Method for detecting or quantifying target biomolecules using temperature-responsive fluorescent particles

Abstract

To provide a method and apparatus that can detect or quantify target biomolecules at a low concentration in a simple manner and short time.SOLUTION: Temperature-responsive probe particles have been prepared by modifying surfaces of temperature-responsive fluorescent particles and a detection method using the same, which is a modified ELISA method, has been invented. The temperature-responsive probe particles exhibit fluorescent properties when the temperature of the temperature-responsive probe particles becomes equal to or more than a specific temperature and have strong fluorescent emission. By taking advantage of this property and using a transparent inspection container 1 that transmits excitation light and fluorescence and a transparent hot plate 2, the temperature-responsive probe particles that are fixed at the bottom in a transparent inspection container are heated. A timing when the temperature of the temperature-responsive probe particles exceeds a phase transition temperature and emits fluorescence is measured by a detection unit 4 disposed below the transparent hot plate 2.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for detecting or quantifying a target biomolecule by using temperature-responsive fluorescent particles. [Background technology]

[0002] In recent years, ELISA has been widely used for the detection and quantification of biomolecules (biomarkers). ELISA utilizes an antigen-antibody reaction, which provides high specificity, but the procedures for washing the reagent and enzyme reactions are complicated and time-consuming, and the detection concentration of biomolecules is limited to about 1 pM.

[0003] In other words, the ELISA method has an essential problem in that the floating photolabeled substances that are not immobilized become background noise, resulting in poor detection sensitivity. Because fluorescent substances that constantly exhibit fluorescent properties regardless of the surrounding environment are used as photolabeled substances, it was not possible to selectively make only the immobilized photolabeled substances emit fluorescence.

[0004] For this reason, a washing process is used to remove the floating photolabeled material that becomes background noise, but this then complicates the testing process.

[0005] In addition, the photolabeling substances used in the ELISA method have a problem in that they emit weak fluorescence, making it difficult to measure with high sensitivity.

[0006] Therefore, biomolecule detection methods that are further developed from the ELISA method have also been developed. For example, in a method called digital ELISA, high sensitivity is achieved by performing an enzyme reaction in a microdroplet (femtoliter) using a devised reaction device (Non-Patent Documents 1 and 2). However, the measurement principle of these methods is the same as that of the ELISA method, and there is a problem that the operations are more complicated and time-consuming than those of the conventional ELISA method.

[0007] Single Molecule Counting (SMCTM) Immunoassay Technology has been developed as a molecular detection technology (Non-Patent Document 3). This technology is also based on the principle of ELISA, but the detection area is miniaturized by improving the detector's optical system, improving the detection sensitivity. However, it requires a dedicated expensive detector, and the measurement takes a long time because the measurement is performed by repeatedly scanning a small area.

[0008] In addition, immunochromatography has been used (Non-Patent Document 4), but it has the disadvantages that it cannot be used for early detection of disease or infection due to its low sensitivity, there are differences in visual sensitivity between examiners when visually assessing, and the false negative rate is high (20-30%).

[0009] Early diagnosis of epidemics and infections using biomarkers requires a detection sensitivity of 1 pM or less, so there is a need for the development of detection methods that are more sensitive, simpler, and can be performed in a shorter period of time.

[0010] Furthermore, although temperature-responsive fluorescent particles with high detection sensitivity have been reported recently (Patent Document 1), no specific method for optimal detection or quantification using these particles has been disclosed. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Rissin DM, et al, Nat Biotechnol. 2010:28(6):595-9. [Non-Patent Document 2] Kim SH, et al, Lab Chip, 2012, 12, 4986-4991. [Non-Patent Document 3] Hwang J., et al, Methods, 2019, 158, 69-79. [Non-Patent Document 4] Hurt CA, et al, J.Clin.Virol., 2007, 39, 132-135. [Patent Document 1] WO2020 / 241830 Summary of the Invention [Problem to be solved by the invention]

[0012] Therefore, as a result of intensive research, the inventors have produced temperature-responsive probe particles in which the surface of a temperature-responsive fluorescent particle is modified, and have invented a detection method, etc., which is an improved ELISA method using the same. Temperature-responsive probe particles exhibit fluorescent properties when the temperature exceeds a certain specific temperature, and have the property of emitting strong fluorescence. By utilizing this property, a transparent inspection vessel and a transparent hot plate that transmit excitation light and fluorescence are used, and the timing at which the temperature-responsive probe particles immobilized on the bottom surface of the transparent inspection vessel are first heated and exceed the phase transition temperature to emit fluorescence is measured in a detection unit located below the transparent hot plate. [Means for solving the problem]

[0013] One aspect of the present invention is a method for detecting or quantifying a target biomolecule contained in a biological sample by contacting a temperature-responsive probe particle having a surface modified with a temperature-responsive fluorescent particle with a biological sample and a capture biomolecule recognition element, and then measuring the fluorescence emission intensity, the method comprising the steps of: (1) preparing a transparent test vessel having a capture biomolecule recognition element fixed to the inside of its bottom surface; (2) contacting the temperature-responsive probe particle with the biological sample and the capture biomolecule recognition element to obtain a measurement target in the transparent test vessel; (3) contacting the temperature-responsive probe particle with the biological sample and the capture biomolecule recognition element to obtain a measurement target in the transparent test vessel; (4) placing the bottom surface of the transparent test vessel on a transparent hot plate; (5) heating the transparent hot plate; and (6) measuring the fluorescence emission intensity near the bottom surface of the transparent test vessel from below the transparent hot plate. Effect of the Invention

[0014] According to the present invention, it is possible to provide a method and an apparatus capable of detecting or quantifying a target biomolecule at a low concentration simply and in a short time. [Brief description of the drawings]

[0015] [Figure 1] 1 is a schematic diagram of a detection device of the present invention; [Diagram 2] FIG. 1 is a perspective view showing a state in which a transparent cuvette 1 of the present invention is placed in a cuvette holder 10. [Diagram 3] 1 is a cross-sectional view showing a state in which a transparent cuvette 1 of the present invention is placed in a cuvette holder 10. FIG. [Figure 4] FIG. 1 is a cross-sectional view of an example of a transparent hot plate 2 of the present invention. [Diagram 5] FIG. 1 is a plan view of a transparent hot sheet 20 used in an example of a transparent hot plate 2 of the present invention. [Figure 6] FIG. 1 is a diagram (1) for explaining an overview of the fluorescence emission intensity measurement in the detection method of the present invention. [Figure 7] FIG. 2 is a diagram for explaining an outline of the fluorescence emission intensity measurement in the detection method of the present invention (2). [Figure 8] FIG. 2 is a diagram illustrating quenching and fluorescence emission of the temperature-responsive fluorescent particles of the present invention. [Figure 9] FIG. 1 is a diagram showing an example of the detection method of the present invention (1). [Figure 10] FIG. 2 is a diagram showing an example of the detection method of the present invention (2). [Figure 11] FIG. 2 is a diagram illustrating the change over time in fluorescence emission intensity in a transparent container 1 when a target biomolecule is present. [Figure 12] FIG. 2 is a diagram illustrating the change over time in fluorescence emission intensity in a transparent container 1 in the absence of a target biomolecule. [Figure 13] FIG. 1 is a diagram illustrating the principle of a detection method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, an embodiment for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiment. Furthermore, the dimensional ratios in the drawings do not necessarily correspond to the actual dimensional ratios.

[0017] (Overview of the detection device of the present invention) An overview of the detection device of the present invention will be described with reference to Fig. 1. The detection device 100 is entirely light-shielded by a housing 9 to prevent noise light from entering from the outside during detection. A transparent hot plate 2 is installed at the top inside the detection device 100. A light source unit 3 and a detection unit 4 are arranged below the transparent hot plate 2. A filter 5 may be arranged to make it easier to measure the fluorescence emitted from the detection unit 4.

[0018] The light source unit 3 is disposed so that light emitted therefrom reaches the transparent hot plate 2 and the transparent inspection container 1 to be placed above it. The detection unit 4 measures the fluorescence emission intensity near the bottom surface inside the transparent inspection container 1 through the transparent hot plate 2, and is therefore installed substantially below the transparent hot plate 2 in the direction of gravity. The light source unit 3 needs to be disposed at a position that does not interfere with the measurement by the detection unit 4.

[0019] (Outline of the detection method of the present invention) The measurement is performed by placing a transparent inspection container 1 containing the object to be measured on a transparent hot plate 2, heating the transparent inspection container 1 from the bottom, emitting excitation light from a light source unit 3, and measuring the fluorescence emission intensity near the bottom inside the transparent inspection container by a detection unit 4.

[0020] (Transparent inspection container) The material of the transparent inspection container 1 used in the present invention is preferably a material such as transparent plastic or glass that does not block or absorb the excitation light emitted by the light source unit 3 and the fluorescence emitted by the measurement target. The shape of the transparent inspection container 1 is, for example, a cylindrical container with an open top, but is not limited to this.

[0021] (Test vessel holder) The transparent test vessel 1 may be placed directly on the transparent hot plate 2, but may also be fixed to a test vessel holder 10 as shown in Figs. 1 and 2. This allows many transparent test vessels 1 to be used for measurement at the same time. In order not to interfere with the detection method of the present invention, it is preferable that the test vessel holder 10 is structured so as not to cover the bottom of the transparent test vessel 1 as shown in Fig. 3(a), or that the bottom is made of a transparent material as shown in Fig. 3(b). In addition, it is preferable that the basic members constituting the test vessel holder 10 are black so as not to interfere with the measurement of the fluorescence emission intensity due to light reflection.

[0022] As an example of the former, it is possible to provide a protuberance on the top of the transparent test vessel 1 and to fix it by hooking it into a hole in the test vessel holder 10. When the test vessel holder 10 is placed on the transparent hot plate 2, the transparent test vessel 1 may be designed to be in contact with the transparent hot plate 2, or may be designed to be floated by about several millimeters. An example of the latter is a transparent bottom 11 made of transparent plastic, glass, or the like that does not block or absorb the excitation light emitted by the light source unit 3 or the fluorescence emitted by the measurement target.

[0023] (Transparent hot plate) The transparent hot plate 2 is preferably made of a material such as transparent plastic or glass that does not block or absorb the excitation light emitted by the light source unit 3 or the fluorescence emitted by the measurement target. Glass is particularly preferable from the viewpoint of rapid and uniform temperature transfer.

[0024] 4 shows an example of the structure of the transparent hot plate 2. The transparent hot sheet 20 is a sheet that is transparent overall, but generates heat by conducting electricity. One example of the structure of the transparent hot plate 2 employs a three-layer structure in which the transparent hot sheet 20 is sandwiched between glass plates 21.

[0025] FIG. 5 is a plan view of the transparent hot sheet 20. The transparent hot sheet 20 is constructed by arranging a plurality of thin heating wires 22 on a transparent film 24 serving as a substrate, and connecting the heating wires 22 to an electrode 23. The transparent film 24 may be made of any material that does not block or absorb the excitation light emitted by the light source unit 3 and the fluorescence emitted by the measurement target. The heating wires 22 are thin wires made of heating elements such as nichrome wires, and are embedded in or attached to the transparent film 24. The heating wires 22 generate heat when electricity is applied to the electrode 23. Although a plurality of heating wires 22 are arranged on the transparent film 24, they are thin wires and do not interfere with the measurement.

[0026] In addition to the above, the transparent hot plate 2 may be a plate made of glass or plastic alone, heated from the edge to heat the entire plate by heat transfer. There are no other limitations on the configuration of the transparent hot plate 2.

[0027] (Light source part) Any light source capable of outputting the excitation wavelength of the photolabeling substance can be used as the light source unit 3. For example, a light emitting diode capable of outputting a specific wavelength, or a laser light source capable of outputting a wide range of wavelengths, with a filter that allows only a specific wavelength to pass, installed in the middle of the laser light path, can be considered.

[0028] (Detection unit) In addition to a typical commercially available fluorometer, a general-purpose digital camera, a built-in camera of a smartphone or tablet, etc. can be used as the detection unit 4. According to the inspection method of the present invention, since the fluorescence of the temperature-responsive fluorescent particles is strong, detection can be practically performed even with general-purpose devices such as smartphones and tablets, depending on the conditions.

[0029] (filter) In the present invention, in order to limit the light entering the detection unit 4, a filter 5 may be provided above the detection unit 4. The filter 5 is a film that cuts out light that becomes noise other than fluorescent emission, such as excitation light.

[0030] (Outline of the fluorescence emission intensity measurement of the present invention) 6 is a diagram for explaining an outline of the fluorescence emission intensity measurement in the method of the present invention. The light source unit 3 irradiates the transparent hot plate 2 with excitation light corresponding to the temperature-responsive fluorescent particles from below the transparent hot plate 2 in the approximate direction of gravity, and the excitation light passes through the transparent hot plate 2 and the bottom surface of the transparent testing container 1 to be irradiated to the measurement target.

[0031] At this time, as shown in Fig. 7, the transparent hot plate 2 gradually heats the transparent inspection container 1 from its bottom, which creates a temperature distribution in the liquid in the transparent inspection container 1, with the liquid being warmer closer to the bottom. When the temperature-responsive fluorescent particles fixed to the bottom exceed a phase transition temperature (described later), the temperature-responsive fluorescent particles exhibit fluorescent properties and emit fluorescence (Fig. 8). In other words, by adjusting the temperature distribution, it is possible to create a timing at which the temperature-responsive fluorescent particles fixed to the bottom exhibit fluorescent properties and the floating temperature-responsive fluorescent particles do not exhibit fluorescent properties. 6, the detection unit 4 measures the intensity of the fluorescence emitted at this time. At the above timing, only the fluorescence emitted by the temperature-responsive fluorescent particles immobilized on the bottom surface can be detected.

[0032] (Temperature-responsive fluorescent particles) The temperature-responsive fluorescent particles contain at least one type of fluorescent molecule in a molecular assembly that contains at least one type of amphipathic molecule. That is, the temperature-responsive fluorescent particles of the present invention utilize the phenomenon that the quenched state caused by the aggregation of fluorescent molecules is dissociated and becomes a luminous state with a phase transition (i.e., a transition from a solid phase to a liquid phase, or a transition from a gel phase to a liquid crystal phase) (Figure 8).

[0033] Therefore, the temperature-responsive fluorescent particles used in the present invention can be used without any particular limitation as long as they undergo a phase transition from a solid state to a liquid state while maintaining the molecular assembly form. The molecular assembly preferably has a monolayer or bilayer, and more preferably has a lipid bilayer (also called a lipid bilayer).

[0034] The amphipathic molecules (e.g., lipid molecules) are not limited to lipids derived from living organisms, but include amphipathic molecules that can be synthesized using raw materials such as semi-synthetically or synthetically produced low molecules such as phospholipids and their derivatives, polymers, peptides, amino acids, and carboxylic acids.

[0035] The temperature-responsive fluorescent particles undergo a solid-liquid phase transition in response to temperature, in which the fluorescent molecules dissociate and emit fluorescence when the molecular assembly is in the liquid phase, and aggregate and quench when the molecular assembly is in the solid phase, thereby enabling the fluorescent molecules to reversibly switch between fluorescence emission and quenching in response to temperature (Figure 8).

[0036] That is, the molecular assembly exhibits a solid-liquid phase transition in response to temperature, has dissociated fluorescent molecules in the liquid phase and emits fluorescence, and has aggregated fluorescent molecules in the solid phase and quenches the fluorescence due to aggregation-caused quenching. When the molecular assembly is a molecular assembly having a lipid bilayer, the solid phase is a gel phase and the liquid phase is a liquid crystal phase.

[0037] The fluorescent molecules are preferably amphiphilic or lipophilic, since they are arranged in hydrophobic regions constructed in a molecular aggregate state in an aqueous phase, for example, in regions surrounded by a monolayer or in bilayer membranes such as lipid bilayers. Generally, fluorescent molecules have the property of quenching by aggregation, so long as the fluorescent molecules show aggregation and dissociation in response to a solid-liquid phase transition (for example, a gel-liquid crystal phase transition of a lipid bilayer membrane), quenching and emission can be converted.

[0038] An example of the fluorescent molecule is a compound represented by the following general formula (A).

[0039] [ka]

[0040] Some biomolecules, such as nicotinamide adenine dinucleotide phosphate, flavins, proteins, and amino acids, absorb light with a wavelength of 600 nm or less and emit fluorescence (autofluorescence) with a wavelength of 600 nm or less. For this reason, when a fluorescent probe with a maximum absorption wavelength and a maximum fluorescence emission wavelength of 600 nm or less is used to detect a biomolecule, interference due to the autofluorescence of contaminant molecules may become a problem in fluorescence measurement. Therefore, the compound represented by the general formula (A) of the present invention is preferably a fluorescent molecule with a maximum absorption wavelength and a maximum fluorescence emission wavelength of 600 nm or more, and further, considering that when light with a wavelength of 980 nm or more is irradiated to water, heat generated by absorption by water acts on the temperature-responsive particles, it is more preferable that the compound is a fluorescent molecule with a maximum absorption wavelength and a maximum fluorescence emission wavelength in the range of 600 to 900 nm.

[0041] More specific examples of the fluorescent molecule of the compound of formula (A) include squaric acid derivatives.

[0042] Examples of the squaric acid derivative include the compound represented by the following general formula (I).

[0043] [ka]

[0044] Here, R1 and R2 each independently represent a hydrophobic group, which may be the same or different.

[0045] The compounds represented by general formula (A) or general formula (I) include not only free forms but also salts thereof. The salts vary depending on the type of compound, but examples thereof include inorganic base salts such as alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salts, and ammonium salts, as well as base addition salts such as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine, or inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, and acid addition salts such as organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate.

[0046] In the squaric acid derivative, R1 and R2 may be straight-chain hydrocarbons, branched-chain hydrocarbons, saturated hydrocarbons, or unsaturated hydrocarbons. A preferred example is a straight-chain saturated hydrocarbon, but is not limited thereto as long as the fluorescent molecules dissociate in the liquid crystal phase of the molecular aggregate and aggregate in the gel phase.

[0047] More specific examples of R1 and R2 include, for example, a hydrocarbon group having 2 to 10 carbon atoms, and more specifically, can be selected from an n-butyl group, an n-pentyl group, or an n-hexyl group, but are not limited thereto as long as the group dissociates in the liquid crystal phase of the molecular assembly and aggregates in the gel phase.

[0048] An example of the amphipathic molecule is a phospholipid.

[0049] There is no particular limitation on the type of phospholipid, but glycerophospholipids (also called diacyl phospholipids) are preferred, and in particular, to form a stable lipid bilayer membrane that exhibits phase transition, diacyl phospholipids having a phosphocholine group in the hydrophilic part are preferred. The acyl chain length of the diacyl phospholipids may be the same or different. Diacyl phospholipids having a phosphocholine group include saturated phospholipids and unsaturated phospholipids, and any of these may be used in the present invention, or these may be used in combination. As the saturated phosphocholine, synthetic or semi-synthetic phospholipids, for example, natural phospholipids such as hydrogenated egg yolk lecithin and hydrogenated soybean lecithin with a hydrogenation rate close to 100%, and derivatives thereof, as well as dimyristoyl phosphocholine, dipentadecanoyl phosphocholine, dipalmitoyl phosphocholine, diheptadecanoyl phosphocholine, distearoyl phosphocholine, etc. may be used.

[0050] Examples of the molecular assembly include emulsions and lipid nanoparticles having a hydrophobic core surrounded by a monolayer (also called a single layer), and vesicles, which are small vesicles having a membrane structure closed in a spherical shell shape. Examples of such vesicles include, but are not limited to, lipid bilayer vesicles (also called liposomes). Particles that can be used as the temperature-responsive fluorescent particles of the present invention, that is, particles that can be used as the molecular assembly, are not limited by whether they are multilayer or single layer, particle size, etc., as long as they are molecular assemblies that undergo a solid-liquid phase transition or a gel-liquid crystal phase transition at a specific temperature.

[0051] In order to prevent aggregation or fusion of molecular assemblies such as lipid vesicles, amphiphilic molecules with charged residues or polymer chains in the hydrophilic part can be included as components. The molecular assemblies such as lipid vesicles obtained in this way have high dispersion stability due to electrostatic repulsion due to the charge and steric exclusion effect of the polymer chains.

[0052] Examples of amphipathic molecules having a charged residue, when the molecule is a lipid molecule, include anionic phospholipids having phosphatidylglycerol, phosphatidylserine, or phosphatidic acid in the hydrophilic portion, carboxylic acid type lipids such as L-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA), and amino acid type lipids having an amino acid in the hydrophilic group.

[0053] In addition, an example of an amphiphilic molecule having a polymer chain, when the molecule is a lipid molecule, is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-monomethoxy poly(ethylene glycol). There is no restriction on the molecular weight of poly(ethylene glycol), but a molecular weight of 1000 to 5000 is preferable to prevent aggregation of lipid vesicles.

[0054] In addition, when the amphipathic molecule is a lipid molecule, the amphipathic molecule may be 1,2-dimyristoyl-sn-glycero-3-phosphocholine (PC14), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (PC15) (manufactured by Avanti Polar Lipids Inc.), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC17) (manufactured by Avanti Polar Lipids Inc.), 1,2-distearoyl-sn-glycero-3-phosphocholine (PC18), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC16 (also called DPPC)), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,5-dihexadecyl-N-succiny-L-gluta mate (DHSG), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), etc. can also be used.

[0055] The aggregation-induced quenching of fluorescent molecules in a molecular assembly such as the lipid vesicle depends on the concentration in the molecular assembly, and the aggregation-induced quenching decreases with a decrease in the concentration of the fluorescent molecules. Therefore, the temperature-responsive fluorescent particle of the present invention contains the compound represented by the general formula (I) at a concentration of 0.3 mol % or more, preferably 0.7 mol % or more, and more preferably 1.0 mol % or more.

[0056] The temperature-responsive fluorescent particles can be produced by allowing the compound of formula (I) to coexist during the production of the molecular assembly. As an example of the production of the molecular assembly, it can be produced according to a known method for producing lipid vesicles (Sheng Dong et al., ACS Appl. Nano Mater. 2018, 1, 1009-1013).

[0057] The temperature-responsive fluorescent particles of the present invention can be used to produce the following temperature-responsive probe particles.

[0058] (Temperature-responsive probe particles) The temperature-responsive probe particle can be obtained by modifying the surface of the temperature-responsive fluorescent particle with a biomolecule recognition element. In this specification, the term "element" means a molecule that exerts a specific function, such as a biomolecule recognition function, and the terms "element" and "molecule" can be used interchangeably. In this specification, "recognition" means recognizing a target molecule and binding to the molecule. Furthermore, the "biomolecular recognition element" includes not only those that directly recognize a biomolecule by themselves, but also those that indirectly recognize a biomolecule through binding with other molecules or molecular complexes (e.g., a complex of streptavidin and a biotinylated antibody that recognizes a biomolecule, etc.).

[0059] Examples of the biomolecule recognition element include an antibody, or the variable region of the antibody, or a fab fragment of the antibody (hereinafter referred to as "antibody, etc."). In this specification, such an element, for example, an antibody, etc., is referred to as a "biomolecular recognition element for detection" as described below, and when the element is an antibody, etc., it is also referred to as a "detection antibody."

[0060] On the other hand, an element for another epitope of the target biomolecule, such as an antibody, may be used for the purpose of fixing the target biomolecule to the bottom surface of the measurement container, etc. In this specification, such an element is referred to as a "capture biomolecule recognition element", and when the element is an antibody, it is also referred to as a "capture antibody".

[0061] The biomolecular recognition element can form a complex between the target biomolecule and the temperature-responsive fluorescent particle by binding to the target biomolecule to be detected or measured.

[0062] When an antibody is used as the biomolecule recognition element as in the conventional sandwich ELISA method, two types of antibodies, a detection antibody and a capture antibody, which bind to different epitopes of the same antigen, are used. The detection antibody binds the antigen to the temperature-responsive fluorescent particle via the antibody. The capture antibody has the role of capturing and fixing the target biomolecule, which is the subject of the test, to the bottom surface of the measurement container such as a well by fixing the antibody to the bottom surface of the measurement container.

[0063] As in the sandwich ELISA method, in the temperature-responsive probe particles of the present invention, a detection antibody is used as an antibody for binding to the temperature-responsive fluorescent particles, and a capture antibody is used as an antibody for fixing a target biomolecule to the bottom surface of a measurement container such as a well.

[0064] That is, when the biomolecule recognition element is an antibody, (a) a temperature-responsive probe particle composed of the temperature-responsive fluorescent particle and a detection antibody, (b) a target biomolecule that is an antigen of the detection antibody and the capture antibody, and (c) a capture antibody fixed to the bottom of a measurement container such as a well form a complex of temperature-responsive probe particle (temperature-responsive fluorescent particle with a detection antibody modified on the membrane surface)-target biomolecule (antigen)-capture antibody-measurement container, and the temperature-responsive probe particle is fixed to the bottom of the measurement container. In this state, when the bottom of the measurement container is heated, the temperature of the complex is rapidly increased by heat conduction from the bottom of the measurement container (Figure 11 (b)). At this time, the temperature-responsive probe particle in the complex undergoes a phase transition from solid phase to liquid phase, and the fluorescent molecules in the temperature-responsive fluorescent particle that were aggregated and quenched in the solid phase are dissociated and emit fluorescence. On the other hand, when the target biomolecule, i.e., the antigen, is not present in the test sample, the complex is not formed. Therefore, the temperature-responsive probe particles cannot adhere to the bottom of the measurement container, and even if the bottom of the measurement container is heated, the temperature rise is only slow due to heat transfer through the solvent, so that the solid phase state is maintained for a longer period of time and the quenching state is maintained (Figure 12(b)).

[0065] The temperature-responsive probe particle of the present invention, i.e., the temperature-responsive fluorescent particle whose surface is modified with a biomolecular recognition element, can be obtained by producing a lipid vesicle modified with a detection antibody according to a known method (International Publication Pamphlet WO2020 / 241830, WO2000 / 064413, JP 2003-73258 A, JP 58-134032 A, Manjappa SA, et al, J. Controlled Release 2011, 150, 2-22, Li T., et al, Nanomed.: Nanotechnol. Biol. Medicine, 2017, 13, 1219-1227).

[0066] In addition, the temperature-responsive fluorescent particles and temperature-responsive probe particles of the present invention can be produced by known methods (such as Scientific Reports volume 10, Article number: 18086 (2020)).

[0067] The measurement procedure of the conventional ELISA method requires an operation for separating the antibody-enzyme conjugate bound to the target biomolecule from the antibody-enzyme conjugate not bound to the target biomolecule, and / or an operation for washing the antibody-enzyme conjugate not bound to the target biomolecule. On the other hand, the present invention utilizes the difference between the rapid temperature rise caused by heat conduction to the temperature-responsive probe particles fixed near the heat source by utilizing the antigen-antibody reaction to the epitope of the target biomolecule, and the slow temperature change caused by heat conduction via the solvent to the temperature-responsive fluorescent particles not bound to the target biomolecule, and therefore the target biomolecule can be detected simply, in a short time, and with high sensitivity by measuring the fluorescence emission intensity without performing the separation and washing procedures required for the ELISA method.

[0068] On the other hand, in the sandwich ELISA method, measurement kits for various biomolecules are already commercially available. That is, the kit includes a measurement microwell plate in which a capture antibody is fixed to the bottom of the well as a measurement container, and a detection antibody. Therefore, for example, such a commercially available sandwich ELISA measurement kit can be used. The present invention can be carried out by producing the temperature-responsive probe particle of the present invention and obtaining the detection antibody contained in such a kit and a microwell plate in which a capture antibody is fixed to the bottom of the well.

[0069] In addition, in the sandwich ELISA method, a measurement kit is commercially available that measures an antigen by forming a complex between a capture antibody immobilized on the bottom surface of a well of a microwell plate, an antigen (target biomolecule), a biotinylated detection antibody, and horseradish peroxidase (HRP)-labeled streptavidin (see FIG. 9(a)). Therefore, as the temperature-responsive probe particle of the present invention, a temperature-responsive fluorescent particle with a biotin-labeled surface is manufactured, and a capture antibody immobilized on the bottom surface of a well of a microplate, an antigen (target biomolecule), a biotinylated detection antibody, streptavidin, and a temperature-responsive fluorescent particle with a biotin-labeled surface are mixed to form a complex, and the bottom surface of the well of the microplate is heated with a heat source to rapidly cause a phase transition of the temperature-responsive fluorescent particle to emit fluorescence, thereby measuring the antigen (target biomolecule) in a test sample (see FIG. 9(b)). That is, in this case, the biorecognition element is not a detection antibody alone, but a biotin-streptavidin-biotinylated detection antibody complex modified on the surface of the temperature-responsive fluorescent particle. Alternatively, a complex produced by modifying the surface of a lipid vesicle with a maleimide group by introducing a maleimide-terminated polyethylene glycol type lipid, and reacting the thiol group of Fab', which is a fragmented antibody obtained by reducing the detection antibody after treating it with pepsin, can also be said to be a complex of a temperature-responsive fluorescent lipid vesicle-fragmented detection antibody. In this way, the biorecognition element is not limited to an antibody alone, nor is it limited to biotin-streptavidin, and the temperature-responsive probe particle of the present invention can be produced and used by utilizing various molecules that form a complex having strong binding properties by molecular recognition or the like.

[0070] Examples of molecular species that can be used to form a complex through molecular recognition include, but are not limited to, a combination of a sugar chain and a lectin, a complementary base sequence of a nucleic acid, a combination of a receptor and a ligand molecule, a nucleic acid aptamer, and a peptide aptamer.

[0071] (Method for detecting or quantifying target biomolecules) The method for detecting or quantifying a target biomolecule of the present invention is completely different from conventional detection methods, such as a method in which fluorescent probe particles or the fluorescent molecules contained in the particles are transported to a cell membrane by endocytosis or membrane fusion to detect a cell, or a method in which fluorescence generated by the release of fluorescent molecules from the fluorescent probe particles is detected.

[0072] As described above, the temperature-responsive probe particles and the temperature-responsive fluorescent particles can detect or measure target biomolecules easily and with high sensitivity in a short time by measuring the fluorescence emission intensity without performing a separation operation or a washing operation. Alternatively, biomolecules can be detected or quantified by a washing operation instead of heating. In addition, by forming the test sample into microdroplets, it can also be used as a molecular detection method for the target biological sample.

[0073] (Step (1) of the detection method of the present invention) Step (1) of the detection method of the present invention is a step of preparing a transparent test vessel in which a capture biomolecule recognition element is fixed on the inside of the bottom surface. The method for fixing the capture biomolecule recognition element is a known method. Note that, for step (1), a commercially available transparent test vessel in which a capture biomolecule recognition element is fixed on the inside of the bottom surface can also be used.

[0074] (Step (2) of the detection method of the present invention) Step (2) of the detection method of the present invention is a step of contacting the temperature-responsive probe particles, the biological sample, and the capture biomolecule recognition element to obtain a measurement target in the transparent test container.

[0075] The concentration of the temperature-responsive probe particles used in the method for detecting or quantifying biomolecules of the present invention is not particularly limited, but it is preferable that the temperature-responsive probe particles are contained in the solution at a concentration of 1.0 μg / ml or more, more preferably 2.0 μg / ml or more, and even more preferably 5.0 μg / ml or more when contacting the capture biomolecule recognition element.

[0076] Examples of biological samples used in the detection or quantification method of the biomolecule of the present invention include samples collected from animals or cells. The sample may be a sample known to contain a target biomolecule, or may be a sample whose presence or absence of a target biomolecule is unknown. Examples of such animals or cells include mammals such as mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, and humans, or cells derived from such animals. Examples of samples derived from animals include blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal secretions, semen, pleural fluid, gastrointestinal secretions, cerebrospinal fluid, interstitial fluid, and lymphatic fluid, and are preferably blood, serum, or plasma. These samples can be obtained by methods known per se. For example, serum and plasma can be prepared by collecting blood from a test animal according to a conventional method and separating the liquid components, and cerebrospinal fluid can be collected by known means such as spinal puncture.

[0077] The method of contacting the temperature-responsive probe particle and the biological sample with the capture biomolecule recognition element fixed to the substrate is not particularly limited. For example, the solution containing the biological sample and the temperature-responsive probe particle can be added to the substrate in this order, but is not limited to this method. Alternatively, a solution prepared by mixing the biological sample and the temperature-responsive probe particle in advance may be added to the substrate (i.e., the biological sample and the temperature-responsive probe particle may be contacted with the substrate at the same time). The conditions for the above contact are not particularly limited, but the contact is usually made at a temperature of 0 to 45°C, preferably at a temperature of 0 to 40°C, more preferably at a temperature of 4 to 37°C, and even more preferably at a temperature of 25 to 37°C. The contact time is also not particularly limited, but is typically 6 hours or less, preferably 2 hours or less, and more preferably 1 hour or less. The lower limit of the contact time is also not particularly limited, but is typically 10 seconds or more, preferably 1 minute or more, and more preferably 10 minutes or more.

[0078] In addition, the same biomolecule recognition element (e.g., biotin, etc.) as that possessed by the temperature-responsive probe particle may be added to the biomolecule recognition element for detection, so that the temperature-responsive probe particle and the biomolecule recognition element for detection may be bonded via an element (e.g., streptavidin, etc.) that recognizes the biomolecule recognition element. Thus, in one embodiment, step (2) may be a step of contacting the temperature-responsive probe particle, the element that recognizes the biomolecule recognition element, the biomolecule recognition element for detection, and the biological sample with the biomolecule recognition element for capture immobilized on the substrate. The contact method may be the same as the method described above. For example, the contact method may be performed by adding a solution containing a biological sample, a biomolecule recognition element for detection, an element that recognizes the biomolecule recognition element, and a solution containing the temperature-responsive probe particle in this order onto the substrate, but is not limited to this order. In addition, the biomolecule recognition element for detection, the element that recognizes the biomolecule recognition element, and the temperature-responsive probe particle may be contacted with the biomolecule recognition element for capture separately. Alternatively, a solution prepared by mixing a biological sample, a detection biomolecule recognition element, an element that recognizes the biomolecule recognition element, and a temperature-responsive probe particle in advance may be added onto the substrate (i.e., a biological sample, an element that recognizes the temperature-responsive fluorescent probe biomolecule recognition element, and a detection biomolecule recognition element may be simultaneously brought into contact with the substrate). Schematic diagrams of this method are shown in Fig. 10(a) to (c). According to this method, as shown in Fig. 10(c), a plurality of temperature-responsive probe particles are bound to one biological molecule, thereby improving the detection sensitivity. In a preferred embodiment, the biomolecule recognition element is biotin, and the element that recognizes the biomolecule recognition element is streptavidin, avidin, or neutravidin.

[0079] (Step (3) of the detection method of the present invention) Step (3) of the detection method of the present invention is a step of placing the bottom surface of the transparent inspection vessel on the transparent hot plate. In this embodiment, one or more transparent inspection vessels may be placed directly on the transparent hot plate, or may be placed in a vessel holder and then placed on the transparent hot plate. As long as the heat of the transparent hot plate is transferred to the transparent inspection vessel, there is no limitation, and there is no problem even if the transparent inspection vessel is floating above the transparent hot plate or a transparent sheet is sandwiched between the transparent inspection vessel and the transparent hot plate. However, it is necessary that the embodiment does not block or absorb the excitation light emitted by the light source unit 3 and the fluorescence emitted by the measurement target.

[0080] (Step (4) of the detection method of the present invention) Step (4) of the detection method of the present invention is a step of heating the transparent hot plate. The timing of heating may be after the bottom surface of the transparent test container is placed on the transparent hot plate, or may be before the bottom surface is placed on the transparent hot plate.

[0081] The transparent hot plate can be one that generates heat by itself, one that generates heat by the action of electricity or light, one that obtains heat by thermal conduction from another heat source, etc. The temperature (phase transition temperature) at which the temperature-responsive fluorescent particles used undergo phase transition (i.e., transition from solid phase to liquid phase, or transition from gel phase to liquid crystal phase) must be measured in advance, and the transparent hot plate must be heated to the phase transition temperature or higher.

[0082] In this case, the greater the difference between the set temperature of the transparent hot plate and the phase transition temperature, the faster the bottom of the transparent inspection vessel heats up and the faster the phase transition occurs. If the difference is too large, the temperature rises rapidly and the entire transparent inspection vessel exceeds the phase transition temperature quickly, so the time difference between the temperature-responsive probe particles to be measured that are captured on the bottom of the transparent inspection vessel and the temperature-responsive probe particles that are not captured and are floating in the transparent inspection vessel and that exceed the phase transition temperature becomes small, resulting in a problem that it becomes difficult to measure the fluorescence emission intensity. On the other hand, if the difference is too small, the temperature-responsive probe particles to be measured that are captured on the bottom surface of the transparent test container do not heat up easily, and it takes too long to measure the fluorescence emission intensity. For this reason, it is preferable to set the transparent hot plate to a temperature at which the measurement of the fluorescence emission intensity can be completed within a few seconds to one minute.

[0083] (Step (5) of the detection method of the present invention) Step (5) of the detection method of the present invention is a step of measuring the fluorescence emission intensity in the vicinity of the bottom surface inside the transparent test container from below the transparent hot plate.

[0084] There is no particular limitation on the method for detecting or measuring the fluorescence emission, but it can be detected or measured using a general optical fiber type fluorescence detector, a microplate reader, a fluorescence microscope, a detector equipped with a camera (e.g., a camera equipped with a CMOS element) (e.g., a gel imager), etc. Furthermore, similar to the method of the ELISA method, a calibration curve can be created using a sample (standard product) with a known concentration of the target biomolecule, and the concentration of the target biomolecule in a biological sample can be quantified using the calibration curve. Furthermore, it can also be quantified by the biomolecule quantification method described below. In other words, the biomolecule detection method of the present invention can be applied as a biomolecule quantification method.

[0085] The basic principle of detection is that excitation light is directed into the transparent testing container from a light source located below the transparent hot plate, causing the temperature-responsive probe particles to selectively emit fluorescence when the phase transition temperature is exceeded and the quenched state caused by the aggregation of fluorescent molecules is dissociated, resulting in an emitting state, and the intensity of the fluorescence is measured in the detection section.

[0086] In this case, since the temperature-responsive probe particles to be measured are captured on the bottom surface of the transparent inspection vessel, the transparent hot plate causes them to exceed the phase transition temperature at an earlier time than the temperature-responsive probe particles that are not captured and are floating in the transparent inspection vessel.

[0087] Figure 11 shows the relationship between the passage of time and the fluorescence intensity when the target biomolecule is present in the biological sample. (a) At the moment when the transparent test vessel is placed on the transparent hot plate, the temperature of the temperature-responsive probe particles to be measured is below the phase transition temperature, so even if excitation light is applied from the light source, no fluorescence is emitted. After that, (b) when the transparent test vessel is heated by the transparent hot plate, the temperature-responsive probe particles to be measured that have been captured on the bottom surface of the transparent test vessel are heated and exceed the phase transition temperature, causing fluorescence emission. However, at this point, the temperature-responsive probe particles that are not captured and floating in the transparent test vessel do not emit fluorescence because they have not yet exceeded the phase transition temperature. After that, (c) the temperature-responsive probe particles that are not captured and floating in the transparent test vessel are also heated and exceed the phase transition temperature, causing fluorescence emission. When this is measured from the lower detection section, it becomes like the graph at the bottom of Figure 11.

[0088] Figure 12 shows the relationship between the passage of time and the fluorescence intensity when the target biomolecule is not present in the biological sample. Compared to Figure 11, since there are no temperature-responsive probe particles to be measured trapped on the bottom surface of the transparent inspection container, most of the temperature-responsive probe particles in the transparent inspection container are in a floating state, and the fluorescence emission intensity is weak even in the state where only the bottom surface of the transparent inspection container is heated (b). After that, when the temperature-responsive probe particles that are not trapped and are floating in the transparent inspection container are also heated, the fluorescence emission intensity reaches a maximum value (c).

[0089] Figure 13 is a superposition of the bottom graphs of Figures 11 and 12. The fluorescence emission intensity when the temperature-responsive probe particles floating uncaptured in the transparent test vessel are also heated is the same whether or not the target biomolecule is present in the biological sample. However, when the target biomolecule is present in the biological sample, the temperature-responsive probe particles to be measured are captured and localized on the bottom surface of the transparent test vessel, and the fluorescence emission intensity becomes stronger at an earlier timing than when the target biomolecule is not present. By measuring this time difference, it is possible to determine whether the target biomolecule is present. Additionally, the amount of target biomolecule present can be quantified by calibration using samples known to contain a particular amount of the target biomolecule. [Explanation of symbols]

[0090] 1 Transparent inspection container 2 transparent hot plates 3 Light source section 4. Detection section 5. Filters 9. Cabinet 10 Test vessel holder 40 antigens 41 Capture antibody 42 Photolabeling substances

Claims

1. A method for detecting or quantifying a target biomolecule contained in a biological sample by contacting a temperature-responsive probe particle having a surface-modified temperature-responsive fluorescent particle with a biological sample and a capture biomolecule recognition element, and then measuring the fluorescence emission intensity, comprising: preparing a transparent test vessel having the capture biomolecular recognition element immobilized on the inside of the bottom surface; a step of contacting the temperature-responsive probe particle, the biological sample, and the capture biomolecule recognition element to obtain a measurement target in the transparent test container; placing a bottom surface of the transparent test vessel on a transparent hot plate; heating the transparent hot plate; measuring the fluorescent emission intensity in the transparent test container from below the transparent hot plate; Including, The method is characterized in that the step of measuring the fluorescence emission intensity is performed at a time when the temperature-responsive probe particles immobilized on the inside of the bottom surface of the transparent inspection vessel are heated faster than the temperature-responsive probe particles that are not immobilized and are floating in the transparent inspection vessel, exceeding their phase transition temperature and emitting fluorescence.

2. 2. The method according to claim 1, wherein the step of obtaining a measurement target in the transparent cuvette does not include a step of cleaning the inside of the transparent cuvette when obtaining the measurement target.

3. The method according to claim 1, wherein the transparent hot plate is made of glass.

4. A detection device for detecting a target biomolecule contained in a biological sample, comprising a light source unit, a transparent hot plate, and a detection unit arranged below the transparent hot plate, for use in the method described in any one of claims 1 to 3.

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