Patent Publication Number: US-2019170758-A1

Title: Method of detection with a fluorescent labeling particle

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a Continuation of copending U.S. application Ser. No. 15/088,758, filed on Apr. 1, 2016, which is a Continuation of PCT International Application No. PCT/JP2014/076257 filed on Oct. 1, 2014, which claims priority under 35 U.S.C. § 119 (a) to Japanese Patent Application No. 2013-207388 filed in Japan on Oct. 2, 2013. Each of the above applications is hereby expressly incorporated by reference, in its entirety, into the present application. 
    
    
     TECHNICAL FIELD 
     The present invention relates to a fluorescent labeling particle. 
     BACKGROUND ART 
     Fine particles on which various kinds of biological substances are immobilized have been widely utilized in medical diagnosis, a biotechnology research field and the like. Fine particles that are formed of a variety of materials and have various particle diameters, forms and functions have been studied and proposed therein. Among them, there exists a technology in which magnetic particles are utilized. Specific examples include a technology in which magnetic particles on which an antibody is immobilized are used to perform affinity column chromatography. At this time, in latex beads, an antigen is purified by repeating centrifugal separation and washing operation. Therefore, a large amount of solvent is required. On the other hand, when the magnetic particles are used, the complicated operation as described above is not required because particles can be easily separated by means of a magnet. That is, in comparison with the latex beads having no magnetism, the magnetic particles have an advantage of capability of rapidly and simply purifying the antigen. Moreover, the magnetic particles are also adapted to automation using a machine because such centrifugal separation and application of the large amount of solvent become unnecessary. For the above reason, immunological magnetic particles have been frequently utilized as a carrier in immunodiagnosis (sandwich method) in recent years. 
     In order to improve molecular recognition properties of the magnetic particles, a surface area thereof is preferably increased. In this regard, in order to further increase the surface area per unit volume for magnetic beads, further reduction of a particle diameter is effective. On the other hand, if the particle diameter of the magnetic particles falls in a nanometer order, the magnetism of the particles is weakened. Further, magnetic separation by means of the magnet becomes significantly difficult under the influence of Brownian motion in water. In order to solve this problem, proposals have been made on arts of using particles in which a thermoresponsive polymer is immobilized on a surface layer of magnetic nanoparticles having a particle diameter of several tens of nanometers (see Patent Literatures 1 and 2). A target substance is detected depending on a change of colors (into transparency) of magnetic materials by aggregating thermoresponsive magnetic particles by heating therein. Specifically, absorbance of a reaction liquid over time is monitored by heating the liquid after a reaction with the target substance, and existence of the target substance (analyte) is judged by the absorbance being gradually decreased. 
     Patent Literature 1: WO 2008/001868 pamphlet 
     Patent Literature 2: Japanese Patent No. 4518767 
     SUMMARY OF INVENTION 
     Technical Problem 
     It is certain that detection sensitivity thereof is considered to be improved by the above-mentioned thermoresponsive magnetic particles to compensate for the low magnetism caused by micronized particles. However, a further improvement in the detection sensitivity is desired in order to realize a further microanalysis or securely prevent a false negative or a false positive in medical diagnosis. 
     Therefore, the present invention is contemplated for providing a labeling particle capable of realizing high detection sensitivity in detection of a target substance utilizing a thermoresponsive magnetic particle, and a method of producing the same. 
     Solution to Problem 
     The problems are solved by the following means.
     [1] A fluorescent labeling particle for detecting a target substance, containing:
       a composite particle having a phase containing a magnetic material and a phase containing a fluorescent material; and   a thermoresponsive polymer on a surface of the composite particle.   
       [2] The fluorescent labeling particle as described in the above item [1], further containing a biomolecule having properties of binding with the target substance on the surface of the composite particle.   [3] The fluorescent labeling particle as described in the above item [1] or [2], wherein the phase containing a fluorescent material is constituted of a continuous phase formed of a transparent material and a dispersed phase formed of the fluorescent material.   [4] The fluorescent labeling particle as described in any one of the above items [1] to [3], wherein the phase containing a magnetic material forms a core, and the phase containing a fluorescent material is arranged in the form of coating the core.   [5] The fluorescent labeling particle as described in any one of the above items [1] to [3], wherein the phase containing a magnetic material and the phase containing a fluorescent material are arranged in a mixed manner in a sea-island form or an indefinite form, and at least of a part of the phase containing a fluorescent material is exposed onto the surface of the composite particle.   [6] The fluorescent labeling particle as described in any one of the above items [1] to [5], wherein the thermoresponsive polymer causes a change in morphologies thereof at a critical solution temperature (CST) as a border, and aggregation properties of the labeling particle change at the critical solution temperature (CST) as the border.   [ 7 ] The fluorescent labeling particle as described in the above item [6], wherein the fluorescent labeling particle easily aggregates upon exceeding the critical solution temperature (CST), and becomes hard to aggregate at the critical solution temperature (CST) or lower, and
 
wherein the critical solution temperature (CST) is in the range of 10° C. to 100° C.
   [8] The fluorescent labeling particle as described in any one of the above items [1] to [7], wherein an average particle diameter of the composite particle is less than 1 μm.   [9] The fluorescent labeling particle as described in any one of the above items [3] to [8], wherein the transparent material contains silica or polystyrene.   [10] The fluorescent labeling particle as described in any one of the above items [1] to [9], wherein the magnetic material is magnetite, nickel oxide, ferrite, cobalt iron oxide, barium ferrite, carbon steel, tungsten steel, KS steel, a rare earth cobalt magnet or hematite.   

     “Having on the surface of particle” means existence in the form of directly contacting with the surface and also existence outward of the surface through a different material. 
     Advantageous Effects of Invention 
     The labeling particle of the present invention realizes high detection sensitivity in detection of a target substance by utilizing a thermoresponsive magnetic particle. According to a production method of the present invention, a labeling particle having the above-described excellent performance can be preferably produced. 
     Other and further features and advantages of the invention will appear more fully from the following description, appropriately referring to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a side view schematically showing a thermoresponsive fluorescent particle constituting a labeling particle of the present invention. 
         FIG. 2  is a side view schematically showing a change between aggregation and dispersion of thermoresponsive fluorescence particles by heating. 
         FIG. 3  is a side view schematically showing one embodiment of a fluorescent labeling particle of the present invention. 
         FIG. 4  is an explanatory view showing a state in which a fluorescent labeling particle forms a linked structure together with a target substance and a dispersant. 
         FIG. 5  is an explanatory view showing an example in which fluorescent labeling particles of the present invention are applied to detection of a target substance. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     The labeling particle of the present invention is a labeling particle for detecting a target substance, and have a thermoresponsive polymer on a surface of a composite particle containing a magnetic material and a fluorescent material. Herein, the labeling particle preferably has a binding material (biomolecule or the like) having properties of binding with the target substance. 
       FIG. 1  is a side view schematically showing a thermoresponsive fluorescent particle  10  constituting a labeling particle of the present invention. In the present embodiment, a composite particle  11  has a form in which a transparent material phase (fluorescent silica phase)  2  forming a shell coats a surface of a magnetic material phase (magnetic material particle)  1  forming a core. Then, thermoresponsive polymers  3  are arranged on a surface of the composite particle  11 . In the present embodiment, a core/shell-type composite particle is illustrated, but the present invention is not limited thereto. For example, the phases may be in a form into a sea-island form or in a state in which the phases are indefinitely mixed. However, even at this time, at least a part of the fluorescent material phase  2  is preferably exposed on the surface of the composite particle, and a greater part of the particle surface is more preferably constituted of the fluorescent material phase so that the fluorescence can be detected from outside. Accordingly, as the aspect of the sea-island form, the aspect is preferable in which the magnetic material phase constitutes an island portion, and the fluorescent material phase constitutes a sea portion. Specific examples of a similar aspect include a core-distributed particle inside which a number of cores formed by the above-described magnetic material phases exist. 
     The thermoresponsive polymers  3  are shown in the aspect in which the polymers  3  are radially extended from the surface of the composite particle  2  outward for convenience of illustration, but the present invention shall not be limited and construed by this aspect. Aspects related to adsorption of the polymer onto the surface are diversified, and generally indefinite. The polymers may have complicatedly entangled structure, or may be immobilized on the surface of the composite particle in the aspect of the particle form in which each molecule or several molecules are shrunk. However, the polymers are preferably in the state in which the fluorescence emitted from the composite particle may reach the outside. In and after  FIG. 2 , the phases of this thermoresponsive polymers are simplified by hatching and shown. 
       FIG. 2  is a side view schematically showing a change between aggregation and dispersion of thermoresponsive fluorescence particles  10  by heating.  FIG. 2  ( 1 ) on a left-hand side in the figure shows a state before heating, in which the thermoresponsive fluorescence particles  10  are dispersed with each other in the system. In contrast, the particles  10  transition to an aggregated state by heating as in  FIG. 2  ( 2 ) on a right-hand side. The reason why the change between aggregation and dispersibility is caused by heating as described above is not always clear. To describe the reason including presumption, it is considered that the polymers are shrunk by heating and approachable distances of adjacent composite particles become shorter, and therefore the aggregation is promoted. In the present embodiment, an example is shown in which the aggregation is promoted upon exceeding the critical solution temperature (CST) by heating, and dispersion is promoted below the critical solution temperature, but may be in a reverse manner. That is, an aspect may be formed in which the dispersion is promoted upon exceeding the critical solution temperature (CST), and the aggregation is promoted below the critical solution temperature. 
       FIG. 3  is a side view schematically showing one embodiment of a fluorescent labeling particle of the present invention. In the present embodiment, an example is shown in which linking materials  4  are provided on a surface of the composite particle  11 , and binding substances  5  are immobilized therein. In an illustrated aspect, although the linking materials  4  are shown as when the linking materials  4  are arranged in an outside of the thermoresponsive polymer  3  (polymer leading end), which is only for convenience of illustration, and is not necessary to be in such an arrangement state. In actual fluorescent labeling particles, the thermoresponsive polymers  3  rather exist in an indefinite manner, and only need to arbitrarily exist in an entangled state or an adsorbed state therein, or the like. Conversely, when a material having a satisfactory molecular design is applied, the linking material may be introduced into the leading end of the thermoresponsive polymer as described above. As a modified aspect, the binding substances  5  may be introduced into the thermoresponsive polymer  3  without using the linking materials  4 . Further, when the thermoresponsive polymer has properties of binding with the target substance, or the like, specific examples include fluorescent labeling particles in which no binding substance is used. 
     Herein, the binding substance  5  is referred to as a particle-side binding substance  5  in distinction from a dispersant-side binding substance  6  as mentioned later in several cases. 
       FIG. 4  is an explanatory view showing a state in which a fluorescent labeling particle  20  of the present invention forms a linked structure  100  together with a target substance S and a dispersant  30 . The particle-side binding substance  5  introduced into the fluorescent labeling particle has the properties of binding with the target substance S. On the other hand, in the present embodiment, a dispersant  30  is introduced into the system. The dispersant  30  is composed of a dispersant base  7  and a dispersant-side binding substance  6 . This dispersant-side binding substance  6  also has the properties of binding with the target substance S. Accordingly, when the fluorescent labeling particle  20 , the target substance S and the dispersant  30  coexist in the system, structure (linked structure  100 ) is formed in which the fluorescent labeling particle  20  and the dispersant  30  are linked through the target substance S. This linked structure  100  exhibits dispersibility in the system due to an effect of the dispersant  30  to prevent the fluorescent labeling particle  20  from being aggregated. Herein,  FIG. 4  shows only one linking material  4  and one binding substance  5 , but is simplified and preferably a large number thereof are provided typically as shown in  FIG. 3 . 
       FIG. 5  is a schematic view showing one example of a detection technique in which an effect of the dispersant  30  as mentioned above is utilized.  FIG. 5  ( 1 ) shows a case where the target substances S exist. At this time, as shown in  FIG. 4  to be mentioned later, a linked structure  100  through the target substance S is formed, and dispersed without being aggregated in an analyte liquid. On the other hand, in a case where no target substance S exists ( FIG. 5  ( 2 )), any dispersant  30  and any fluorescent labeling particle  20  are not linked, and each of the fluorescent labeling particles  20  exists independently within a system. At this time, when the liquid is heated and the liquid temperature is adjusted to a critical solution temperature (CST) or higher, the fluorescent labeling particles  20  are aggregated. In the present embodiment, a magnet M is installed on a right side plane of a reaction vessel B. The fluorescent labeling particles  20  that are aggregated and strongly magnetized are attracted by magnetic force of this magnet, and accumulated in the vicinity of the magnet M. On the other hand, when the state of  FIG. 5  ( 1 ) in which the particles  20  exist in the form of the linked structures  100  having high dispersibility, aggregation of the fluorescent labeling particles  20  is not promoted, and the magnetism thereof is limited in a small state. Accordingly, even when the magnet M is placed laterally to the vessel B, the particles  20  are not attracted thereto, and a dispersion state is maintained. 
     In the present embodiment, the reaction liquid (vessel B) in which existence or non-existence of the target substances S is distinguished in the state of  FIG. 5  described above is irradiated with the excitation light. Then, in  FIG. 5  ( 1 ) in the state in which the target substances S is contained, fluorescence is limited to the emission from each of the fluorescent labeling particles  20 , and is not intensified significantly, although somewhat emission is found in the liquid as a whole. On the other hand, in  FIG. 5  ( 2 ) in which no target substance S is contained, when the liquid is irradiated with the excitation light, the fluorescent labeling particles  20  that are heated, and attracted in the vicinity of the magnet and in the aggregated state emit the fluorescence. The fluorescence results in the fluorescence emission of the fluorescent labeling particles  20  in the aggregated state and concentrated in a part, and the fluorescence can be detected in a significantly marked state. 
     Aggregation of the particles and accumulation thereof in the vicinity of the magnet are a phenomenon that occurs in conventional thermoresponsive magnetic particles in which the fluorescent material phase  2  is not used, according to which an orange-colored concentrated portion is observed, for example. However, the dispersion liquid in which no aggregation is caused is also slightly orange-colored, and when an amount of the target substance is small or the like, the color is not developed as a significantly distinct difference in several cases. In contrast, according to the fluorescent labeling particle of the present invention, the difference can be detected by the fluorescence, and therefore a marked change of the fluorescence emission can be detected. When the change is expressed by a difference in detection sensitivity (difference in absorbance or fluorescence intensity caused by existence or non-existence of the target substance), according to a preferred embodiment of the present invention, the difference can be improved by as much as 10 times to several hundred times in comparison with a conventional means. (However, the present invention shall not be limited and construed by exemplification of this improvement effect.) 
     Further, in the present embodiment, the fluorescent material phase  2  that constitutes the fluorescent labeling particle  20  is formed of silica (continuous phase)  2   a  containing a fluorescent dye (dispersion layer)  2   b.  A detail thereof is mentioned later, but a silica raw material ordinarily has smaller specific gravity in comparison with a magnetic material, and becomes easily floatable in a medium of the analyte liquid, such as water. The effect is also produced, and a change of the dispersion and aggregation states becomes sharp, further leading to an improvement in the detection sensitivity. In the following, the materials used in the above-described fluorescent labeling particle having the excellent effect according to the present invention are described in more detail. 
     In illustrated aspects, each material is schematically or exaggeratedly shown for ease of understanding, and the present invention shall not be limited and construed by the aspects shown in these figures. 
     [Composite Particle] 
     (Magnetic Material) 
     The magnetic material that can be used in the present invention is not particularly limited, but is preferably in a particle form. Specifically, the material has an average particle diameter of preferably 0.5 nm or more and less than 1,000 nm, and particularly preferably 3 nm or more and less than 200 nm. Specific examples of the magnetic materials (raw materials) include fine particles of magnetite, nickel oxide, ferrite, cobalt iron oxide, barium ferrite, carbon steel, tungsten steel, KS steel, a rare earth cobalt magnet and hematite. 
     (Fluorescent Material) 
     In the present invention, the phase containing the fluorescent material is preferably constituted of a continuous phase  2   a  formed of the transparent material and a dispersed phase  2   b  formed of the fluorescent material. In this case, the transparent material preferably contains silica or polystyrene. 
     Silica Phase 
     A method of forming the fluorescent silica phase as the above-described fluorescent material phase is not particularly limited, and the phase obtained by an arbitrary production method may be applied. Examples of the method include a sol-gel method described in Journal of Colloid and Interface Science, vol. 159, p. 150-157 (1993). In the present invention, a method of producing colloid silica particles containing fluorescent dye compounds as described in WO 2007/074722 A1 also serves as reference. 
     Specifically, an example in which a fluorescent dye as a fluorescent material is used is described. The silica phase containing a fluorescent dye can be prepared by reacting a fluorescent dye with a silane coupling agent and performing polycondensation of a product obtained through a covalent bond, ionic bond, or other chemical bonds or by adsorption with one or more of silane compounds to form a siloxane bond. Accordingly, the silica phase consisting of the organosiloxane component and the siloxane component that are bound to each other via siloxane bond are obtained. As one example, production can be made by reacting a fluorescent dye having or provided with an active group such as an N-hydroxysuccinimide (NHS) ester group, a maleimide group, an isocyanate group, an isothiocyanate group, an aldehyde group, a para-nitrophenyl group, a diethoxy methyl group, an epoxy group, and a cyano group with a silane coupling agent having a substituent which reacts with those active groups (e.g., an amino group, a hydroxy group, and a thiol group), and condensing and polymerizing the product obtained by forming a siloxane bond after forming a covalent bond with one or more types of silane compounds. 
     The following example relates to a case in which APS and tetraethoxy silane (TEOS) are used as a silane coupling agent and a silane compound, respectively. 

 
     Specific examples of the functional compound having or provided with an active group may include NHS ester group-containing fluorescence dye compounds such as 5- (and -6)-carboxytetramethylrhodamine-NHS ester (trade name, manufactured by emp Biotech GmbH), DY550-NHS ester or DY630-NHS ester represented as follows (each trade name, manufactured by Dyomics GmbH). 
     
       
         
         
             
             
         
       
     
     Examples of the substituent-containing silane-coupling agent include an amino group-containing silane-coupling agent such as γ-aminopropyltriethoxysilane (APS), 3-[2-(2-aminoethylamino)ethylamino]-propyltriethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, and 3-aminopropyltrimethoxysilane. Among them, APS is preferable. 
     The silane compound to be condensed and polymerized is not particularly limited, and examples thereof include TEOS, γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS), 3-thiocyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3-[2-(2-aminoethylamino)ethylamino]propyl-triethoxysilane. Among them, TEOS is preferable from the point of view of forming the siloxane component to be contained in the silica particles, and besides MPS and APS are preferable from the point of view of forming the organosiloxane component to be contained in the silica particles. 
     For example, spherical or almost spherical magnetic material/silica composite particles can be produced by using the above-described materials and providing the above-described magnetic particles into a reaction system so as to form a core. Meanwhile, the almost spherical particles mean particles having a major axis/minor axis ratio of 2 or less. For obtaining silica particles having a desirable average particle diameter, it is possible to remove particles having an excessively large particle diameter or an excessively small particle diameter by ultrafiltration by using an ultrafiltration membrane such as YM-10 or YM-100 (each trade name, manufactured by Millipore Corporation) or by recovering only a supernatant or precipitates after performing centrifugal separation with suitable acceleration of gravity. 
     The average particle diameter of the fluorescent composite particle is not specifically limited, but preferably 1 nm or more, and more preferably 20 nm or more. The upper limit thereof is preferably less than 1 μm, and more preferably 500 nm or less. 
     In the present invention, the average particle diameter is an average diameter of the circle (average circle-equivalent diameter) obtained by measuring the total projected area of 100 pieces of randomly-selected labeling reagent silica particles for example in an image obtained under transmission electron microscope (TEM) or scanning electron microscope (SEM) using an image processing equipment, dividing the total area with the number of the labeling reagent silica particles (100 pieces), and determining the circle having an area equivalent to that. 
     Further, the “average particle diameter” indicates an average particle diameter of particles consisting of only primary particles, which is different from the “particle size according to a dynamic light scattering method” described below having a concept including secondary particles formed by aggregation of primary particles. 
     As described herein, the “particle size according to the dynamic light scattering method” is measured by the dynamic light scattering method, and it is a concept including secondary particles formed by aggregation of primary particles as well as primary particles, different from average particle diameter. This particle size is an indicator for evaluating dispersion stability of the complex particles described above. 
     Examples of a device for measuring the particle size according to the dynamic light scattering method include Zetasizer Nano (trade name, manufactured by Malvern Instruments Ltd.). According to the method, fluctuation in light scattering intensity over time that is caused by light scatterers such as fine particles is measured, the speed of the light scatterers in Brownian motion is calculated based on an autocorrelation function, and the particle size distribution of the light scatterers is determined based on the results. 
     The fluorescent silica particles preferably have monodispersion as a granular substance. The variation coefficient, so-called CV value, of the particle size distribution is not specifically limited, but preferably 10% or less, and more preferably 8% or less. 
     Latex Phase 
     In the present invention, it is preferable to use the above-described silica fine particles because the effect thereof is significant, but in addition thereto or in place thereof, a latex phase may be used as the continuous phase of the fluorescent material phase in the composite particles. Examples of the material for constituting the latex phase include synthetic polymer particles consisting of polystyrene, styrene-sulfonic acid (salt) copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-sulfonic acid copolymer, vinyl chloride-acrylic acid ester copolymer, or vinyl acetate-acrylic acid ester copolymer. Further, as for the method of introducing a dye into the latex phase, methods disclosed in JP-A-2000-178309 (“JP-A” means unexamined published Japanese patent application), JP-A-10-48215, JP-A-8-269207, JP-A-6-306108, or the like can be used. Immobilization of a fluorescent substance (labeling substance) for those kinds of particles can be suitably performed according to a common method. For example, reference can be made to Japanese JP-T-2005-534907 (“JP-T” means published searched patent publication), JP-A-2010-156642 and JP-A-2010-156640, or the like. 
     An amount of the fluorescent material in the phase of the fluorescent material is not particularly limited in the present invention, but is preferably 0.05 part by mass or more, and more preferably 0.1 part by mass or more when the amount of the transparent material (continuous phase) is taken as 100 parts by mass. An upper limit is preferably 1.0 part by mass or less, and more preferably 0.5 part by mass or less. 
     At the ratio of the phase of the magnetic material to the phase of the transparent material, the fluorescence particles are preferably gathered due to action of the phase of the magnetic material in a predetermined portion for detection. On the other hand, in the phase of the transparent material, the fluorescent material preferably exists in the transparent material at a specific proportion to allow effective measurement of fluorescence. Accordingly, the ratio of the phase of the transparent material to the phase of the fluorescent material, and the ratio of the phase of the magnetic material to the phase of the transparent material are preferably designed so as to satisfy the above-described purposes. 
     Moreover, the ratio of the phase of the magnetic material to the phase of the fluorescent material can be determined from the ratio of the phase of the transparent material to the phase of the fluorescent material, and the ratio of the phase of the magnetic material to the phase of the transparent material. 
     (Thermoresponsive Polymer) 
     The thermoresponsive polymer preferably causes no structural change, even if the polymer is bounded with a molecule having an electric charge. As the thermoresponsive polymer, a polymer having the critical solution temperature (hereinafter, also referred to as CST) is preferable. This critical solution temperature (CST) means a temperature serving as a border at which characteristics or morphologies of the polymer are changed. The thermoresponsive polymer may be a polymer exhibiting a lower critical solution temperature (LCST) or an upper critical solution temperature (UCST). When the thermoresponsive polymer exhibits the lower critical solution temperature (LCST), specific examples include a polymer that causes aggregation when the liquid temperature is increased, and redispersion when the temperature is decreased. Conversely, when the thermoresponsive polymer exhibits the upper critical solution temperature (UCST), specific examples include a polymer that causes aggregation when the liquid temperature is decreased, and redispersion when the temperature is increased. The critical solution temperature (CST) is preferably 10° C. or higher, and more preferably 30° C. or higher. An upper limit is preferably 100° C. or lower, and more preferably 50° C. or lower. 
     Herein, the lower critical solution temperature and the upper critical solution temperature can be determined as described below, for example. First, a sample is placed in a cell of an absorptiometer and heated at a rate of 1° C./min. During heating, a change of transmittance at 550 nm is recorded. Here, the transmittance when the polymer is melted in a transparent state is taken as 100% and the transmittance when the polymer is completely aggregated is taken as 0%. Then, a temperature at which the transmittance becomes 50% is determined as LCST. 
     Specific examples of the polymer having the lower critical solution temperature include polymers composed of N-substituted (meth)acrylamide derivatives such as N-n-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, N-n-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N,N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine and N-methacryloylmorpholine; polyoxyethylene alkylamine derivatives such as hydroxypropylcellulose, a polyvinyl alcohol partially acetylated product, polyvinyl methyl ether, a (polyoxyethylene-polyoxypropylene) block copolymer and polyoxyethylene lauryl amine; polyoxyethylene sorbitan ester derivatives such as polyoxyethylene sorbitan laurate; (polyoxyethylene alkylphenyl ether)(meth)acrylates such as (polyoxyethylene nonylphenyl ether) acrylate and (polyoxyethylene octylphenyl ether)methacrylate; and polyoxyethylene (meth)acrylic acid ester derivatives of (polyoxyethylene alkyl ether)(meth)acrylates, such as (polyoxyethylene lauryl ether)acrylate and (polyoxyethylene oleyl ether)methacrylate. Further, a polymer thereof or a copolymer formed of at least two kinds of monomers thereof can also be used. Moreover, a copolymer of N-isopropylacrylamide and N-t-butyl acrylamide can also be used. When the polymer containing the (meth)acrylamide derivative is used, any other copolymerizable monomer may be copolymerized with this polymer in the range in which the resultant copolymer has the lower critical solution temperature. 
     Among them, a polymer composed of at least one kind of monomers selected from the group consisting of N-n-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, N-n-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N,N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine and N-methacryloylmorpholine, or a copolymer of N-isopropylacrylamide and N-t-butylacrylamide can be preferably used. 
     As the polymer having the upper critical solution temperature, a polymer formed of at least one kind of monomer selected from the group consisting of acroylglycinamide, acroylnipecotamide, acryloylasparaginamide, acryloyl glutaminamide and the like can be used. Moreover, the polymer may be a copolymer formed of at least two kinds of these monomers. With these polymers, any other copolymerizable monomer such as acrylamide, acetylacrylamide, biothionol acrylate, N-bithionyl-N′-methacroyltrimethylene amide, acroylsarcosine amide, methacrylicsarcosine amide and acroylmethyluracil may be copolymerized in the range in which the resultant copolymer has the upper critical solution temperature. 
     As the thermoresponsive polymer, a polymer described in Japanese Patent No. 4518767, for example can be preferably used. 
     (Immobilization of Thermoresponsive Polymer Onto Composite Particle) 
     The thermoresponsive polymer can be immobilized thereonto through polyhydric alcohol or a polyhydric alcohol derivative. This thermoresponsive polymer may be immobilized thereonto by being subjected to graft polymerization with the polyhydric alcohol or the polyhydric alcohol derivative immobilized on the surface of the composite particle, or by binding a functional group in a terminal or a side chain of the polymer with a functional group of the polyhydric alcohol or the polyhydric alcohol derivative. An average particle diameter of the composite particle on which the thermoresponsive polymer is immobilized (thermoresponsive fluorescent particle) becomes larger by a portion of the thermoresponsive polymer in comparison with the above-described composite particle, but a preferred range thereof is substantially similar to the range specified as the composite particle. 
     The polyhydric alcohol preferably has at least two hydroxy groups in a constitutional unit. Specific examples include dextran, polyvinyl alcohol, mannitol and sorbitol. Moreover, a compound that has an epoxy group and forms a polyhydric alcohol structure after ring opening, such as a glycidyl methacrylate polymer, can also be used. As the polyhydric alcohol derivative, polyhydric alcohol into which a reactive functional group or polymerizable group, such as carboxyl, amino, epoxy, thiol, methacrylic or acrylic is introduced by modification can be used. 
     (Linking Material) 
     The linking material is an arbitrary material, and is preferably selected and used appropriately when such a material is necessary for introduction of the binding substance. A kind thereof is not particularly limited, but a material that can be incorporated into the thermoresponsive polymer and has the properties of binding with the binding substance is preferable. In taking into account use of a biological substance in the binding substance, specific examples include biotin, avidin, streptoavidin, a primary antibody and a secondary antibody. 
     As a method of incorporating the linking material into the thermoresponsive polymer, the description in WO 01/09141 or Japanese Patent No. 4518767 can serve as reference. Specifically, biotin or the like is bound with a polymerizable functional group such as a methacrylic group and an acrylic group into an addition-polymerizable monomer, and the monomer is copolymerized with any other monomer to perform incorporation. As an alternative method, such a method can be utilized, in which a monomer having a functional group such as carboxylic acid, an amino group or an epoxy group is copolymerized with any other monomer during polymerization of a polymer to bind an antibody-affinity substance (for example, melon gel, protein A, protein G) with the polymer through this functional group according to an ordinary method. Alternatively, a monomer having a functional group such as carboxylic acid, an amino group or an epoxy group may be copolymerized with any other monomer during polymerization of a polymer to directly bind an antibody (binding substance) against an antigen of a detection object with these functional groups according to an ordinary method. Thus, use of the linking substance can be omitted. 
     (Particle-Side Binding Substance) 
     The particle-side binding substance is an arbitrary material, and only needs to be appropriately selected when necessary according to a kind of target substance. As a biomolecule (binding substance) to be combined or absorbed on the surface of the composite particle, there includes antigens, antibodies, DNAs, RNAs, sugars, polysaccharides, ligands, receptors, proteins, peptides and the like. Here, the term “ligand” means a substance capable of specifically binding to a protein, and examples thereof include substrates capable of binding to enzyme, coenzymes, regulatory factors, hormones, neurotransmitters, and the like. Thus, the ligands include low-molecular weight molecules or ions as well as high-molecular weight substances. The antibody used herein may be any type of immunoglobulin molecules, or immunoglobulin molecule fragments having an antigen binding site, such as Fab. Moreover, the antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably two kinds of monoclonal antibodies having different antigen recognition sites. A technique for binding the binding substance with the linking substance is not particularly limited, and binding can be performed by an ordinary method in this kind of technologies. 
     Preferred materials are shown in Table A below in conformity with an example shown in  FIG. 4 . 
     
       
         
           
               
               
               
             
               
                 TABLE A 
               
               
                   
               
             
            
               
                 Labeling 
                 Magnetic material 
                 Magnetite, Nickel oxide, Ferrite or the like 
               
               
                 particle 
                 Fluorescent 
                 5- (and -6)-carboxytetramethylrhodamine- 
               
               
                   
                 material (dye) 
                 NHS ester 
               
               
                   
                   
                 DY550-NHS ester and the like 
               
               
                   
                 Transparent 
                 Silica, Latex (polystyrene) 
               
               
                   
                 material 
                   
               
               
                   
                 Linking material 
                 Streptavidin 
               
               
                   
                 Binding substance 
                 Biotin-introduced antibody 
               
            
           
           
               
               
            
               
                 Target substance 
                 TSH antigen 
               
            
           
           
               
               
               
            
               
                 Dispersant 
                 Dispersant base 
                 Polyacrylic acid 
               
               
                   
                 Dispersant-side 
                 Antibody 
               
               
                   
                 binding substance 
               
               
                   
               
            
           
         
       
     
     [Dispersant] 
     (Dispersant Base) 
     The dispersant base is a hydrophilic high molecular weight compound, for example, and is preferably a polyanion or a polycation. The polyanion means a substance having a plurality of anion groups, and the polycation means a substance having a plurality of cation groups. Specific examples of the polyanion include nucleic acid such as DNA and RNA. These nucleic acids have properties of the polyanion due to existence of plural pieces of phosphodiester groups along a skeleton of the nucleic acid. Moreover, the polyanion includes polypeptide containing a large number of carboxylic acid functional groups (polypeptide composed of amino acid such as glutamic acid and aspartic acid), polyacrylic acid, polymethacrylic acid, a polymer containing, as a polymerization component, acrylic acid and methacrylic acid, and a polysaccharide such as carboxymethylcellulose, hyaluronic acid and heparin. On the other hand, specific examples of the polycation include polylysine, polyarginine, polyornithine, polyalkylamine, polyethyleneimine and polypropylethyleneimine. In addition, the number of functional groups in the polyanion (carboxyl group) or the polycation (amino group) is preferably 25 or more. 
     (Dispersant-Side Binding Substance) 
     The dispersant-side binding substance only needs to be appropriately selected when necessary according to the kind of target substance. A kind thereof is preferably selected in taking into account introduction into the dispersant base. Specific examples are identical with the substances exemplified as the above-described particle-side binding substance. 
     The method of binding the binding substance with the dispersant base is not particularly limited, and specific examples include a method in which mutual affinity substances (for example, avidin and biotin, glutathione and glutathione S transferase) are bound to both to indirectly bind the dispersant base and the binding substance through these substances. When both are directly bound, both may be bound through a functional group. When such a functional group is used, for example, both can be bound according to maleimide-thiol coupling in the method of Ghosh, et al (Ghosh et al: Bioconjugate Chem., 1, 71-76, 1990). Specific examples include two methods described below. In a first method thereof, while a mercapto group (alias, a sulfhydryl group) is introduced first into a 5′ terminus of nucleic acid, a maleimide group is introduced into an antibody by allowing 6-maleimide hexanoic acid succinimide ester (for example, “EMCS (trade name)” (manufactured by Dojindo Laboratories)) to react with the antibody. Next, two kinds of these substances are bound through the mercapto group and the maleimide group. In a second method, a mercapto group is introduced first into a 5′ terminus of nucleic acid in a manner similar to the first method, and a maleimide group is further introduced into a 5′ terminus of nucleic acid by allowing N,N-1,2-phenylenedimaleimide being a homo bifunctional reagent to react with this mercapto group. Meanwhile, a mercapto group is introduced into an antigen. Next, two kinds of these substances are bound through the mercapto group and the maleimide group. 
     In addition thereto, as a method of introducing nucleic acid into protein, for example, methods described in Nucleic Acids Research, Vol. 15, p. 5275 (1987), and Nucleic Acids Research, Vol. 16, p. 3671 (1988) are known. These technologies can be applied to binding of the antibody with the nucleic acid. 
     [Detection Method] 
     In a preferred detection method utilizing the fluorescent labeling particle of the present embodiment, fluorescent labeling particles  20 , dispersants  30 , and analytes containing target substances S are mixed. Subsequently, presence or absence of dispersion of the thermoresponsive polymers is judged under conditions in which the thermoresponsive polymers are aggregated. At this time, the analytes are irradiated with the excitation light, and the dispersion is detected by the fluorescence emission. Behavior of the fluorescent labeling particles  20 , the dispersant  30 , and the target substance S in this case was previously described based on  FIG. 5 . 
     In the present embodiment, presence or absence of dispersion ( FIGS. 5  ( 1 ),  5  ( 2 )) is judged by irradiation with the excitation light and detection of the fluorescence excited by the irradiation. From a viewpoint of detecting the fluorescence visually or with a general-purpose sensor, the above-described excitation light source preferably emits the excitation light in the following wavelength region. Examples of the excitation light source include a mercury lamp, a halogen lamp, and a xenon lamp. In the present invention, excitation light illuminated from a laser diode or light emitting diode is particularly preferably used. The detection system is preferably equipped with a filter for selectively transmitting light of specific wavelength from the excitation light source. Further, from the viewpoint of detecting only the fluorescence by visual observation or the like, it is more preferably equipped with a filter which is capable of removing the excitation light and transmitting only the fluorescence. In particular, the detection system preferably contains a photomultiplier tube or CCD detector capable of receiving the fluorescence. Accordingly, fluorescence with visually undeterminable intensity or wavelength can be detected, and further quantification of target substances can be made as its fluorescence intensity can be measured, enabling detection and quantification with high sensitivity. 
     The wavelength of the excitation light is preferably 300 nm or more, more preferably 400 nm or more, and particularly preferably 500 nm or more. The upper limit thereof is preferably 700 nm or less, more preferably 600 nm or less, and particularly preferably 550 nm or less. The fluorescence in the following range can be efficiently generated by adjusting the wavelength to the excitation light in this range, and such a case is preferable. 
     The wavelength of the fluorescence is preferably 350 nm or more, more preferably 450 nm or more, and particularly preferably 530 nm or more. The upper limit thereof is preferably 800 nm or less, more preferably 750 nm or less, and particularly preferably 580 nm or less. Detection by visual observation or with a generally used detector is facilitated by adjusting the wavelength in this range, and such a case is preferable. 
     In the following, the above-described fluorescent labeling particles that produce the excellent effects are described in more detail. 
     (Target Substance) 
     As the target substance, a substance used in clinical diagnosis is exemplified, and specific examples include, as contained in body fluid, urine, sputum, feces and the like, human immunoglobulin G, human immunoglobulin M, human immunoglobulin A, human immunoglobulin E, human albumin, human fibrinogen (fibrin and a decomposition product thereof), α-fetoprotein (AFP), C-reactive protein (CRP), myoglobin, a carcinoembryonic antigen, a hepatitis virus antigen, human chorionic gonadotropin (hCG), human placental lactogen (HPL), an HIV virus antigen, allergen, bacteriotoxin, a bacteria antigen, an enzyme, hormone (for example, human thyroid stimulating hormone (TSH), insulin and the like) and a medical agent. 
     EXAMPLES 
     The present invention will be described in more detail based on Examples given below, but the invention is not limited to that. 
     A system shown in  FIG. 5  was reproduced using materials shown in Table A. The labeling particles were particles in which magnetic particles were coated with silica particles as shown in Table A, and an average particle diameter thereof was about 200 nm. The particles were incorporated into a test liquid containing analytes, and the resultant material was heated at about 50° C. for 15 minutes. Then, the resultant material was irradiated with excitation light (550 nm), and a target substance contained in the above-described analytes was detected depending on a state of light emission (580 nm) of the above-described labeling particles. At this time, it was confirmed that, under existence of the analyte, dispersion was maintained because a dispersant was bound with magnetic fluorescent thermoresponsive particles through the target substance, resulting in causing no change in fluorescence intensity in comparison with the intensity before heating. To the contrary, under non-existence of the analyte, the magnetic fluorescent thermoresponsive particles were aggregated by heating and then precipitated, resulting in causing reduction of the fluorescence intensity in composition with the intensity before heating. 
     Having described our invention as related to the present embodiments, it is our intention that the invention not be limited by any of the details of the description, unless otherwise specified, but rather be construed broadly within its spirit and scope as set out in the accompanying claims. 
     REFERENCE SIGNS LIST 
     
         
           1  Magnetic material phase (magnetic material particle, core) 
           2  Transparent material phase (fluorescent silica phase, shell) 
           2   a  Transparent material (continuous phase) 
           2   b  Fluorescent material (fluorescent dye, dispersion phase) 
           3  Thermoresponsive polymer (thermoresponsive polymer phase) 
           4  Linking material 
           5  Particle-side binding substance 
           6  Dispersant-side binding substance 
           7  Dispersant base 
           10  Thermoresponsive fluorescence particle 
           11  Composite particle 
           20  Fluorescent labeling particle (magnetic fluorescent thermoresponsive particle) 
           30  Dispersant 
           100  Linked structure 
         S Target substance 
         M Magnet