Method for simultaneous detection of biomolecules using temperature-responsive fluorescent particles

Abstract

The purpose of the present invention is to provide a method for simultaneously detecting a plurality of target biomolecules in one execution by using temperature-responsive fluorescent particles. The aforementioned problem is overcome by providing a method for detecting or quantifying biomolecules contained in a biological sample, the method including the following steps (1) to (3): 　(1) bringing a biological sample, temperature-responsive fluorescent probe particles, and magnetic particles to which a capturing biomolecule recognition element is bonded into contact with a magnetic substrate; (2) heating the magnetic substrate; and (3) measuring fluorescence emission.

Description

Simultaneous detection of biomolecules using temperature-responsive fluorescent particles

[0001] The present invention relates to a method for simultaneously detecting biomolecules using temperature-responsive fluorescent particles.

[0002] In recent years, ELISA has become widely used for the detection and quantification of biomolecules (biomarkers). Because ELISA utilizes an antigen-antibody reaction, it has high specificity. However, the procedures for washing reagents and enzyme reactions are time-consuming and complicated, and the detection concentration of biomolecules is limited to approximately 1 pM.

[0003] Furthermore, biomolecule detection methods that are further developments of ELISA have also been developed. For example, a method called digital ELISA achieves high sensitivity by performing an enzyme reaction in a microdroplet (femtoliter) using a modified reaction device (Non-Patent Documents 1 and 2). However, while these methods use the same measurement principle as ELISA, they require more time-consuming and complicated procedures than conventional ELISA.

[0004] Single Molecule Counting (SMC™) 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 by improving the detector's optical system, the detection area is made smaller, improving detection sensitivity. However, it requires a dedicated, expensive detector, and the measurement takes time because the measurement requires repeated scanning of a small area.

[0005] Furthermore, immunochromatography has been used (Non-Patent Document 4), but it has drawbacks such as low sensitivity that makes it unusable for early detection of disease or infection, differences in visual sensitivity among examiners due to visual judgment, and a high false negative rate (20 to 30%).

[0006] The present inventors have previously reported that they have developed temperature-responsive fluorescent particles whose fluorescence intensity changes depending on the temperature, and have succeeded in developing a method for detecting low concentrations of target biomolecules (biomarkers) in a short period of time using the particles (Patent Document 1). However, this method has the drawback that it must be performed individually for each target biomolecule contained in a biological sample, and is not suitable for simultaneously detecting multiple target biomolecules in a single run.

[0007] Patent No. 7284470

[0008] Rissin DM, et al, Nat Biotechnol. 2010:28(6):595-9.Kim SH, et al, Lab Chip, 2012, 12, 4986-4991.Hwang J., et al, Methods, 2019, 158, 69-79.Hurt CA et al, J.Clin.Virol., 2007, 39, 132-135

[0009] An object of the present invention is to provide a method for simultaneously detecting multiple target biomolecules in a single run using temperature-responsive fluorescent particles.

[0010] The present inventors have discovered a compound of formula I:

[0011]

[0012] (where R1 and R2 each independently represent a hydrophobic group, which may be the same or different.) We have confirmed that when lipid vesicles (temperature-responsive fluorescent particles) containing fluorescent squaric acid derivatives represented by the formula (I) are mixed and heated, the fluorescence intensity increases stepwise depending on the heating time. Therefore, the present inventors contacted a plurality of magnetic particles each having antibodies targeting the target substance on their surface, a plurality of temperature-responsive fluorescent particles each having antibodies targeting the target substance on their surface and each having different lipid acyl chain lengths (temperature-responsive fluorescent probe particles that emit fluorescence at different temperatures), and a biological sample on the same magnetic substrate, heated, and observed the change in fluorescence intensity over time. As a result, we confirmed that the presence or absence of each target substance corresponds to a change in fluorescence intensity that depends on the heating time, and that the range of change in fluorescence intensity correlates with the concentration of the target substance, thereby completing the present invention.

[0013] That is, the present invention is as follows: [1] A method for detecting or quantifying biomolecules contained in a biological sample, comprising the following steps (1) to (3): (1) a step of contacting a magnetic particle having a capture biomolecule recognition element bound thereto, a biological sample, and temperature-responsive fluorescent probe particles with a magnetic substrate, (2) a step of heating the magnetic substrate, and (3) a step of measuring fluorescence emission, wherein the temperature-responsive fluorescent probe particles are temperature-responsive fluorescent particles having a detection biomolecule recognition element bound thereto, the temperature-responsive fluorescent particles contain at least one fluorescent molecule in a molecular assembly comprising at least one amphiphilic lipid molecule, the fluorescent molecule is quenched when the molecular assembly is in the solid phase and emits fluorescence when in the liquid phase due to a solid-liquid phase transition responsive to temperature, thereby causing a reversible conversion between quenching and fluorescence emission of the fluorescent molecule in response to temperature, the molecular assembly has aggregated fluorescent molecules in the solid phase and dissociated fluorescent molecules in the liquid phase, [2] A method for detecting or quantifying a biomolecule, wherein the fluorescent molecule is at least one compound selected from the group consisting of squaric acid derivatives, fluorescein derivatives, rhodamines, coumarins, oxazines, pyrenes, anthracenes, phthalocyanines, cyanines, azo dyes, carbopyronines, NBD derivatives, dansyl derivatives, BODIPY fluorophores, ALEXA fluorophores, rhodol dyes, naphthalimides, and porphyrins. [3] The method for detecting or quantifying a biomolecule according to [1], wherein the step (1) comprises the following steps: (1') contacting magnetic particles bound to a capture biomolecule recognition element with a magnetic substrate, (2') contacting a biological sample with the magnetic particles, and (3') contacting temperature-responsive fluorescent probe particles with the magnetic particles. [3] The method for detecting or quantifying a biomolecule according to [1], wherein the step (1) comprises the following steps: (1'') contacting a biological sample with magnetic particles bound to a capture biomolecule recognition element; (2'') contacting the complex obtained in step (1'') with a magnetic substrate; (3'') contacting the complex with temperature-responsive fluorescent probe particles; and (4'') contacting the complex obtained in step (3'') with a magnetic substrate.[4] The method for detecting or quantifying a biomolecule according to [1], wherein step (1) comprises the following steps: (1''') contacting a temperature-responsive fluorescent probe particle with a biological sample; (2''') contacting the complex obtained in step (1''') with magnetic particles to which a capture biomolecule recognition element is bound; and (3''') contacting the complex obtained in step (2''') with a magnetic substrate. [5] The method for detecting or quantifying a biomolecule according to any one of [1] to [4], wherein the method comprises washing the magnetic particles between steps (1) and (2). [6] The method for detecting or quantifying a biomolecule according to any one of [1] to [5], wherein the biomolecules to be detected or quantified are two or more types; the magnetic particles are two or more types to which different capture biomolecule recognition elements are bound; and the temperature-responsive fluorescent probe particles are two or more types to which different detection biomolecule recognition elements are bound and which have different solid-liquid phase transition temperatures. [7] The method for detecting or quantifying a biomolecule according to any one of [1] to [6], wherein the molecular assembly has a lipid bilayer, and undergoes a gel-liquid crystal phase transition in response to temperature, causing the fluorescent molecule to quench when the molecular assembly is in the gel phase and to fluoresce when the molecular assembly is in the liquid crystal phase, thereby reversibly converting between quenching and fluorescence emission of the fluorescent molecule in response to temperature. [8] The method for detecting or quantifying a biomolecule according to any one of [1] to [7], wherein the amphiphilic molecule is a phospholipid. [9] The method for detecting or quantifying a biomolecule according to [8], wherein the molecular assembly is a bilayer membrane vesicle.

[10] The method for detecting or quantifying a biomolecule according to [9], wherein the bilayer membrane vesicle contains 0.3 mol % or more of the compound represented by general formula I.

[11] The method for detecting or quantifying a biomolecule according to any one of [1] to

[10] , wherein the biomolecule recognition element is an antibody, a variable region of the antibody, or a Fab fragment of the antibody.

[12] A kit for detecting or quantifying a biomolecule contained in a biological sample, comprising the following (1) to (2): (1) a magnetic particle to which a capture biomolecule recognition element is bound, and (2) a temperature-responsive fluorescent probe particle which is a temperature-responsive fluorescent particle to which a detection biomolecule recognition element is bound, wherein the temperature-responsive fluorescent particle contains at least one fluorescent molecule in a molecular assembly comprising at least one amphipathic lipid molecule, and the fluorescent molecule is quenched when the molecular assembly is in the solid phase and emits fluorescence when the molecular assembly is in the liquid phase due to a solid-liquid phase transition in response to temperature, thereby reversibly converting between quenching and fluorescence emission of the fluorescent molecule in response to temperature, and the molecular assembly has aggregated fluorescent molecules in the solid phase and dissociated fluorescent molecules in the liquid phase,

[13] The kit for detecting or quantifying a biomolecule according to

[12] , further comprising a microchannel chip.

[0014] According to the present invention, multiple target biomolecules contained in a biological sample can be simultaneously detected or quantified in a single run, making it possible to process a large amount of target biomolecules in a shorter time than conventional methods.

[0015] Figure 1 shows the structural formula of SQR22. Figure 2 shows the UV-visible absorption spectrum of SQR23 (ethanol solution). Figure 3 shows the fluorescence emission spectrum of SQR23 (ethanol solution). Figure 4 shows a schematic diagram of temperature-responsive liposomes containing SQR dye. Figure 5 shows the fluorescence emission characteristics of temperature-responsive fluorescent liposomes with different phase transition temperatures. Figure 6 shows the temperature-responsive fluorescence emission characteristics of B-TLip. Figure 7 shows the change in fluorescence intensity over time when a B-TLip dispersion is heated on a hot plate. Figure 8 shows a schematic diagram of binding detection by biotin-streptavidin molecular recognition using temperature-responsive liposomes (TLip) and magnetic particles. Figure 9 shows the detection of binding by biotin-streptavidin molecular recognition using temperature-responsive liposomes (TLip) and magnetic particles. The negative control used magnetic particles without streptavidin modification, while the other controls were streptavidin-modified magnetic particles blocked with different amounts of biotin before binding to biotinylated TLip. Figure 10 shows a schematic diagram of antigen detection by antigen-antibody sandwich using temperature-responsive liposomes (TLip) and magnetic particles. 1 shows the effect of reaction time on antigen detection using an antigen-antibody sandwich with temperature-responsive liposomes (TLip) and magnetic particles. This is a design diagram of a microfluidic device for detection. This is a schematic diagram showing the injection and flow of solutions into a microfluidic device for detection. This is a schematic diagram showing the process of antigen detection in a microfluidic device using a detection antibody-labeled IgG-TLip and a capture antibody-labeled magnetic particle. This is a diagram showing the change in fluorescence intensity over time when an IgG-TLip bound to magnetic particles via an antigen is captured with a magnet and heated. This is a diagram showing the detection results of SARS-CoV-2 recombinant nucleocapsid protein (N antigen). NC indicates the negative control. This is a diagram showing the plot of the logarithm of the N antigen concentration and the difference in fluorescence intensity before and after heating. This is a schematic diagram showing the simultaneous detection of different antigens using temperature-responsive fluorescent liposomes with different gel-liquid crystal phase transition temperatures (emission temperatures). This is a schematic diagram showing the waveform pattern in the simultaneous detection of different antigens using temperature-responsive fluorescent liposomes with different gel-liquid crystal phase transition temperatures (emission temperatures). FIG. 1 shows the time-dependent changes in fluorescence intensity when B-TLip(DMPC), B-TLip(DPPC), and their mixture, which have different gel-liquid crystal phase transition temperatures, are heated from room temperature.Figure 1 shows the detection of B-TLip (DMPC), B-TLip (DPPC), and a mixture thereof bound to streptavidin-modified magnetic particles based on the difference in luminescence time. Figure 2 shows the detection of influenza A recombinant nucleocapsid protein (N antigen) using the influenza A detection reagent (DMPC) and confirmation of interference due to the coexistence of the SARS-CoV-2 detection reagent (DPPC). Figure 3 shows the detection of influenza A recombinant nucleocapsid protein (N antigen) using the influenza A detection reagent (DMPC) and confirmation of interference due to the coexistence of the SARS-CoV-2 N antigen. This figure shows the change in fluorescence intensity over time when magnetic particles bound to influenza A capture antibodies and magnetic particles bound to SARS-CoV-2 capture antibodies were mixed and treated with (a) influenza A antigen only, (b) SARS-CoV-2 antigen only, (c) both influenza A antigen and SARS-CoV-2 antigen, or (d) no antigen added, followed by treatment with both influenza A detection reagent (DMPC) and SARS-CoV-2 detection reagent (DPPC) and heating (three replicates per condition). This figure shows the change in fluorescence intensity over time when influenza A detection reagent (DMPC) and SARS-CoV-2 detection reagent (DPPC) bound to magnetic particles via influenza A and SARS-CoV-2 N antigens were heated. This figure shows the simultaneous detection of influenza A and SARS-CoV-2 N antigens using influenza A detection reagent (DMPC) and SARS-CoV-2 detection reagent (DPPC). This shows the change in the difference in fluorescence intensity when the concentration of influenza A N antigen is changed while the concentration of SARS-CoV-2 N antigen is fixed. This shows the simultaneous detection of influenza A and SARS-CoV-2 N antigens using an influenza A detection reagent (DMPC) and a SARS-CoV-2 detection reagent (DPPC). This shows the change in the difference in fluorescence intensity when the concentration of influenza A N antigen is fixed while the concentration of SARS-CoV-2 N antigen is changed. This shows the simultaneous detection of influenza A and SARS-CoV-2 N antigens using a microfluidic device.

[0016] The present invention provides a method for detecting or quantifying a biomolecule contained in a biological sample (hereinafter referred to as the method for detecting or quantifying a biomolecule of the present invention).

[0017] Examples of biological samples used in the biomolecule detection or quantification method of the present invention include samples derived from animals or cells. Furthermore, the sample may be a sample known to contain the target biomolecule, or a sample whose presence or absence 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 animal-derived samples include blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal secretions, semen, pleural effusion, gastrointestinal secretions, cerebrospinal fluid, interstitial fluid, nasal swabs, pharyngeal swabs, and lymph, with blood, serum, or plasma being preferred. Examples of cell-derived samples include proteins and nucleic acids (DNA, RNA) contained within cells or cell membranes obtained by disrupting cell membranes. These samples can be obtained by methods known per se. For example, serum and plasma can be prepared by collecting blood from animals and separating the liquid components according to standard methods, and cerebrospinal fluid can be collected by standard procedures such as spinal tap. Nasal and pharyngeal swabs can be collected from the mucosal walls of the nasal cavity and pharynx using a cotton swab according to standard methods. Cells can be disrupted by standard methods (e.g., freeze-thawing, sonication, surfactant treatment, etc.) to recover proteins and nucleic acids (DNA, RNA).

[0018] The method for detecting or quantifying a biomolecule of the present invention comprises the following steps: (1) contacting a magnetic substrate with magnetic particles bound to a capture biomolecule recognition element, a biological sample, and temperature-responsive fluorescent probe particles (step (1) of the present invention), (2) heating the magnetic substrate (step (2) of the present invention), and (3) measuring fluorescence emission (step (3) of the present invention).

[0019] In step (1) of the present invention, magnetic particles to which a capture biomolecule recognition element is bound, a biological sample, and temperature-responsive fluorescent probe particles are brought into contact with a magnetic substrate.

[0020] In step (1) of the present invention, the magnetic particles are not particularly limited as long as they are magnetic particles, and examples include metal particles and magnetic beads. The particle diameter of the magnetic particles is typically 1 μm to 10 μm. The surface of the magnetic particles may also be coated with a hydrophilic polymer so that reactive functional groups can be introduced. Examples of reactive functional groups include maleimide groups, thiol groups, amino groups, hydroxy groups, carboxy groups, propargyl groups, and azide groups.

[0021] A capture biomolecular recognition element is bound to the magnetic particle. In this specification, "element" refers to a molecule that exhibits a specific function, such as a biomolecular recognition function, and the terms "element" and "molecule" can be used interchangeably. In this specification, "recognition" refers to recognizing and binding to a target molecule. Furthermore, the term "biomolecular recognition element" encompasses not only elements that directly recognize biomolecules, but also elements that indirectly recognize biomolecules through binding with other molecules or molecular complexes (e.g., complexes of streptavidin and biotinylated antibodies that recognize biomolecules).

[0022] Examples of the biomolecule recognition element include an antibody, the variable region of the antibody, or a Fab fragment of the antibody (hereinafter referred to as "antibody, etc."). Herein, for the purpose of immobilizing a target biomolecule to a magnetic particle, an element for an epitope of the target biomolecule, such as an antibody, may be used. 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." On the other hand, an element for a different epitope of the target biomolecule recognized by the capture biomolecule recognition element, such as an antibody, may be used. Herein, such an element is referred to as a "detection biomolecule recognition element," and when the element is an antibody, it is also referred to as a "detection antibody." The "different epitope" may be an epitope on the target biomolecule other than the epitope to which the "capture biomolecule recognition element" actually binds. Therefore, for example, if there are multiple epitopes of the same type on a target biomolecule, the same epitopes other than the epitope to which the "capture biomolecule recognition element" actually binds are encompassed by the "different epitope." Other examples of the biomolecular recognition element 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.

[0023] The binding of magnetic particles to capture biomolecule recognition elements is achieved by a chemical reaction between reactive functional groups on the surface of the magnetic particles and reactive functional groups on the capture biomolecule recognition elements. For example, the magnetic particles and capture biomolecule recognition elements are linked by a combination of a maleimide group and a thiol group, an amino group or a hydroxyl group and a carboxyl group, a propargyl group and an azide group, etc. The binding of magnetic particles to capture biomolecule recognition elements may also be via a non-covalent bond, for example, by mixing magnetic particles with streptavidin bound to the surface and a capture biomolecule recognition element with biotin chemically bound thereto, or conversely, by mixing magnetic particles with biotin bound to the surface and a capture biomolecule recognition element with streptavidin chemically bound thereto.

[0024] In step (1) of the present invention, the temperature-responsive fluorescent probe particles are temperature-responsive fluorescent particles to which a detection biomolecule recognition element is bound.

[0025] The temperature-responsive fluorescent particles contain at least one fluorescent molecule in a molecular assembly comprising at least one amphipathic molecule. Specifically, the present invention utilizes the phenomenon in which the quenched state of the fluorescent molecules due to aggregation dissociates and becomes luminescent upon thermal transition (i.e., transition from a solid phase to a liquid phase or a gel phase to a liquid crystal phase). Therefore, the temperature-responsive fluorescent particles used in the present invention can be used without particular limitations as long as they undergo a phase transition from a solid phase to a liquid phase while maintaining the molecular assembly form. The molecular assembly preferably has a monolayer or bilayer membrane, and more preferably has a lipid bilayer membrane (also called a lipid bilayer).

[0026] The amphipathic molecules (e.g., lipid molecules) are not limited to lipids derived from living organisms, but also include amphipathic molecules that can be synthesized using raw materials such as semi-synthetic or synthetically produced small molecules such as phospholipids and their derivatives, polymers, peptides, amino acids, and carboxylic acids.

[0027] The temperature-responsive fluorescent particles undergo a solid-liquid phase transition in response to temperature, such that when the molecular assembly is in the liquid phase, the fluorescent molecules dissociate and emit fluorescence, and when the molecular assembly is in the solid phase, they aggregate and quench, thereby enabling the fluorescent molecules to reversibly switch between fluorescence emission and quenching in response to temperature.

[0028] That is, the molecular assembly exhibits a temperature-responsive solid-liquid phase transition, 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 (ACQ). 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.

[0029] Since the fluorescent molecules are arranged in a hydrophobic region constructed in a molecular aggregate state in an aqueous phase, for example, a region surrounded by a monolayer or in a bilayer membrane such as a lipid bilayer, amphiphilic or lipophilic fluorescent molecules are suitable. Fluorescent molecules usually have the property of quenching upon aggregation, so fluorescent molecules that exhibit aggregation and dissociation in response to a solid-liquid phase transition (e.g., a gel-liquid crystal phase transition of a lipid bilayer) can convert quenching and emission.

[0030] Many fluorescent molecules that exhibit fluorescence emit light from an excited state and then return to the ground state in dilute solutions, but the fluorescence efficiency drops significantly in highly concentrated solutions or solids. This drop in fluorescence efficiency is thought to be due to disordered energy transfer in the excited state caused by aggregation of fluorescent molecules, and to thermal dissipation of energy (non-radiative deactivation) due to interactions between excitons and with molecules in the ground state, a phenomenon known as aggregation-caused quenching (ACQ).

[0031] Many fluorescent molecules are known to exhibit ACQ because π-conjugated compounds, which are highly planar and have extended π-conjugated systems, have high aggregation properties due to strong intermolecular π-π stacking. References 1 to 8 describe the quenching of fluorescence by aggregation in a wide variety of fluorescent molecules, including squaric acid derivatives, fluorescein derivatives, rhodamines, coumarins, oxazines, pyrenes, anthracenes, phthalocyanines, cyanines, BODIPY fluorophores, naphthalimides, and porphyrins. While ACQ has traditionally been considered a problem for fluorescent molecules, the present invention utilizes the ACQ property of fluorescent molecules due to the transition from a quenched state in a solid phase (including a gel phase) of molecular aggregates in an aqueous phase to a liquid phase (including a liquid crystal phase). Therefore, any of the various conventional fluorescent molecules known to exhibit ACQ can be used.

[0032] To support these fluorescent molecules in molecular assemblies in the aqueous phase, highly hydrophobic fluorescent chromophores can be directly introduced into the hydrophobic portion of molecular assemblies formed by amphiphilic molecules (e.g., lipid bilayer membranes). Alternatively, various fluorescent chromophores can be incorporated into lipid molecular assemblies formed by amphiphilic molecules by chemically linking a hydrophobic group to the fluorescent chromophore, as shown in the following general formula (A) (References 9-12).

[0033] In one embodiment, an example of the fluorescent molecule is a compound represented by the following general formula (A):

[0034]

[0035] In general formula (A), the hydrophobic group is not particularly limited as long as it can be linked to the fluorescent chromophore via a covalent bond, and examples thereof include linear saturated hydrocarbons, linear unsaturated hydrocarbons, branched saturated hydrocarbons, branched unsaturated hydrocarbons, saturated fatty acids, unsaturated fatty acids, diacylglycerol, diacylphosphatidylethanolamine, N,N-dialkylaniline, aromatic hydrocarbons, amino acid derivatives, sterols, and terpenes. Examples of their structures are shown below. In the following formula, F represents a fluorescent chromophore, and n is an integer of 1 or greater.

[0036]

[0037] Some biomolecules, such as nicotinamide adenine dinucleotide phosphate, flavins, proteins, and amino acids, absorb light at wavelengths of 600 nm or less and emit fluorescence (autofluorescence) at wavelengths of 600 nm or less. Therefore, when a fluorescent probe with a maximum absorption wavelength and maximum fluorescence emission wavelength of 600 nm or less is used to detect a biomolecule, interference from the autofluorescence of contaminating molecules can become a problem in fluorescence measurement. Therefore, the compound represented by general formula (A) of the present invention is preferably a fluorescent molecule with a maximum absorption wavelength and maximum fluorescence emission wavelength of 600 nm or more. Furthermore, considering that when water is irradiated with light with a wavelength of 980 nm or more, heat generated by water absorption acts on the temperature-responsive particles, it is more preferable that the compound be a fluorescent molecule with a maximum absorption wavelength and maximum fluorescence emission wavelength in the range of 600 to 900 nm.

[0038] In one embodiment, the fluorescent molecule represented by general formula (A) is preferably a squaric acid derivative, a fluorescein derivative, a rhodamine, a coumarin, an oxazine, a pyrene, an anthracene, a phthalocyanine, a cyanine, an azo dye, a carbopyronine, an NBD derivative, a dansyl derivative, a BODIPY fluorophore, an ALEXA fluorophore, a rhodol dye, a naphthalimide, or a porphyrin.

[0039] Examples of the squaric acid derivative include compounds represented by the following general formula (I):

[0040]

[0041] (wherein R and R each independently represent a hydrophobic group, which may be the same or different.)

[0042] The compounds represented by general formula (A) or general formula (I) encompass not only free forms but also salts thereof. While the salts vary depending on the type of compound, examples include inorganic base salts such as alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), aluminum salts, and ammonium salts, as well as base addition salts such as organic base salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine, as well as inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, and organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate.

[0043] In the squaric acid derivative, R and R may be straight-chain hydrocarbons, branched-chain hydrocarbons, saturated hydrocarbons, or unsaturated hydrocarbons. Preferably, straight-chain saturated hydrocarbons are used, but there is no limitation thereto as long as the fluorescent molecules dissociate in the liquid crystal phase of the molecular aggregate and aggregate in the gel phase.

[0044] More specific examples of R1 and R2 include hydrocarbon groups having 2 to 10 carbon atoms, and more specifically, they 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 they dissociate in the liquid crystal phase of the molecular aggregate and aggregate in the gel phase.

[0045] Fluorescein, carboxyfluorescein, and calcein are known water-soluble fluorescent molecules that exhibit ACQ (Reference 1). Fluorescein derivatives incorporating hydrophobic groups include those represented by general formula (2), in which fluorescein is the fluorescent chromophore, and general formula (3), in which fluorescein isothiocyanate is the fluorescent chromophore. In general formulas (2) and (3), R can be selected from the aforementioned hydrophobic groups, preferably linear hydrocarbons or diacylphosphatidylethanolamines.

[0046]

[0047] Rhodamines include those represented by general formula (4) in which rhodamine B is the chromophore, general formula (5) in which rhodamine B isothiocyanate is the fluorescent chromophore, general formula (6) in which sulforhodamine B is the chromophore, and general formula (7) in which Texas Red is the fluorescent chromophore. Furthermore, in general formulas (4), (5), and (6), the ethyl group of rhodamine B may be replaced with a proton, resulting in rhodamine 110, or tetramethylrhodamine, in which the ethyl group is replaced with a methyl group. In general formulas (4), (5), (6), and (7), R can be selected from the hydrophobic groups listed above, preferably a linear saturated hydrocarbon or diacylphosphatidylethanolamine. Reference 4 discloses a method for measuring membrane fusion by utilizing the concentration quenching property of a fluorescent molecule in which rhodamine B has been modified with an octadecyl group, a linear saturated hydrocarbon, in lipid bilayer membranes. It has been shown that rhodamines with hydrophobic groups aggregate and quench in lipid bilayer membranes.

[0048]

[0049] Examples of coumarins include those represented by general formula (8) in which coumarin serves as a fluorescent chromophore, and in general formula (8), R can be selected from the above-mentioned hydrophobic groups.

[0050]

[0051] Examples of oxazines include Nile Red and general formula (9) in which resorufin is used as a fluorescent chromophore, and in general formula (9), R can be selected from the above-mentioned hydrophobic groups.

[0052]

[0053] Pyrene and anthracene derivatives are highly hydrophobic and can be directly introduced into the hydrophobic portion of a molecular assembly. Alternatively, they can be chemically introduced into a hydrophobic group as shown in general formulas (10) and (11), where R can be selected from the above hydrophobic groups.

[0054]

[0055] Phthalocyanines are compounds based on the phthalocyanine skeleton, and metal complexes with various metals (M) can be used as fluorescent molecules. Examples of central metals include iron, cobalt, silicon, zinc, copper, and vanadium. Various phthalocyanine derivatives in which the protons of the benzyl groups of phthalocyanine are substituted, as well as naphthalocyanine and naphthalocyanine metal complexes, can also be used as fluorescent molecules.

[0056]

[0057] Examples of cyanine fluorescent molecules include commercially available lipophilic carbocyanine dyes such as DiIC12(3) (trade name), DiD (trade name), DiO (trade name), DiI (trade name), and DiR (trade name).

[0058]

[0059] Examples of azo dyes include Sudan III and Oil Red O, which are used to stain neutral fats.

[0060]

[0061] Carbopyronines are fluorescent molecules whose basic structure is represented by the general formula (12), where R can be selected from the hydrophobic groups listed above. Atto 633 (trade name) is an example of a carbopyronine.

[0062]

[0063] The NBD derivatives include those represented by general formula (13) in which NBD is the chromophore, and in general formula (13), R can be selected from the above-mentioned hydrophobic groups.

[0064]

[0065] The dansyl derivative is represented by the general formula (14), and in the general formula (14), R can be selected from the above-mentioned hydrophobic groups.

[0066]

[0067] As BODIPY fluorophores, BODIPY 493 / 503 (trade name) and BODIPY 505 / 515 (trade name) can be introduced into the hydrophobic portion of the molecular assembly. Alternatively, they can be chemically introduced into the hydrophobic group as shown in general formula (15), where R can be selected from the above hydrophobic groups.

[0068]

[0069] Commercially available ALEXA fluorophores include Alexa 350, Alexa 405, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 561, Alexa 568, Alexa 594, Alexa 647, Alexa 660, Alexa 680, Alexa 700, and Alexa 750 (all trade names). These ALEXA fluorophores can be used as amphiphilic fluorescent molecules by conjugating them to hydrophobic groups such as amino groups, thiol groups, aldehyde ketones, carboxylates or phosphates, azide groups, and alkyne groups.

[0070] Examples of rhodol dyes include those represented by the general formulae (16) and (17), and in the general formulae (16) and (17), R can be selected from the hydrophobic groups described above.

[0071]

[0072] Examples of naphthalimides include those represented by the general formula (18), in which R can be selected from the above-mentioned hydrophobic groups.

[0073]

[0074] Porphyrins are compounds with a porphyrin skeleton, and metal complexes with various metals (M) can be used as fluorescent molecules. Examples of central metals include iron, cobalt, silicon, zinc, copper, and vanadium. They can also be used as amphiphilic molecules in which porphyrin is chemically introduced into the hydrophobic moiety.

[0075]

[0076] In contrast to fluorescent molecules that exhibit ACQ, Reference 13 describes aggregation-induced emission (AIE) fluorescent molecules that exhibit strong luminescence properties upon aggregation. When using molecular assemblies incorporating such AIE fluorescent molecules, it is possible to construct a detection system in which AIE fluorescent molecules aggregate in a solid or gel phase and then luminesce, dissociating and quenching upon transition to a liquid or liquid crystal phase due to an increase in temperature. However, it is known that detection systems that rely on a change from an luminescent state to a quenched state (turn off) have lower detection sensitivity than systems that rely on a change from a quenched state to an luminescent state (turn on). Additionally, because fluorescent molecules generally exhibit a tendency for their fluorescence intensity to decrease with increasing temperature, detection systems using AIE fluorescent molecules as fluorescent molecules exhibit similar fluorescence temperature characteristics, making them indistinguishable from each other. For these reasons, AIE fluorescent molecules are not suitable for use in the present invention. Furthermore, fluorescent molecules whose fluorescence emission properties do not change upon aggregation and dissociation cannot be used to construct a detection system for the present technology that simultaneously measures multiple target biomolecules using the changes in the fluorescence emission temperature and light emission intensity of temperature-responsive fluorescent particles as indicators, and therefore are not encompassed by the present invention.

[0077] An example of the amphipathic molecule is a phospholipid.

[0078] While the type of phospholipid is not particularly limited, glycerophospholipids (also known as diacyl phospholipids) are preferred, and diacyl phospholipids having a phosphocholine group in the hydrophilic portion are particularly preferred for forming stable lipid bilayers that exhibit phase transition. The acyl chain lengths of the diacyl phospholipids may be the same or different. Diacyl phospholipids having a phosphocholine group include saturated phospholipids and unsaturated phospholipids, and either can be used in the present invention, or a combination of these can be used. Saturated phosphocholines can include synthetic and semi-synthetic phospholipids, such as natural phospholipids and derivatives thereof, such as hydrogenated egg yolk lecithin and hydrogenated soybean lecithin, with a hydrogenation rate close to 100%, as well as dimyristoylphosphocholine, dipentadecanoylphosphocholine, dipalmitoylphosphocholine, diheptadecanoylphosphocholine, and distearoylphosphocholine.

[0079] 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 spherical, closed membrane structure. 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, i.e., the molecular assembly, are not limited by their multilayer or single layer structure, particle size, or other characteristics, 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.

[0080] To prevent aggregation and fusion of molecular aggregates such as lipid vesicles, amphiphilic molecules with charged residues or polymer chains in the hydrophilic part can be included as constituents. The resulting molecular aggregates, such as lipid vesicles, have high dispersion stability due to electrostatic repulsion caused by the charge and steric exclusion effect of the polymer chains.

[0081] 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 lipids such as L-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA), and amino acid lipids having an amino acid in the hydrophilic group.

[0082] Furthermore, an example of an amphiphilic molecule with 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 preferred to prevent aggregation of lipid vesicles.

[0083] When the amphiphilic molecule is a lipid molecule, it is preferably 1,2-dimyristoyl-sn-glycero-3-phosphocholine (PC 14 ), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (PC 15 ) (manufactured by Avanti Polar Lipids Inc.), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC 17 ) (manufactured by Avanti Polar Lipids Inc.), 1,2-distearoyl-sn-glycero-3-phosphocholine (PC 18Other lipid bilayers that can be used include 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,5-dihexadecyl-N-succiny-L-glutamate (DHSG), and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). The gel-liquid crystal phase transition temperature of the bilayer membrane formed by lipid molecules can be changed by the number of carbon atoms in the acyl chains of the constituent lipid molecules. When detecting two or more types of biomolecules simultaneously, lipid vesicles composed of lipid molecules with different numbers of carbon atoms in the acyl chains, such as two types of DMPC (phase transition temperature 23°C) and DPPC (phase transition temperature 41°C), two types of PC15 (phase transition temperature 35°C) and PC17 (phase transition temperature 50°C), or three types of DMPC (phase transition temperature 23°C), DPPC (phase transition temperature 41°C), and DSPC (phase transition temperature 55°C), can be combined and used as temperature-responsive fluorescent particles.

[0084] The aggregation-induced quenching of fluorescent molecules in molecular assemblies such as lipid vesicles depends on their concentration in the molecular assemblies, and the aggregation-induced quenching decreases as the concentration of the fluorescent molecules decreases. Therefore, the temperature-responsive fluorescent particles of the present invention contain the compound represented by 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.

[0085] Such temperature-responsive fluorescent particles can be produced by adding the compound of formula (I) to the molecular assembly during production. For example, the molecular assembly can be produced according to a known method for producing lipid vesicles (Sheng Dong et al., ACS Appl. Nano Mater. 2018, 1, 1009-1013).

[0086] The temperature-responsive fluorescent particles of the present invention obtained as described above can be obtained as temperature-responsive fluorescent probe particles by binding a detection biomolecule recognition element.

[0087] The temperature-responsive fluorescent particles are bonded to the biomolecule recognition element for detection by chemical reaction between reactive functional groups on the surface of the temperature-responsive fluorescent particles and reactive functional groups on the biomolecule recognition element for detection, for example, by combinations such as maleimide and thiol groups, amino or hydroxyl groups and carboxyl groups, and propargyl and azide groups. Alternatively, temperature-responsive fluorescent particles bound to biomolecular recognition elements can be obtained by producing lipid vesicles modified with detection antibodies according to known methods (International Publication WO2000 / 064413, JP2003-73258A, JP58-134032A, Manjappa SA, et al., J. Controlled Release 2011, 150, 2-22, Li T., et al., Nanomed.: Nanotechnol. Biol. Medicine, 2017, 13, 1219-1227).

[0088] The magnetic particles bind to the target biomolecule to be detected or measured via the capture biomolecule recognition element, and the temperature-responsive fluorescent particles described below bind to the target biomolecule to be detected or measured via the detection biomolecule recognition element, thereby forming a complex consisting of the magnetic particles, the target biomolecule, and the temperature-responsive fluorescent particles described below. In step (1) of the present invention, the magnetic particles to which the capture biomolecule recognition element is bound, the biological sample, and the temperature-responsive fluorescent probe particles are brought into contact with a magnetic substrate, whereby the temperature-responsive fluorescent particles can be fixed to the magnetic substrate via the formed complex.

[0089] For example, when the biomolecule recognition element is an antibody, (a) a temperature-responsive probe particle composed of a 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 bound to a magnetic particle form a complex of the temperature-responsive fluorescent probe particle (temperature-responsive fluorescent particle bound to the detection antibody)-target biomolecule (antigen)-capture antibody-magnetic particle, and the temperature-responsive fluorescent particle becomes fixed to the magnetic substrate.

[0090] The magnetic substrate used in step (1) of the present invention is not particularly limited as long as it is a magnetic substrate, and examples thereof include metal plates (microplates, dishes, etc.). Furthermore, when magnetism is generated at a specific location using a magnet or the like, the substrate does not necessarily have to be metal, and examples of insoluble substrates include polymers such as polystyrene, glass beads, microplates, immunochromatography filter paper, and glass filters. From the perspective of ease of handling, a microplate is preferred. The microplate may include inlets for introducing magnetic particles bound to a capture biomolecular recognition element, a biological sample, temperature-responsive fluorescent probe particles, or a buffer solution, a flow path for forming a complex between the magnetic particles, the biological sample, and the temperature-responsive fluorescent probe particles, a site for immobilizing the magnetic particles using a magnet, and a detection port for detecting fluorescence.

[0091] The manner in which the magnetic particles bound to the capture biomolecule recognition element, the biological sample, and the temperature-responsive fluorescent probe particles are brought into contact with the magnetic substrate in step (1) of the present invention is not particularly limited, as long as the temperature-responsive fluorescent particles can be fixed to the magnetic substrate via the formed complex. In one embodiment, step (1) of the present invention may include, for example, the following steps: (1') a step of bringing the magnetic particles bound to the capture biomolecule recognition element into contact with the magnetic substrate (step (1') of the present invention); (2') a step of bringing the biological sample into contact with the magnetic substrate (step (2') of the present invention); and (3') a step of bringing the temperature-responsive fluorescent probe particles into contact with the magnetic substrate (step (3') of the present invention).

[0092] In step (1') of the present invention, the magnetic particles bound to the capture biomolecule recognition element are brought into contact with a magnetic substrate. By bringing the magnetic particles into contact with the magnetic substrate, the magnetic particles are immobilized on the magnetic substrate. Between steps (1') and (2') of the present invention, a step of washing the magnetic substrate to remove unimmobilized magnetic particles may be further included.

[0093] In step (2') of the present invention, the biological sample is brought into contact with the magnetic substrate to which the magnetic particles have been immobilized in step (1') of the present invention. By bringing the biological sample into contact with the magnetic substrate, the target biomolecules in the biological sample bind to the capture biomolecule recognition elements bound to the magnetic particles, resulting in the target biomolecules being immobilized on the magnetic substrate. Between steps (2') and (3') of the present invention, a step of washing the magnetic substrate to remove unimmobilized target biomolecules may be further included.

[0094] In step (3') of the present invention, temperature-responsive fluorescent probe particles are brought into contact with the magnetic substrate to which target biomolecules have been immobilized in step (2') of the present invention. By bringing the temperature-responsive fluorescent probe particles into contact with the magnetic substrate, the target biomolecules bind to the detection biomolecule recognition elements bound to the temperature-responsive fluorescent particles, resulting in the temperature-responsive fluorescent particles being immobilized on the magnetic substrate. A step of washing the magnetic substrate to remove any unimmobilized temperature-responsive fluorescent particles may be further included between step (3') and step (2) of the present invention.

[0095] In another embodiment, step (1) of the present invention may include the following steps: (1'') contacting a biological sample with magnetic particles having a capture biomolecular recognition element bound thereto, (2'') contacting the complex obtained in step (1'') with a magnetic substrate, (3'') contacting the complex with temperature-responsive fluorescent probe particles, and (4'') contacting the complex obtained in step (3'') with a magnetic substrate.

[0096] In step (1") of the present invention, a biological sample is brought into contact with magnetic particles to which a capture biomolecule recognition element is bound. By bringing the biological sample into contact with the magnetic particles, the target biomolecule in the biological sample binds to the capture biomolecule recognition element bound to the magnetic particles, resulting in the formation of a complex consisting of the target biomolecule and the magnetic particle to which the capture biomolecule recognition element is bound.

[0097] In step (2") of the present invention, the complex obtained in step (1") is contacted with a magnetic substrate. By contacting the complex obtained in step (1") with the magnetic substrate, the magnetic particles are immobilized on the magnetic substrate, and as a result, the target biomolecule is immobilized on the magnetic substrate. Between steps (2") and (3") of the present invention, a step of washing the magnetic substrate may be further included to remove the unimmobilized complex, magnetic particles, and target biomolecule.

[0098] In step (3") of the present invention, temperature-responsive fluorescent probe particles are brought into contact with the complex of target biomolecules and magnetic particles formed in step (2") of the present invention. At this time, the complex may remain fixed to the magnetic substrate. However, removing the magnetic field and mixing and contacting the temperature-responsive fluorescent probe particles with the complex increases the contact area, promoting binding between the detection biomolecule recognition element bound to the temperature-responsive fluorescent particles and the target biomolecule, resulting in the formation of a complex of temperature-responsive fluorescent particles and magnetic particles via the target biomolecule. In step (4") of the present invention, the complex obtained in step (3") is brought into contact with a magnetic substrate, thereby fixing the temperature-responsive fluorescent particles to the magnetic substrate. Between step (4") and step (2") of the present invention, a step of washing the magnetic substrate may be further included to remove any unfixed temperature-responsive fluorescent particles.

[0099] In yet another embodiment, step (1) of the present invention may include the following steps: (1''') contacting a temperature-responsive fluorescent probe particle with a biological sample, (2''') contacting the complex obtained in step (1''') with magnetic particles having a capture biomolecular recognition element bound thereto, and (3''') contacting the complex obtained in step (2''') with a magnetic substrate.

[0100] In step (1''') of the present invention, the temperature-responsive fluorescent probe particles are brought into contact with a biological sample. By bringing the biological sample into contact with the temperature-responsive fluorescent probe particles, target biomolecules in the biological sample are bound to the temperature-responsive fluorescent probe particles, resulting in the formation of complexes consisting of the temperature-responsive fluorescent probe particles and the target biomolecules.

[0101] In step (2''') of the present invention, the complex obtained in step (1''') is contacted with magnetic particles to which a capture biomolecule recognition element is bound. By contacting the complex obtained in step (1''') with the magnetic particles to which a capture biomolecule recognition element is bound, the target biomolecule in the biological sample binds to the capture biomolecule recognition element bound to the magnetic particles, resulting in the formation of a complex consisting of the magnetic particles to which the capture biomolecule recognition element is bound, the target biomolecule, and the temperature-responsive fluorescent probe particles.

[0102] In step (3''') of the present invention, the composite obtained in step (2''') is brought into contact with a magnetic substrate. By bringing the composite obtained in step (2''') into contact with the magnetic substrate, the magnetic particles in the composite are fixed to the magnetic substrate, and as a result, the composite is fixed to the magnetic substrate.

[0103] The concentration of the magnetic particles used in step (1), step (1'), step (1") or step (2'") of the present invention is not particularly limited, but it is preferable that the magnetic particles be contained in the solution at a concentration of 5 μg / ml or more, more preferably 10 μg / ml or more, even more preferably 100 μg / ml or more, and particularly preferably 500 μg / ml or more at the time of contact. The upper limit is not particularly limited, but is preferably 10 mg / ml or less, more preferably 5 mg / ml or less, and even more preferably 1 mg / ml or less.

[0104] The concentration of the temperature-responsive fluorescent probe particles used in step (1), step (3'), step (3"), or step (1"') of the present invention is not particularly limited, but it is preferable that at the time of contact, the fluorescent molecules contained in the probe particles are contained in the solution at a concentration of 0.55 μg / ml or more, more preferably 1.0 μg / ml or more, and even more preferably 5.0 μg / ml or more. The upper limit is not particularly limited, but is preferably 50 μg / ml or less, more preferably 40 μg / ml or less, and even more preferably 30 μg / ml or less.

[0105] The magnetic particles, biological samples, temperature-responsive fluorescent probe particles, or complexes formed therefrom, bound to the capture biomolecular recognition element, may be dispersed in an aqueous solvent when contacted with the magnetic substrate. The aqueous solvent is not particularly limited, and may be, for example, a buffer solution, such as acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate-buffered saline (PBS), McIlvaine buffer, or a solution containing any of these and a nonionic surfactant (e.g., Tween 20, etc.) (e.g., PBS-T, etc.).

[0106] The conditions for each of the above contacting steps are not particularly limited, but the contacting is usually carried out at a temperature of 0 to 45°C, preferably 0 to 40°C, more preferably 4 to 37°C, and even more preferably 25 to 37°C. The contacting 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 contacting 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.

[0107] The method of the present invention may further include a step of washing the magnetic substrate between steps (1) and (2), (1') and (2'), (2') and (3'), (3') and (2), (2") and (3"), (4") and (2), and (3'") and (2). Washing can remove magnetic particles, biological samples, temperature-responsive fluorescent probe particles, or complexes formed therefrom that are not fixed to the magnetic substrate. The solvent used for washing is not particularly limited, but an aqueous solvent can be used, for example. The aqueous solvent is not particularly limited, but a buffer solution can be used, for example. Examples of the buffer solution include acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate-buffered saline (PBS), McIlvaine buffer, and solutions containing these buffer solutions and a nonionic surfactant (e.g., Tween 20, etc.) (e.g., PBS-T, etc.).

[0108] As described in the examples below, magnetic particles with hydrophilic surfaces on which streptavidin has been immobilized and multiple temperature-responsive fluorescent particles (temperature-responsive fluorescent probe particles that emit fluorescence at different temperatures) bearing avidin on their surfaces and with different lipid acyl chain lengths were contacted with a magnetic substrate, the substrate was heated, and the change in fluorescence intensity over time was observed. As a result, it was confirmed that the fluorescence intensity changes over time depending on the temperature at which the thermal transition of the temperature-responsive fluorescent particles (i.e., the transition from solid to liquid phase or the transition from gel to liquid crystal phase) occurs. Based on the above, the inventors have discovered that in the biomolecule detection or quantification method of the present invention, two or more types of magnetic particles bound to different capture biomolecule recognition elements, or two or more types of temperature-responsive fluorescent particles bound to different detection biomolecule recognition elements and with different lipid acyl chain lengths, can be used to simultaneously detect or quantitate two or more types of biomolecules. Therefore, step (1) of the present invention may include the following features: (i) Two or more types of biomolecules are detected or quantified. (ii) The magnetic particles are two or more types of magnetic particles to which different capture biomolecule recognition elements are bound. (iii) The temperature-responsive fluorescent probe particles are two or more types of temperature-responsive fluorescent particles to which different detection biomolecule recognition elements are bound and which have different lipid acyl chain lengths.

[0109] In step (2) of the present invention, the magnetic substrate to which the temperature-responsive fluorescent particles have been fixed in step (1), step (3'), step (4'') or step (3''') is heated.

[0110] When the magnetic substrate is heated, the temperature of the complex rapidly rises due to heat conduction from the magnetic substrate. At this time, the temperature-responsive fluorescent particles in the complex undergo a phase transition from a gel phase to a liquid crystal phase, causing the fluorescent molecules in the temperature-responsive fluorescent particles, which had aggregated and quenched in the gel phase, to dissociate and emit fluorescence. On the other hand, when the target biomolecule, i.e., the antigen, is not present in the biological sample, the complex is not formed. Therefore, the temperature-responsive fluorescent particles cannot adhere to the magnetic substrate and remain suspended in the solution or are removed by washing, so they maintain their quenched state even when the magnetic substrate is heated.

[0111] In step (2) of the present invention, the method of heating the magnetic substrate includes, for example, placing the substrate on a thermoplate or hot plate set to the desired temperature, or in a water bath, but is not limited to these methods. When using such an apparatus, the set temperature and heating time of the apparatus may be appropriately determined by a person skilled in the art depending on the temperature at which the temperature-responsive fluorescent particles undergo a phase transition from a gel phase to a liquid crystal phase. Furthermore, when the magnetic substrate is not made of metal and the temperature-responsive fluorescent particles are fixed to the substrate by a magnet, it is sufficient to heat only the fixed portion.

[0112] In step (3) of the present invention, the fluorescent molecules in the temperature-responsive fluorescent particles heated in step (2) are dissociated, and the emitted fluorescence is measured.

[0113] In step (3) of the present invention, the method for measuring fluorescence emission is not particularly limited, and measurement can be performed using a general optical fiber fluorescence detector, a microplate reader, a fluorescence microscope, a detector equipped with a camera (e.g., a CCD camera, etc.) (e.g., Celvin, etc.), etc. Furthermore, as in the ELISA method, a calibration curve can be prepared using a sample (standard) 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.

[0114] An example of a reactor for continuously carrying out steps (1) to (3) of the present invention is a microchannel chip. The microchannel chip used preferably has inlets for the sample, detection reagent, and washing solution, and a structure including a magnetic particle washing tank, a stirring / mixing tank, and a detection tank arranged in this order along the direction of liquid flow in the channel. Fluorescence is measured while the magnetic particles collected in the detection tank are heated using a magnetic substrate.

[0115] The present invention also provides a kit for detecting or quantifying a biomolecule contained in a biological sample (hereinafter referred to as the kit for detecting or quantifying a biomolecule of the present invention).

[0116] The kit for detecting or quantifying biomolecules of the present invention includes (1) magnetic particles bound to a capture biomolecule recognition element, and (2) temperature-responsive fluorescent probe particles, which are temperature-responsive fluorescent particles bound to a detection biomolecule recognition element, and may further include a microfluidic chip as a detection device. The (1) magnetic particles bound to a capture biomolecule recognition element and the (2) temperature-responsive fluorescent probe particles, which are temperature-responsive fluorescent particles bound to a detection biomolecule recognition element, may be the same as those used in the biomolecule detection or quantification method of the present invention. The kit of the present invention may also include other reagents in addition to the above (1) and (2). These reagents may be premixed with the above (1) and (2) or stored in separate containers. Examples of the reagents include an aqueous solvent, a standard with a known concentration of the biomolecule, a positive control, a negative control, and instructions describing the protocol. These reagents may also be premixed as needed.

[0117] All documents mentioned herein are incorporated by reference in their entirety. The examples described herein illustrate embodiments of the present invention and should not be construed as limiting the scope of the invention.

[0118] (Example 1) Synthesis of Fluorescent Dye (SQR22) Squaraine fluorescent dye (SQR22) was synthesized according to a previously published method (Sou K. et al., Scientific Reports, 2019, 9, 17991). The structure of SQR22 was identified by NMR and mass spectrometry. The structural formula of SQR22 is shown in Figure 1. 1H NMR (CDCl3, 400 MHz, δ ppm): 0.89 (t, J = 7.3 Hz, 6H, -CH3); 1.25-1.35 (m, 4H, -CH2-); 1.41 (s, 6H, -CH3); 1.50-1.57 (m, 4H, -CH2-); 3.27-3.31 (m, 4H, -CH2-); 5.75 (s, 1H, =CH-); 6.60 (d, J = 9.2 Hz, 2H, ArH), 7.04-7.36 (m, 4H, ArH); 8.03 (d, J = 9.1 Hz, 2H, ArH). 13 C NMR (CDCl3, 400 MHz, δ ppm): 14.12, 20.47, 25.64, 29.61, 50.12, 51.07, 89.87, 111.95, 113.27, 118.85, 122.74, 125.18, 128.82, 130.95, 140.06, 141.59, 151.19, 169.22, 181.33, 181.80, 185.51, 187.28. + ) 443.

[0119] Example 2: Synthesis of Fluorescent Dye (SQR23) The fluorescent dye (SQR23) was synthesized by modifying a previously published method (Dong, S. ACS Appl. Nano Mater. 2018, 1, 1009-1013). The synthetic scheme for SQR23 is shown in Scheme 1. First, the precursors N,N-diheptylaniline 1 and squaryl chloride 2 were synthesized according to a previously published method (Li F., et al. Chem. Eur. J. 20, 9991-9997 (2014); Easwaran A., et al. J. Am. Chem. Soc. 2004, 126, 6590-6598). 1 and 2 were reacted under reflux in toluene for 6 hours to obtain a chloride intermediate. This chloride intermediate was then hydrolyzed in a 2N hydrochloric acid / acetic acid / water (1 / 1) mixed solvent to obtain compound 3. Compound 3 and 2,3,3-trimethylindolenine 4 were reacted in a 4:1 mixture of n-butanol and toluene under reflux, and the reaction product was separated on a silica gel column (mobile phase: hexane / ethyl acetate (2:1)) to give SQR23.1 The structure of SQR23 was identified by 1 H NMR and mass spectrometry. 1 H NMR (CDCl3, 400 MHz, δ ppm): 0.90 (t, 6H, -CH3); 1.25-1.35 (m, 16H, -CH2-); 1.50 (s, 6H, -CH3); 1.60-1.70 (m, 4H, -CH2-); 3.27-3.31 (m, 4H, -CH2-); 5.85 (s,1H, =CH-); 6.70 (d, 2H, ArH), 7.18-7.40 (m, 4H, ArH); 8.10 (d, 2H, ArH). MS m / z[MH] - 525.

[0120]

[0121] (Example 3) Spectroscopic Properties of SQR23 SQR23 was dissolved in ethanol, and the UV-visible absorption spectrum (Fig. 2A) and fluorescence emission spectrum (Fig. 2B) were measured using a UV-visible spectrophotometer (V-670, JASCO Corporation) and a fluorescence spectrophotometer (RF-5300PC, Shimadzu Corporation). The maximum absorption wavelength of SQR23 in ethanol was 633 nm, and the maximum fluorescence emission wavelength was 658 nm.

[0122] Example 4: Introduction of SQR dye into liposomes with different phase transition temperatures. Three amphiphilic molecules: 1,2-diacyl-sn-glycero-3-phosphocholine (PC), L-glutamicacid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA) (Nippon Fine Chemicals Co., Ltd.), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-monomethoxy poly(ethylene glycol) (5000) (PEG-DSPE) (NOF Corporation) were dissolved in t-butanol in a molar ratio of PC / SA / PEG-DSPE = 9 / 1 / 0.06 and lyophilized to obtain a mixed lipid powder. Five types of PCs with different acyl chain lengths were used: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (PC14 or DMPC) (Avanti Polar Lipids Inc.), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (PC15) (Avanti Polar Lipids Inc.), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC16 or DPPC) (Nippon Fine Chemicals), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC17) (Avanti Polar Lipids Inc.), and 1,2-distearoyl-sn-glycero-3-phosphocholine (PC18) (Avanti Polar Lipids Inc.) (Figure 3A). The resulting mixed lipid powder was mixed with a stock solution of SQR22 (ethanol) (mixed lipid / SQR22 = 50 / 1 [weight ratio]), dissolved in t-butanol, and lyophilized to obtain a mixed lipid powder containing SQR22. The mixed lipid powder containing SQR22 was dispersed in phosphate-buffered saline (PBS) (5 mg / mL) and hydrated using a vortex mixer.This dispersion was added to an EXTRUDER (Northern Lipids Inc.) and passed through polycarbonate membrane filters (Merck Millipore) with pore sizes of 0.45 μm, 0.20 μm, and 0.05 μm sequentially at 60°C under pressure to obtain a SQR22-containing liposome dispersion. As shown in Figure 3A, the amphiphilic SQR dye was incorporated into the lipid bilayer membrane that formed the liposome.

[0123] Example 5: Fluorescence Properties of SQR-Incorporated Liposomes. Figure 3B shows the correlation curve between the fluorescence intensity and temperature of SQR22-incorporated liposomes. The fluorescence intensity of SQR22 incorporated into lipid bilayer membranes changes over different temperature ranges depending on the lipid species constituting the bilayer membrane. The gel-liquid crystal phase transition temperatures of lipid bilayer membranes composed of PC14, PC15, PC16, PC17, and PC18 are 24, 35, 40, 49, and 55°C, respectively. The temperature range in which the fluorescence intensity of SQR22 changes rapidly corresponds roughly to the phase transition temperature of the lipid bilayer membrane. This is because SQR22 aggregates and is in a quenched state when the lipid bilayer membrane is in the gel phase. However, when the lipid membrane is heated above the phase transition temperature and transitions to the liquid crystal phase, SQR22 dissociates and transitions to an emissive state. By using SQR-incorporated temperature-responsive liposomes (TLips), a series of temperature-responsive fluorescent particles with different fluorescence emission temperatures can be prepared depending on the gel-liquid crystal phase transition temperature of the lipid bilayer membrane.

[0124] Example 6 Preparation of Biotin-Modified Thermoresponsive Liposomes Three types of lipid molecules: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Nippon Fine Chemicals Co., Ltd.), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) (NOF Corporation), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (ammonium salt) (B-PEG-DSPE) (Avanti Polar Lipids) were dissolved in cyclohexane with SQR23 in a molar ratio of DPPC / DPPA / B-PEG-DSPE / SQR23 = 86.6 / 9.6 / 0.5 / 3.3, and the solution was lyophilized to obtain a SQR23-containing mixed lipid powder. The resulting SQR23-containing mixed lipid powder was dispersed (4 mg / mL) in 0.02% sodium azide phosphate-buffered saline (PBS), and the dispersion was heated to 50°C and stirred for 40 minutes to hydrate and disperse the mixed lipid powder. This dispersion was added to an EXTRUDER (Northern Lipids Inc.) and passed through a 0.20 μm pore size polycarbonate membrane filter (Whatman) twice under pressure at 58°C to obtain a dispersion of temperature-responsive liposomes (B-TLip) carrying biotin on their surfaces.

[0125] Example 7 Temperature-Responsive Fluorescence Properties of B-TLip The fluorescence emission spectrum (excitation wavelength = 570 nm) of a B-TLip dispersion ([SQR23] = 1 μg / mL) was measured using a fluorescence spectrophotometer (RF-5300PC, Shimadzu Corporation). To confirm the temperature-responsive fluorescence emission properties, the dispersion temperature was measured at 25°C, and then the temperature was raised to 50°C. As shown in Figure 4, SQR23 was quenched at 25°C, but strong fluorescence emission was observed when heated to 50°C, above the phase transition temperature of the DPPC bilayer membrane (41°C). When the DPPC bilayer membrane was in the gel phase, SQR23 aggregated and was in a quenched state. However, when heated above the phase transition temperature and the lipid membrane transitioned to the liquid crystalline phase, SQR23 aggregated and dissociated, transitioning to a luminescent state. SQR23-containing liposomes exhibited temperature-responsive fluorescence properties similar to those of the SQR22-containing liposomes shown in Example 5. Next, a B-TLip dispersion ([SQR23] = 1 μg / mL, 60 μL) was poured into a flat-bottom 1 × 8 strip well (Thermo Fisher Scientific), and the strip was placed on a glass plate at 80°C for heating. The time course of the fluorescence intensity when this was measured is shown in Figure 5. As the temperature of the dispersion increases due to heating from the bottom of the strip, the fluorescence intensity rises sharply and then reaches a steady value.

[0126] Example 8: Detection of Binding via Biotin-Streptavidin Molecular Recognition between B-TLip and Streptavidin-Modified Magnetic Particles. 60 μL of Magnosphere™ MS300 / Streptavidin (Medical and Biological Laboratories), magnetic particles with hydrophilic surfaces to which streptavidin was immobilized, was added to a flat-bottom 1 × 8 strip well at a final streptavidin concentration of 0.50 μg / mL. 60 μL of B-TLip was added to a flat-bottom 1 × 8 strip well at a final SQR23 concentration of 1 μg / mL. The well was then incubated at room temperature for 10 minutes. After the reaction, the magnetic particles were pulled to the bottom of the well with a magnet, and the well was washed twice with 200 μL of DPBS to remove any B-TLip that was not bound to the magnetic particles. The flat-bottom 1 × 8 strip well was then heated on a hot plate preheated to 80°C. The fluorescence intensity was then measured over time using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.). A schematic diagram of the detection system is shown in Figure 6A. Magnosphere™ MS300 / Carboxyl magnetic particles (no streptavidin modification) and a sample containing 50 μg / mL streptavidin were compared (n=3) with magnetic particles in which streptavidin had been blocked by adding an excess amount of biotin (100% blocking) as a negative control. As shown in Figure 6B, the fluorescence intensity values ​​for the negative control (no streptavidin modification) and the sample in which streptavidin had been blocked 100% with an excess amount of biotin were similar, indicating that streptavidin on the magnetic particles had been blocked and that unbound B-TLip had been removed by washing. Reducing the streptavidin blocking rate to 50% increased the difference in fluorescence intensity, and the difference in fluorescence intensity increased even more without blocking. This indicates that the binding of TLip to magnetic particles through molecular recognition between biotin and streptavidin can be detected by the increase in fluorescence intensity upon heating, and that the magnitude of the increase in TLip fluorescence intensity reflects the amount of TLip bound to the magnetic particles.

[0127] (Example 9) Preparation of liposomes for detecting SARS-CoV-2 Three types of lipid molecules: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (manufactured by Nippon Fine Chemicals Co., Ltd.), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) (manufactured by Nippon Oil & Fats Co., Ltd.), and N-[(3-Maleimide-1-oxopropyl)aminopropyl polyethyleneglycol-carbamyl] distearoylphosphatidyl-ethanolamine (MAL-PEG-DSPE, SUNBRIGHT DSPE-020MA) (manufactured by NOF Corporation), and SQR23 were dissolved in cyclohexane in a molar ratio of DPPC / DPPA / MAL-PEG-DSPE / SQR23 = 86.6 / 9.6 / 0.5 / 3.3, and then lyophilized to obtain a mixed lipid powder containing SQR23. The resulting SQR23-containing mixed lipid powder was dispersed (4 mg / mL) in 0.02% sodium azide phosphate-buffered saline (PBS) and heated to 50°C while stirring for 40 minutes to hydrate the mixed lipid powder. This dispersion was added to an EXTRUDER (Northern Lipids Inc.) and passed twice through a 0.20 μm pore size polycarbonate membrane filter (Whatman) at 58°C under pressure to obtain a dispersion of SQR23-containing temperature-responsive liposomes (Mal-TLip) bearing maleimide groups on their surface. Next, sulfhydryl groups were introduced into the IgG using 2-iminothiolane hydrochloride (Traut's reagent) to conjugate a detection monoclonal antibody (IgG-9) that specifically recognizes the SARS-CoV-2 nucleocapsid protein. 2-Iminothiolane hydrochloride was dissolved in Trout's solution (25 mM HEPES, 140 mM NaCl, 3 mM EDTA) and added to the IgG solution (IgG / 2-iminothiolane hydrochloride = 1 / 20 (molar ratio)), and the mixture was shaken at room temperature for 1 hour to react.To remove unreacted 2-iminothiolane hydrochloride, the reaction solution was passed through a gel filtration chromatography column (PD-10 column, Cytiva) to collect the fraction containing sulfhydryl-functionalized IgG (IgG-SH). Because the resulting IgG-SH solution was diluted, it was concentrated using a centrifugal ultrafiltration filter (50 kDa Amicon Ultrafilter, Millipore) before use in the next reaction. A 0.2-fold volume of IgG-SH solution (IgG-SH concentration: 0.7 mg / mL) was added to the Mal-TLip dispersion (SQR23 concentration: 80 μg / mL) and incubated overnight at room temperature with gentle shaking. Subsequently, 0.8 mM N-acetyl-L-cysteine ​​solution was added to the reaction mixture and the mixture was shaken at room temperature for 10 minutes to block the unreacted maleimide groups. The reaction mixture was passed through a chromatography column packed with gel filtration media (Sephacryl s-300 HR, GE Healthcare) (mobile phase: PBS containing 0.02% sodium azide), and the blue liposome fraction of SQR23 was collected. IgG-SH and N-acetyl-L-cysteine ​​that did not bind to Mal-TLip were eluted in a different fraction from the blue liposome fraction of SQR23 and were therefore separated.

[0128] Example 10: Analysis of antibody-labeled temperature-responsive liposomes containing SQR23. The particle size distribution and zeta potential of the recovered liposome fraction were measured using dynamic light scattering and electrophoretic light scattering (Zeta-Sizer Nano ZS, Malvern Panalytical Ltd). The SQR23 concentration in the dispersion was determined from the absorbance (631 nm) of SQR23 in ethanol. DPPC concentration was measured using a commercially available phospholipid measurement kit (Phospholipid C-Test Wako, Fujifilm Wako Pure Chemical Corporation) based on the choline oxidase-DAOS method. Fluorescamine, which reacts with the amino group of IgG to emit fluorescence, was used to measure IgG concentration. As shown in Table 1, the liposome particle size was controlled to approximately 200 nm, confirming the formation of temperature-responsive liposomes (IgG-TLip) carrying the detection monoclonal antibody IgG-9.

[0129]

[0130] Example 11: Preparation of Magnetic Particles Labeled with Capture Antibodies. Following the protocol provided with the product, a capture antibody was immobilized on Magnosphere™ MS300 / Carboxyl magnetic particles (particle diameter: 3 μm) coated with a hydrophilic polymer and incorporating carboxyl groups as reactive functional groups. First, a Magnosphere™ MS300 / Carboxyl dispersion (particle content: 1%) was stirred for 10 seconds using a vortex mixer to uniformly disperse the particles. Then, 1 mL of the dispersion (equivalent to 10 mg of particles) was dispensed into a reaction microtube. The magnetic particles were captured using a magnetic stand and the supernatant was removed. Then, 1 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.0) was added as a reaction buffer, and the mixture was stirred for 10 seconds using a vortex mixer to disperse the magnetic particles. Next, 100 μL of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, a condensation agent dissolved in 0.1 M MES buffer at 10 mg / mL, was added and the mixture was allowed to react for 30 minutes at room temperature while mixing on a shaker. Furthermore, 100 μg of a capture monoclonal antibody (IgG-7) that specifically binds to the SARS-CoV-2 nucleocapsid protein was added to the reaction solution, and the mixture was stirred for 10 seconds on a vortex mixer, followed by mixing by inversion at room temperature for 3 hours. After the reaction was complete, the magnetic particles were captured using a magnetic stand, the supernatant was removed using a pipette, and the mixture was washed with PBS-T (10 mM PO4 3- After adding 1 mL of PBS-T (pH 7.4, 0.14 M NaCl, 2.7 mM KCl, 0.05% Tween 20) and stirring for 10 seconds on a vortex mixer to disperse the particles, this washing cycle was repeated three times in total, and finally the magnetic particles labeled with the capture monoclonal antibody IgG-7 were resuspended in 1 mL of PBS-T and stored in a refrigerator at 4°C until use.

[0131] Example 12: Detection of SARS-CoV-2 Recombinant N Antigen Using Capture Antibody-Labeled Magnetic Particles IgG-TLip Figure 7 shows a schematic diagram of antigen detection using magnetic particles and IgG-TLip in microwells. Magnetic particles labeled with capture antibody IgG-7 were diluted to a solids concentration of 1 mg / mL and 60 μL was added to a flat-bottom 1 × 8 strip well. Next, 60 μL of SARS-CoV-2 recombinant N antigen was added to final concentrations of 0, 10, 100 pg / mL, 1, and 100 ng / mL, and the mixture was incubated at room temperature for 0.5, 1, 2.5, 5, and 10 minutes. The beads were attracted using a magnet, and unbound antigen was removed by washing with 200 μL of PBS. Then, 60 μL of IgG-TLip was added to the sample to achieve a final concentration of [SQR23] = 0.5 μg / mL. The mixture was incubated for the same time as the recombinant N antigen reaction to allow the IgG-TLip to bind to the antigen. After removing unbound IgG-TLip with 200 μL of PBS using a magnet, the mixture was heated on a hot plate and the fluorescence intensity was measured over time using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.) (n=3). Figure 8 shows the difference in fluorescence intensity before and after heating for each incubation time and antigen concentration for the antigen-antibody reaction, along with its standard deviation. The results indicate that the difference in fluorescence intensity increases with increasing antigen concentration in the sample. However, there is little change in the difference in fluorescence intensity between 30 seconds and 10 minutes for the same antigen concentration. Therefore, it is believed that the capture of antigen by magnetic particles and the binding of IgG-TLip to the captured antigen proceed rapidly, reaching equilibrium within 30 seconds of incubation.

[0132] Example 13: Creation of Microchannels To create microchannels, acrylic plates were cut and excavated using a laser processing machine. Holes were drilled in the upper acrylic plate (top plate) according to the design shown in Figure 9, and the lower acrylic plate (channel substrate) was excavated according to the design shown. After processing the acrylic plates, acrylic cutting dust generated by the laser cutter was removed using ultrasonic cleaning, and the plates were dried under reduced pressure. Next, the two acrylic plates, the top plate and the channel substrate, were welded together with an adhesive to create a channel. The two bonded acrylic plates were left pressed together in a vice to dry and harden overnight. A schematic diagram showing the injection and flow of solutions into the microfluidic device is shown in Figure 10.

[0133] Example 14: Antigen Detection Using Microchannels Figure 11 shows the process for antigen detection using a microchannel constructed using IgG-T Lip and capture antibody-labeled magnetic particles. Detection of SARS-CoV-2 recombinant N antigen was performed according to this schematic diagram. First, a syringe pump was connected to the channel via a pipette port and tubing. 60 μL of sample solution (SARS-CoV-2 recombinant N antigen) was mixed with 60 μL of a capture antibody-labeled magnetic particle dispersion diluted to a solids concentration of 1 mg / mL in a 1.5 mL tube, and then the mixture was introduced into the channel using a syringe pump. After capturing the magnetic particles with a magnet, PBS (400 μL, 400 μL / min) was flowed through the channel to remove unbound recombinant N antigen. Next, the magnet capturing the magnetic particles was removed, and IgG-T Lip ([SQR23] = 1 μg / mL, 100 μL) was flowed through the channel at 400 μL / min, capturing the magnetic particles with the magnet in the detection section. Next, PBS (400 μL) was passed through the flow channel at 400 μL / min to wash away unbound IgG-TLip. After heating the detection section, fluorescence was detected using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.) (n=3). As shown in Figure 12, the fluorescence intensity increased with heating, and the difference in fluorescence intensity before and after heating was analyzed. As shown in Figures 13A and 13B, the difference in fluorescence intensity increased with increasing N antigen concentration, demonstrating a good linear relationship between the logarithm of the N antigen concentration and the difference in fluorescence intensity. Based on the standard deviation (σ) of the negative control, the detection limit (3σ) was calculated to be 0.21 pg / mL, and the quantification limit (10σ) was calculated to be 2.34 pg / mL. The use of magnetic particles and a microfluidic device improved the sensitivity of SARS-CoV-2 detection using TLip-LISA and demonstrated quantitative detection. Furthermore, the use of magnetic particles accelerated the antigen-antibody reaction, shortening the total detection time to 10 minutes. This detection method enables highly sensitive quantitative antigen testing in a test time comparable to that of commercially available qualitative antigen test kits based on the immunochromatography method (requiring a reading of approximately 15 minutes).

[0134] Example 15: Simultaneous Detection of Different Antigens by Using Different Response Temperatures of TLips As shown in Example 5, by incorporating an SQR dye into liposomes that exhibit a gel-liquid crystal phase transition, a series of temperature-responsive fluorescent particles can be prepared that exhibit fluorescence emission in response to the phase transition temperature of the lipid bilayer. Taking advantage of this property, we devised a method for detecting different antigens in a single measurement. A schematic diagram illustrating this method is shown in Figure 14A. TLipA (phase transition temperature < 30°C) is labeled with detection antibody A, which recognizes antigen A, and magnetic particle A is labeled with capture antibody A, which recognizes antigen A. Therefore, if antigen A is present in the sample, TLipA and magnetic particle A will bind via antigen A. On the other hand, TLipB (phase transition temperature > 40°C) is labeled with detection antibody B, which recognizes antigen B, and magnetic particle B is labeled with capture antibody B, which recognizes antigen B. If antigen B is present in the sample, TLipB and magnetic particle B will bind via antigen B. Figure 14A(a) shows the case where both antigen A and antigen B are present in the sample, and TLipA and TLipB bound to the magnetic particles can be collected at the detection site using a magnet. Figure 14A(b) shows the case where antigen A is present but antigen B is not in the sample. Because detection antibody B and capture antibody B do not react with antigen A, only TLipA binds to the magnetic particles. Conversely, Figure 14A(c) shows the case where antigen A is absent but antigen B is present in the sample. Because detection antibody A and capture antibody A do not react with antigen B, only TLipB binds to the magnetic particles. When neither antigen A nor antigen B is present in the sample, neither TLipA nor TLipB binds to the magnetic particles, as shown in Figure 14A(d). Therefore, by collecting magnetic particles at the detection site using a magnet and obtaining the information (a) both TLipA and TLipB are detected, (b) only TLipA is detected, (c) only TLipB is detected, or (d) neither TLipA nor TLipB is detected, the presence or absence of antigen A and antigen B can be simultaneously determined. The fluorescence emission temperature of TLips varies depending on the phase transition temperature of the liposome. For example, TLipA labeled with a detection antibody for antigen A can be made primarily of DMPC, which has a phase transition temperature of 23°C, while TLipB labeled with a detection antibody for antigen B can be made primarily of DPPC, which has a phase transition temperature of 41°C.Because TLipA and TLipB have different emission temperatures, the time until fluorescence emission differs when the fluorescence intensity is measured while heating. As shown in Figure 14B, the fluorescence emission at time T1 corresponds to the fluorescence emission of TLipA, and the fluorescence emission at time T2 corresponds to the fluorescence emission of TLipB. By analyzing this fluorescence emission time, the presence or absence of antigen A and antigen B can be detected in a single measurement.

[0135] Example 16: Differences in Emission Time Due to Differences in TLip Response Temperatures To more specifically demonstrate the method described in Example 15, two types of TLip with different phase transition temperatures were prepared. B-TLip(DPPC) composed of DPPC, DPPA, B-PEG-DSPE, and SQR22 was prepared according to the method described in Example 7. B-TLip(DMPC) composed of DMPC, DPPA, B-PEG-DSPE, and SQR22 was prepared in a similar manner. The resulting B-TLip dispersion ([SQR22] = 2.5 μg / mL, 120 μL) was poured into a flat-bottom 1 × 8 strip well. The sample was heated from room temperature (20 °C) on a hot plate heated to 65 °C, and the time course of fluorescence intensity was measured using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.). As shown in Figure 15, the fluorescence intensity of B-TLip(DMPC) increased immediately after the start of heating (Figure 15a), whereas that of B-TLip(DPPC) increased 80 seconds after the start of heating (Figure 15b). This difference in the time from the start of heating to light emission was due to the fact that the gel-liquid crystal phase transition temperature of B-TLip (DMPC) is 23°C, whereas that of B-TLip (DPPC) is 41°C. In addition, in a mixture ([SQR22] = 5 μg / mL, 60 μL) of B-TLip(DMPC) and B-TLip(DPPC) ([SQR22] = 5 μg / mL, 60 μL) ([SQR22] = 5 μg / mL, 120 μL), an increase in fluorescence intensity due to B-TLip(DMPC) was observed immediately after the start of heating, followed by an increase in fluorescence intensity due to B-TLip(DPPC) approximately 80 s after heating (Fig. 15c). This indicates that even when B-TLips composed of DMPC or DPPC with different phase transition temperatures are mixed, each can be detected in a single measurement due to the difference in emission time.

[0136] Example 17: Simultaneous detection of multiple items based on the fluorescence emission time of TLips Using two types of B-TLips with different phase transition temperatures as described in Example 16, the simultaneous detection method exemplified in Example 15 was examined using the fluorescence emission of B-TLips composed of DMPC or DPPC bound to streptavidin-modified magnetic particles via biotin-streptavidin molecular recognition as an example. Magnosphere magnetic particles with hydrophilic surfaces to which streptavidin was immobilized were used according to the method described in Example 8. TMSixty μL of MS300 / Streptavidin (Medical and Biological Laboratories) (to a final streptavidin concentration of 0.50 μg / mL) and 60 μL of B-TLip(DMPC) or B-TLip(DPPC) (to a final SQR22 concentration of 1 μg / mL) were added to a flat-bottom 1 × 8 strip well and incubated at room temperature for 10 min. Next, (a) 60 μL of the B-TLip(DMPC) reaction mixture + 60 μL of untreated magnetic particles, (b) 60 μL of the B-TLip(DPPC) reaction mixture + 60 μL of untreated magnetic particles, or (c) 60 μL of the B-TLip(DMPC) reaction mixture + 60 μL of the B-TLip(DPPC) reaction mixture were added to a 384-well plate. Unbound B-TLip was removed by washing twice with 100 μL of DPBS while the magnetic particles were pulled to the bottom of the wells with a magnet. Next, magnetic particles were dispersed in DPBS (40 μL), and the bottom of a 384-well plate was heated on a hot plate preheated to 65°C. The time course of fluorescence intensity was measured using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.). As shown in Figure 16, B-TLip(DMPC) bound to magnetic particles via molecular recognition between biotin and streptavidin exhibited an increase in fluorescence intensity immediately after heating (Figure 16a), whereas B-TLip(DPPC) exhibited an increase in fluorescence intensity after 90 seconds (Figure 16b). There was a clear difference in the time from the start of heating to light emission between B-TLip(DMPC) and TLip(DPPC) bound to magnetic particles. Furthermore, a clear difference in the light emission time between B-TLip(DMPC) and TLip(DPPC) was observed in a mixture of B-TLip(DMPC) and TLip(DPPC) bound to magnetic particles (Figure 16c). The method illustrated in Example 9 allows TLips to be labeled with any desired detection antibody, while the method illustrated in Example 11 allows magnetic particles to be labeled with any desired capture antibody. Thus, for example, two detection antibodies for two different antigens, A and B, are bound to the surfaces of two TLips, A and B, respectively, which have different phase transition temperatures, and two capture antibodies for two different antigens are bound to the surfaces of two magnetic particles, respectively. By simultaneously using the resulting two TLips and two magnetic particles, it was demonstrated that different target antigens can be simultaneously detected based on analysis of fluorescence emission times, as illustrated in Figures 14A and 14B.As shown in Figure 3B of Example 5, it is possible to prepare temperature-responsive liposomes that emit light at various temperatures. Since the fluorescent dye is common, various antigens can be detected simultaneously using a detector equipped with one light source and one light receiver.

[0137] Example 18: Preparation of liposome reagent and magnetic particles for influenza A detection. Three types of lipid molecules: DMPC, DPPA, and MAL-PEG-DSPE, and SQR23 were dissolved in cyclohexane in a molar ratio of DMPC / DPPA / MAL-PEG-DSPE / SQR23 = 86.6 / 9.6 / 0.5 / 3.3, and then lyophilized to obtain an SQR23-containing mixed lipid powder. A liposome reagent carrying a detection monoclonal antibody (HL1103, GeneTex) that binds to influenza A nucleocapsid was prepared according to the method described in Example 9. Magnetic particles carrying a capture monoclonal antibody (HL1089, GeneTex) that binds to influenza A nucleocapsid were also prepared according to the method described in Example 11.

[0138] Example 19: Detection of Influenza A N Antigen (Nucleocapsid Protein) Recombinant influenza A N antigen was detected using the influenza A detection TLip (DMPC) prepared in Example 18 and magnetic particles. 30 μL of magnetic particles (solid concentration: 10 μg / mL) labeled with a capture antibody and 30 μL of TLip dispersion ([SQR23] = 20 μg / mL) carrying a detection antibody were mixed in a flat-bottom 384-well plate. Recombinant influenza A N antigen (origin: Puerto Rico / 8 / 1934 H1N1) was added to this mixture to a final concentration of 50-400 ng / mL and allowed to stand at room temperature for 5 minutes. The magnetic particles were attracted using a magnet, the solution was removed, and the plate was washed with PBS. The magnetic particles were then dispersed in 30 μL of PBS. The fluorescence intensity was measured over time using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.) while the bottom of the well plate was heated on a hot plate (70 °C). As shown in Figure 17A, the difference in fluorescence intensity before and after heating increased with increasing influenza A N antigen concentration, demonstrating quantitative detection of antigen concentration. The presence of SARS-CoV-2 antibody-loaded TLip (DPPC) did not affect the fluorescence intensity difference, confirming that the presence of SARS-CoV-2 TLip (DPPC) for influenza A detection was minimally affected by the presence of SARS-CoV-2 N antigen on influenza A antigen detection using TLip (DMPC) for influenza A detection. Furthermore, as shown in Figure 17B, the presence of SARS-CoV-2 N antigen did not affect influenza A antigen detection using TLip (DMPC).

[0139] (Example 20) Detection of SARS-CoV-2 and Influenza A N Antigen (Nucleocapsid Protein) 60 μL of magnetic particles labeled with a capture antibody for SARS-CoV-2 (solid concentration: 1 mg / mL) and 60 μL of magnetic particles labeled with a capture antibody for influenza A (solid concentration: 1 mg / mL) were added to a flat-bottom microwell, and the mixture was diluted with 1. Influenza A recombinant N antigen (origin: Puerto Rico / 8 / 1934 H1N1) (1 μg / mL, 60 μL) + DPBS (60 μL), 2. SARS-CoV-2 recombinant N antigen (200 ng / mL, 60 μL) + DPBS (60 μL), and 3. Recombinant influenza A N antigen (origin: Puerto Rico / 8 / 1934 H1N1) (1 μg / mL, 60 μL) + recombinant SARS-CoV-2 N antigen (200 ng / mL, 60 μL), 4. DPBS (200 μL) was added and the mixture was left at room temperature for 2 minutes to capture the antigen onto the capture antibody on the magnetic particles. The supernatant was removed by pulling the magnetic particles with a magnet, and the magnetic particles were washed twice with DPBS (200 μL). The washed magnetic particles were dispersed in a TLip(DMPC) dispersion ([SQR23] = 1 μg / mL, 60 μL) loaded with an influenza A detection antibody and a TLip(DPPC) dispersion ([SQR23] = 1 μg / mL, 60 μL) loaded with a SARS-CoV-2 detection antibody. The particles were then allowed to stand at room temperature for 2 minutes to allow TLip to bind to the antigen captured by the magnetic particles via the detection antibody. Unbound TLip was then removed by pulling the magnetic particles with a magnet, and the magnetic particles were washed twice with DPBS (200 μL). After adding 120 μL of DPBS to the wells, the bottom of the well plate was heated on a hot plate (65°C) while the fluorescence intensity was measured over time using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.). As shown in Figure 18, in the system to which only influenza A antigen was added, only an increase in fluorescence intensity due to the phase transition of TLip(DMPC) was observed immediately after the start of heating (Figure 18a). Because TLip(DMPC) corresponds to the influenza A detection antibody, only influenza A antigen was detected.In contrast, in the system containing only the SARS-CoV-2 antigen, no increase in fluorescence intensity was observed immediately after the start of heating. After approximately 90 seconds, only an increase in fluorescence intensity was observed, which was due to the phase transition of TLip(DPPC), which corresponds to the SARS-CoV-2 detection antibody (Figure 18b). Furthermore, in the system containing both influenza A and SARS-CoV-2 antigens, a two-step increase in fluorescence intensity was observed, which was due to both TLip(DMPC) and TLip(DPPC), and both influenza A and SARS-CoV-2 antigens were detected (Figure 18c). In the system without antigen, no increase in fluorescence intensity was observed for either TLip(DMPC) or TLip(DPPC) (Figure 18d).

[0140] (Example 21) Simultaneous detection of SARS-CoV-2 and influenza A N antigen (nucleocapsid protein) In a 384-well plate, 30 μL of magnetic particles labeled with a capture antibody for SARS-CoV-2 (solid concentration: 10 μg / mL), 30 μL of magnetic particles labeled with a capture antibody for influenza A (solid concentration: 10 μg / mL), 30 μL of a TLip(DPPC) dispersion ([SQR23] = 20 μg / mL) carrying a detection antibody for SARS-CoV-2, and 30 μL of a TLip(DMPC) dispersion ([SQR23] = 20 μg / mL) carrying a detection antibody for influenza A were added and mixed. To this mixture, SARS-CoV-2 recombinant N antigen was added at a final concentration of 100 ng / mL, and influenza A recombinant N antigen (origin: Puerto Rico / 8 / 1934 H1N1) was added at final concentrations of 0-400 ng / mL, and the mixture was left to stand at room temperature for 5 minutes. In addition, influenza A recombinant N antigen was added at a final concentration of 200 ng / mL, and SARS-CoV-2 recombinant N antigen was added at final concentrations ranging from 0-75 ng / mL, and the mixture was left to stand at room temperature for 5 minutes. The magnetic particles were then attracted using a magnet, the solution was removed, and the wells were washed with PBS. The magnetic particles were then dispersed in 30 μL of PBS. The fluorescence intensity was measured over time using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.) while the bottom of the well plate was heated on a hot plate (70°C). As shown in Figure 19, when both SARS-CoV-2 and influenza A antigens were added, an increase in fluorescence intensity due to the phase transition of DMPC corresponding to influenza A was observed for approximately 50 seconds after the start of heating, followed by an increase in fluorescence intensity due to the phase transition of DPPC corresponding to SARS-CoV-2. As shown in Figure 20A, when only the SARS-CoV-2 antigen was added, there was no increase in fluorescence intensity until approximately 50 seconds after the start of heating, and only an increase in fluorescence intensity due to the phase transition of DPPC corresponding to SARS-CoV-2 was observed. As the concentration of influenza A antigen increased, the difference in fluorescence intensity due to the phase transition of DMPC increased.On the other hand, as shown in Figure 20B, when only influenza A antigen was added, there was no increase in fluorescence intensity after 50 seconds; only an increase in fluorescence intensity due to the phase transition of DMPC corresponding to influenza A was observed. When SARS-CoV-2 antigen was added, an increase in fluorescence intensity due to the phase transition of DPPC was observed after 50 seconds, indicating that SARS-CoV-2 was detected. For SARS-CoV-2, the difference in fluorescence intensity did not change even when the antigen concentration was varied from 25 to 75 ng / mL. This is likely due to the significantly higher antigen detectability (pg / mL) of the SARS-CoV-2 capture and detection antibody pair compared to the influenza antibody pair (ng / mL), leading to saturation of the antigen-antibody reaction at antigen concentrations in the ng / mL range. Next, SARS-CoV-2 and influenza A were detected using the microfluidic channel fabricated in Example 13. Magnetic particles (solid concentration: 10 μg / mL) conjugated with each capture antibody and recombinant N antigen (influenza A = 200 ng / mL, SARS-CoV-2 = 25 ng / mL) were mixed and then introduced into the channel using a syringe pump. After capturing the magnetic particles with a magnet, PBS (400 μL, 400 μL / min) was passed through the channel to remove unbound recombinant N antigen. Next, the magnet capturing the magnetic particles was removed, and a mixed dispersion of TLip (DMPC) conjugated with influenza A detection antibodies ([SQR23] = 5 μg / mL) and TLip (DPPC) conjugated with SARS-CoV-2 detection antibodies ([SQR23] = 5 μg / mL) was flowed through the flow channel, and the magnetic particles were captured by the magnet in the detection section. Next, PBS (400 μL) was flowed through the flow channel at 400 μL / min to wash out unbound TLip. Fluorescence was then detected using an optical fiber probe fluorescence detector (FLE 1100, Nippon Sheet Glass Co., Ltd.) while the detection section was heated on a hot plate (65 °C). As shown in Figure 21, an increase in fluorescence intensity due to the phase transition of DMPC (corresponding to influenza A antigen) was observed immediately after heating, and an increase in fluorescence intensity due to the phase transition of DPPC (corresponding to SARS-CoV-2 antigen) was observed after 50 s or more.These results demonstrate that by loading antibodies corresponding to different antigens onto TLips with different phase transition temperatures, it is possible to simultaneously test multiple antigens based on the difference in the time it takes for light to be emitted.

[0141] According to the present invention, multiple target biomolecules contained in a biological sample can be simultaneously detected or quantified in a single run, making it possible to process a large amount of target biomolecules in a shorter time than conventional methods.

[0142] (References) 1. Ji X., et al, Nanoscale, 2023, 15, 9290-9296. 2. Qi J., et al., Advanced Drug Delivery Reviews, 2019, 143, 206-225. 3. Zhang Y., et al., ACS Appl. Mater. Interfaces, 2013, 5, 8710-8717. 4. Hoekstra D. et al., Biochemistry, 1984, 23, 24, 5675-5681. 5. Somijit V. et al., Microporous and Mesoporous Materials, 2021, 328, 111452. 6. Tu L., et al., SmartMat., 2021, 2, 326-346. 7. Teixeira R. et al., Molecules, 2021, 26, 4264. 8. Reisch A. et al., Small, 2016, 12, 1968-1992.9. BaumgartT. Et al., Biochim Biophys Acta 2007, 1768(9), 2182-2194. 10. Klymchenko AS et al., Chem Biol. 2014, 21(1), 97-113. 11. Filipe HAL et al., Molecules 2020, 25, 3424. 12. Demchenko AP et al., Methods Mol Biol. 2015, 1232, 19-43. 13. Mei J. et al., Chem. Rev. 2015, 115, 21, 11718-11940.

Claims

1. A method for detecting or quantifying a biomolecule contained in a biological sample, comprising the following steps (1) to (3): (1) a step of contacting a magnetic particle having a capture biomolecule recognition element bound thereto, a biological sample, and a temperature-responsive fluorescent probe particle with a magnetic substrate; (2) a step of heating the magnetic substrate; and (3) a step of measuring fluorescence emission, wherein the temperature-responsive fluorescent probe particle is a temperature-responsive fluorescent particle having a detection biomolecule recognition element bound thereto, the temperature-responsive fluorescent particle contains at least one fluorescent molecule in a molecular assembly comprising at least one amphipathic lipid molecule, the fluorescent molecule is quenched when the molecular assembly is in the solid phase and emits fluorescence when in the liquid phase due to a solid-liquid phase transition responsive to temperature, thereby causing a reversible conversion between the quenching and fluorescence emission of the fluorescent molecule in response to temperature, the molecular assembly contains aggregated fluorescent molecules in the solid phase and dissociated fluorescent molecules in the liquid phase, The method for detecting or quantifying a biomolecule, wherein the fluorescent molecule is at least one compound selected from the group consisting of squaric acid derivatives, fluorescein derivatives, rhodamines, coumarins, oxazines, pyrenes, anthracenes, phthalocyanines, cyanines, azo dyes, carbopyronines, NBD derivatives, dansyl derivatives, BODIPY fluorophores, ALEXA fluorophores, rhodol dyes, naphthalimides, and porphyrins.

2. A method for detecting or quantifying a biomolecule according to claim 1, wherein the step (1) comprises the following steps: (1') a step of contacting magnetic particles having a capture biomolecule recognition element bound thereto with a magnetic substrate; (2') a step of contacting a biological sample with the magnetic particles; and (3') a step of contacting temperature-responsive fluorescent probe particles with the magnetic particles.

3. The method for detecting or quantifying a biomolecule according to claim 1, wherein the step (1) comprises the following steps: (1'') contacting a biological sample with magnetic particles having a capture biomolecule recognition element bound thereto; (2'') contacting the complex obtained in step (1'') with a magnetic substrate; (3'') contacting the complex with temperature-responsive fluorescent probe particles; and (4'') contacting the complex obtained in step (3'') with a magnetic substrate.

4. The method for detecting or quantifying a biomolecule according to claim 1, wherein the step (1) comprises the following steps: (1''') contacting a temperature-responsive fluorescent probe particle with a biological sample; (2''') contacting the complex obtained in step (1''') with a magnetic particle having a capture biomolecule recognition element bound thereto; and (3''') contacting the complex obtained in step (2''') with a magnetic substrate.

5. A method for detecting or quantifying a biomolecule according to any one of claims 1 to 4, comprising a step of washing the magnetic particles between steps (1) and (2).

6. A method for detecting or quantifying a biomolecule described in any one of claims 1 to 4, characterized in that the biomolecules to be detected or quantified are two or more types, the magnetic particles are two or more types of magnetic particles to which different capture biomolecule recognition elements are bound, and the temperature-responsive fluorescent probe particles are two or more types of temperature-responsive fluorescent particles to which different detection biomolecule recognition elements are bound and which have different solid-liquid phase transition temperatures.

7. A method for detecting or quantifying a biomolecule according to any one of claims 1 to 4, characterized in that the molecular assembly has a lipid bilayer, and undergoes a gel-liquid crystal phase transition in response to temperature, causing the fluorescent molecule to quench when the molecular assembly is in the gel phase and to emit fluorescence when the molecular assembly is in the liquid crystal phase, thereby reversibly converting between quenching and fluorescence emission of the fluorescent molecule in response to temperature.

8. A method for detecting or quantifying a biomolecule according to any one of claims 1 to 4, characterized in that the amphipathic molecule is a phospholipid.

9. The method for detecting or quantifying a biomolecule according to claim 8, wherein the molecular assembly is a bilayer membrane vesicle.

10. The method for detecting or quantifying a biomolecule according to claim 9, wherein the bilayer membrane vesicle contains 0.3 mol % or more of the compound represented by general formula I.

11. A method for detecting or quantifying a biomolecule according to any one of claims 1 to 4, characterized in that the biomolecule recognition element is an antibody, or a variable region of said antibody, or a fab fragment of said antibody.

12. A kit for detecting or quantifying a biomolecule contained in a biological sample, comprising the following (1) and (2): (1) a magnetic particle having a capture biomolecule recognition element bonded thereto; and (2) a temperature-responsive fluorescent probe particle which is a temperature-responsive fluorescent particle having a detection biomolecule recognition element bonded thereto, wherein the temperature-responsive fluorescent particle contains at least one fluorescent molecule in a molecular assembly comprising at least one amphipathic lipid molecule, and the fluorescent molecule is quenched when the molecular assembly is in the solid phase and emits fluorescence when in the liquid phase due to a solid-liquid phase transition responsive to temperature, thereby causing a reversible conversion between quenching and fluorescence emission of the fluorescent molecule in response to temperature, and the molecular assembly has aggregated fluorescent molecules in the solid phase and dissociated fluorescent molecules in the liquid phase, The temperature-responsive fluorescent probe particle, wherein the fluorescent molecule is at least one compound selected from the group consisting of squaric acid derivatives, fluorescein derivatives, rhodamines, coumarins, oxazines, pyrenes, anthracenes, phthalocyanines, cyanines, azo dyes, carbopyronines, NBD derivatives, dansyl derivatives, BODIPY fluorophores, ALEXA fluorophores, rhodol dyes, naphthalimides, and porphyrins.

13. A kit for detecting or quantifying a biomolecule according to claim 12, further comprising a microchannel chip.

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