induced acceleration is possible even in highly viscous samples. This suggests that target DNA9 can be detected even in samples containing impurities.

[0159] As described above, this embodiment employs a heteroprobe system using probe particles 1 containing metal nanoparticles 11 and probe particles 2 on the order of micrometers. This allows for the detection of extremely low concentrations of target DNA 9, on the order of nM, or even on the order of pM if the conditions are appropriately set, in just a few minutes, through the synergistic effects of Brownian motion, dissipative force, and thermal convection. In other words, this embodiment enables highly sensitive and rapid detection of target DNA 9.

[0160] [Aspect] Those skilled in the art will understand that the above exemplary embodiments are specific examples of the following embodiments.

[0161] <Section 1> The steps include preparing a liquid sample comprising a plurality of nanoparticles, each modified by a first host molecule that specifically binds to the substance to be detected, and a plurality of microparticles, each modified by a second host molecule that specifically binds to the substance to be detected, The steps include irradiating the liquid sample with non-resonant light, which is light outside the wavelength range of the electronic resonance of the plurality of nanoparticles and also outside the wavelength range of the electronic resonance of the plurality of microparticles, The step includes detecting the substance to be detected based on an output signal from a photodetector that receives light from the liquid sample, A method for detecting a substance to be detected, comprising the step of combining a plurality of nanoparticles and a plurality of microparticles that differ in size and material from one another, such that each of the plurality of nanoparticles has a size that diffuses in the liquid sample by Brownian motion, each of the plurality of microparticles has a size that is subject to a dissipative force by Mie scattering of non-resonant light, and each of the plurality of nanoparticles contains a material that generates thermal convection in the liquid sample upon irradiation with non-resonant light.

[0162] <Section 2> The method for detecting a substance to be detected according to the first paragraph, wherein the preparation step further includes the step of combining the plurality of nanoparticles and the plurality of microparticles having different concentrations such that the average interparticle distance of the plurality of nanoparticles is shorter than the average interparticle distance of the plurality of microparticles.

[0163] <Section 3> The method for detecting a substance to be detected according to the first or second paragraph, wherein the preparation step further comprises the step of uniformly mixing the plurality of nanoparticles and the plurality of microparticles.

[0164] <Section 4> The steps include: holding the liquid sample on the substrate, A method for detecting a substance to be detected according to any one of the first to third claims, further comprising the step of adjusting the irradiation position of the non-resonant light such that, after the holding step, the focal point of the non-resonant light is located behind the substrate in the propagation direction of the non-resonant light.

[0165] <Section 5> The steps include adjusting the irradiation position of the non-resonant light so that the focal point of the non-resonant light is located behind the substrate in the direction of propagation of the non-resonant light, A method for detecting a substance to be detected according to any one of the first to third claims, further comprising the step of holding the liquid sample on a substrate after the adjustment step.

[0166] <Section 6> The process further includes the step of holding the liquid sample in a well provided on the substrate, The irradiating step includes a step of focusing the non-resonant light using a focusing lens including a correction ring, A method for detecting a substance to be detected according to any one of the first to third claims, further comprising the step of adjusting the correction ring so that spherical aberration correction is performed according to the thickness of the substrate and the depth of the well.

[0167] <Section 7> The substance to be detected is labeled with a fluorescent dye. The aforementioned detection step is, The steps include measuring the fluorescence intensity of the liquid sample, A method for detecting a substance to be detected according to any one of paragraphs 1 to 6, comprising the step of detecting the substance to be detected based on the amount of change in fluorescence intensity associated with irradiation with non-resonant light.

[0168] <Section 8> The substance to be detected is DNA containing multiple bases, The method for detecting a substance to be detected according to paragraph 7, wherein the detection step includes a step of detecting a single nucleotide polymorphism in the DNA.

[0169] <Section 9> The aforementioned detection step is, The steps include measuring the absorbance spectrum of the liquid sample, A method for detecting a substance to be detected according to any one of paragraphs 1 to 6, comprising the step of detecting the substance to be detected based on the amount of change in the absorbance spectrum due to irradiation with non-resonant light.

[0170] <Section 10> The aforementioned detection step is, The steps include: taking an image of the accumulation region in which the plurality of nanoparticles and the plurality of microparticles are accumulated; A method for detecting a substance to be detected according to any one of the first to sixth paragraphs, comprising the step of detecting the substance to be detected based on the area of ​​the accumulation region.

[0171] <Section 11> The aforementioned liquid sample is a highly viscous liquid sample containing impurities. The method for detecting a substance to be detected according to any one of the first to tenth paragraphs, wherein the detection step includes a step of specifically detecting the substance to be detected from the highly viscous liquid sample.

[0172] <Section 12> Multiple nanoparticles, each modified by a first host molecule that specifically binds to the substance to be detected, A plurality of microparticles, each modified by a second host molecule that specifically binds to the substance to be detected, A light source that emits non-resonant light, which is outside the wavelength range of the electronic resonance of the plurality of nanoparticles and also outside the wavelength range of the electronic resonance of the plurality of microparticles, A photodetector that receives light from a liquid sample containing the plurality of nanoparticles and the plurality of microparticles, to which the non-resonant light is irradiated, The system includes a processor that performs a detection process to detect the substance to be detected based on the output signal from the light receiver, The plurality of nanoparticles and the plurality of microparticles are combined such that each of the plurality of nanoparticles has a size that diffuses in the liquid sample by Brownian motion, each of the plurality of microparticles has a size that is subject to dissipative force due to Mie scattering of non-resonant light, and each of the plurality of nanoparticles contains a material that generates thermal convection in the liquid sample upon irradiation with the non-resonant light.

[0173] The embodiments disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of this disclosure is indicated by the claims rather than by the description of the embodiments above, and all modifications within the meaning and scope of the claims are intended to be included. [Explanation of Symbols]

[0174] 1,2 Probe particles, 11 Metal nanoparticles, 12 Host molecules, 13 Interaction sites, 21 Microparticles, 22 Host molecules, 23 Interaction sites, 231 Avidin, 232 Biotin, 3 Aggregates, 4 Detection kit, 41 Substrate, 42 Cover, 43 Spacer, 44 Well, 51 Sample stage, 52 Adjustment mechanism, 53 Laser light source, 54 Focusing lens, 55 Illumination light source, 55A Excitation light source, 56 Objective lens, 57 Camera, 58 Spectrometer, 59 Optical components, 591 Mirror, 592 Half mirror, 593 Lens, 594 Shutter, 595A, 595B Mirror, 596 Dichroic mirror, 597A, 597B Beam splitter, 598 Lens, 6 Controller, 61 Processor, 62 Memory, 63 Input / Output ports, 9 Target DNA, 90 fluorescent dye, 100, 100A detection system.

Claims

1. A method for detecting a substance to be detected that is labeled with a fluorescent dye, The procedure includes the step of preparing a liquid sample comprising a plurality of nanoparticles, each modified by a first host molecule that specifically binds to the substance to be detected, and a plurality of microparticles, each modified by a second host molecule that specifically binds to the substance to be detected, The plurality of nanoparticles and the plurality of microparticles are combined such that each of the plurality of nanoparticles has a size that diffuses in the liquid sample by Brownian motion, each of the plurality of microparticles has a size that is subject to dissipative force due to Mie scattering of non-resonant light, and each of the plurality of nanoparticles contains a material that generates thermal convection in the liquid sample by photothermal effect due to irradiation with the non-resonant light. The non-resonant light is laser light that lies outside the wavelength range of the electronic resonance of the plurality of nanoparticles and outside the wavelength range of the electronic resonance of the plurality of microparticles, and has a wavelength approximately the same as the diameter of the plurality of microparticles. The aforementioned detection method further includes, The steps include irradiating the liquid sample with non-resonant light such that, when the substance to be detected is contained in the liquid sample, the plurality of nanoparticles and the plurality of microparticles combine via the first host molecule, the substance to be detected and the second host molecule to form aggregates, The step includes detecting the substance to be detected based on an output signal from a photodetector that receives light from the liquid sample, The aforementioned detection step is, The steps include measuring the fluorescence intensity of the liquid sample, A method for detecting a substance to be detected, comprising the step of detecting the substance to be detected based on the amount of change in fluorescence intensity associated with irradiation with non-resonant light.

2. The method for detecting a substance to be detected according to claim 1, wherein the preparation step further includes the step of combining the plurality of nanoparticles and the plurality of microparticles having different concentrations such that the average interparticle distance of the plurality of nanoparticles is shorter than the average interparticle distance of the plurality of microparticles.

3. The method for detecting a substance to be detected according to claim 1, wherein the preparation step further comprises the step of uniformly mixing the plurality of nanoparticles and the plurality of microparticles.

4. The steps include: holding the liquid sample on the substrate, A method for detecting a substance to be detected according to any one of claims 1 to 3, further comprising the step of adjusting the irradiation position of the non-resonant light such that, after the holding step, the focal point of the non-resonant light is located behind the substrate in the propagation direction of the non-resonant light.

5. The steps include adjusting the irradiation position of the non-resonant light so that the focal point of the non-resonant light is located behind the substrate in the direction of propagation of the non-resonant light, A method for detecting a substance to be detected according to any one of claims 1 to 3, further comprising the step of holding the liquid sample on a substrate after the adjustment step.

6. The process further includes the step of holding the liquid sample in a well provided on the substrate, The irradiating step includes a step of focusing the non-resonant light using a focusing lens including a correction ring, A method for detecting a substance to be detected according to any one of claims 1 to 3, further comprising the step of adjusting the correction ring so that spherical aberration correction is performed according to the thickness of the substrate and the depth of the well.

7. The substance to be detected is DNA containing multiple bases, The aforementioned detection step includes the step of detecting a single nucleotide polymorphism in the DNA, The method for detecting a substance to be detected according to claim 1, wherein the step of detecting a single nucleotide polymorphism in the DNA includes the step of dissociating the double helix by raising the temperature of the double helix containing non-complementary bases that are not complementary to the plurality of bases contained in the DNA, due to the photothermal effect of the plurality of nanoparticles.

8. The aforementioned detection step is, The steps include measuring the absorbance spectrum of the liquid sample, A method for detecting a substance to be detected according to any one of claims 1 to 3, comprising the step of detecting the substance to be detected based on the amount of change in the absorbance spectrum due to irradiation with non-resonant light.

9. The aforementioned detection step is, The steps include: taking an image of the accumulation region in which the plurality of nanoparticles and the plurality of microparticles are accumulated; A method for detecting a substance to be detected according to any one of claims 1 to 3, comprising the step of detecting the substance to be detected based on the area of ​​the accumulation region.

10. The aforementioned liquid sample is a highly viscous liquid sample containing impurities. The method for detecting a substance to be detected according to any one of claims 1 to 3, wherein the detection step includes a step of specifically detecting the substance to be detected from the highly viscous liquid sample.

11. Multiple nanoparticles, each modified by a first host molecule that specifically binds to a substance to be detected labeled with a fluorescent dye, A plurality of microparticles, each modified by a second host molecule that specifically binds to the substance to be detected, A light source that emits non-resonant light, which is laser light having a wavelength approximately equal to the diameter of the plurality of microparticles, and is outside the wavelength range of the electronic resonance of the plurality of nanoparticles and outside the wavelength range of the electronic resonance of the plurality of microparticles. A photodetector that receives light from a liquid sample containing the plurality of nanoparticles and the plurality of microparticles, to which the non-resonant light is irradiated, The system includes a processor that performs a detection process to detect the substance to be detected based on the output signal from the light receiver, The detection process is a process of detecting aggregates formed when the substance to be detected is contained in the liquid sample, by the binding of the plurality of nanoparticles and the plurality of microparticles via the first host molecule and the substance to be detected and the second host molecule, based on the amount of change in the fluorescence intensity of the liquid sample upon irradiation with non-resonant light. A detection system for a substance to be detected, wherein the plurality of nanoparticles and the plurality of microparticles are combined such that each of the plurality of nanoparticles has a size that diffuses in the liquid sample by Brownian motion, each of the plurality of microparticles has a size that is subject to a dissipative force due to Mie scattering of non-resonant light, and each of the plurality of nanoparticles contains a material that generates thermal convection in the liquid sample by photothermal effect due to irradiation with non-resonant light.

12. The substance to be detected is DNA containing multiple bases, The detection system for a substance to be detected according to claim 11, wherein the detection process includes a process in which the plurality of nanoparticles, by the photothermal effect, raise the temperature of the double helix containing non-complementary bases that are not complementary to the plurality of bases contained in the DNA, thereby causing the double helix to dissociate and detecting single nucleotide polymorphisms in the DNA.

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