JP2015102337A - Monodispersed state maintaining method for particle, and analysis object detecting method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a monodispersed state maintaining method for particles that enables the particles to maintain a monodispersed state longer than by any conventional method, and an analysis object detecting method utilizing particles obtained by the monodispersed state maintaining method.SOLUTION: By a monodispersed state maintaining method for particles, dispersion liquid 10 is formed by dispersing monodispersed particles 14 in a dispersion medium 12; after a supporting body 20 that can support the monodispersed particles 14 is soaked in the liquid, the supporting body 20 is dried to preserve it; and at the time of using the monodispersed particles 14, the preserved supporting body 20 is soaked in a dispersion medium 32 to cause the monodispersed particles 14 supported by the supporting body 20 to elute into the dispersion medium 32.

Description

  The present invention relates to a method for maintaining a monodispersed state of particles and a method for detecting an analyte.

  In recent years, fine particles of about several nm to 1 μm have been applied to various fields and attracted attention. For example, porous silica particles used for adsorbents, catalysts, etc., zeolite particles, carbon black used for pigments, metal oxide particles, inorganic compound particles, metal nanoparticles used for conductive materials, and resin reinforcing agents There are a wide variety of materials and applications for fine particles such as silica particles. The application of such fine particles is expected in various fields. For example, the research is being actively promoted in the fields of biochemistry, new materials, electronic devices, light-emitting display devices, printing, and medicine.

  For example, in the medical field, microparticles are applied to a measurement method using an immune reaction (hereinafter also referred to as “immunological measurement method”) used in clinical examinations. There are many immunological measurement methods such as RIA method, EIA method, immunoturbidimetric method, latex agglutination method, colloidal gold agglutination method, and immunochromatography method. Among them, the latex agglutination method and the colloidal gold agglomeration method are homogeneous systems that do not require separation or washing operation of the reaction solution, and are therefore suitable for measurement automation and measurement in a short time. In particular, gold colloidal particles having a particle size of 5 to 100 nm have a smaller particle size than latex particles usually used in the latex agglutination method, and thus are more suitable for measurement of trace substances.

  As a technique related to the measurement of a trace substance using gold colloid particles, for example, there is one described in Patent Document 1 or Patent Document 2. In Patent Document 1, a substance to be measured and a colloidal gold particle bonded with a substance that can specifically bind to the substance to be measured are reacted in a solution, and the change in absorbance after the start of the reaction is within a range of 610 to 800 nm. Techniques for measuring are described. In Patent Document 2, a specimen and a first reagent containing a hapten-binding protein are mixed, and a second reagent containing an anti-hapten antibody-binding gold colloid is added to and mixed with the mixed solution. A technique is described in which a change in absorbance due to immune reaction aggregation between a hapten-binding protein and an anti-hapten antibody-conjugated gold colloid is measured in the range of 500 to 580 nm.

  When particles dispersed in a liquid (dispersion medium) are bonded to the particle surface, aggregation is likely to occur between the particles due to the influence of the molecules. In particular, nanoscale particles classified as microparticles (so-called nanoparticles) are significantly affected by the particle surface. Aggregated particles may not be suitable for the intended application because the particle size changes. In this regard, there is a method in which aggregated particles are monodispersed by ultrasonic treatment and used as primary particles, as in the method described in Patent Document 3, for example. More specifically, there is a method in which two or more ultrasonic waves having different frequencies are applied to the aggregated nanoparticle liquid to finely disperse the aggregated nanoparticles as in the method described in Patent Document 3.

  Moreover, as a device that does not cause aggregation, there is a method in which a copolymer containing a phosphorylcholine group is attached to the surface of silica nanoparticles, as in the method described in Patent Document 4, for example. More specifically, as in the method described in Patent Document 4, a biomolecule and a copolymer containing a phosphorylcholine group are adsorbed or bound to the surface of silica particles, and the biomolecule is bound to an antigen, antibody, DNA, RNA, sugar, sugar. There is at least one method selected from the group consisting of a chain, a ligand, a receptor, a protein, and a peptide.

JP 2005-283250 A JP 2004-325192 A JP 2006-312130 A JP 2011-252819 A

  However, although the method described in Patent Document 3 can reliably re-aggregate the agglomerated particles, a dedicated device for re-dispersing and a work process related to re-dispersion are separately required. For this reason, the operation becomes complicated and the reproducibility of the particle size distribution may be lowered in the redispersed particles. For this reason, the method described in Patent Document 3 may not be suitable for a test for determining quantitativeness using the immunological measurement method.

Further, in the method described in Patent Document 4, since the particle composition (particle material) is limited to silica, there is a possibility that it cannot cope with various immunological measurement methods.
As described above, the conventional technique has a problem that it is difficult to maintain a monodispersed state of particles for a long period of time.
In view of the above circumstances, the present invention provides a method for maintaining a monodispersed state of particles capable of maintaining the monodispersed state of particles for a long period of time as compared with the prior art, and monodispersed particles obtained by using the method. It is an object of the present invention to provide a method for detecting an analysis object used.

  In one embodiment of the present invention, a dispersion in which monodispersed particles are dispersed in a first dispersion medium is infiltrated into a support capable of supporting the monodispersed particles, and then the support is dried to preserve the support. The stored support is immersed in a second dispersion medium when the monodisperse particles are used, and the monodisperse particles supported on the support are eluted into the second dispersion medium. This is a method for maintaining a monodispersed state of particles.

In the monodispersed state maintaining method, the monodispersed particles may be formed of any one of a simple substance, a compound, and a mixture, and can be dried and solidified.
In the monodispersed state maintaining method, the monodispersed particles may be composed of at least one of metal colloid particles, fluorescent dye-containing particles, and visible dye-containing particles.

In the monodispersed state maintaining method, the metal colloid particles may be composed of at least one of gold colloid particles, silver colloid particles, iron colloid particles, aluminum colloid particles, and platinum colloid particles.
In the monodispersed state maintaining method, the support may be fibrous or porous, and may be formed of an inorganic material or an organic material.

In the monodispersed state maintaining method, the support may be formed of any one of silica gel, glass fiber, quartz fiber, and zeolite.
In the monodispersed state maintaining method, the first dispersion medium may be a buffer containing at least one of a saccharide, a surfactant, and a polymer.
In the monodispersed state maintaining method, the fluorescent dye-containing particles may include a fluorescent dye and a magnetic substance.

  In the monodispersed state maintaining method, the fluorescent dye may be divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, trivalent or tetravalent. Titanium, copper, silver, divalent samarium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, ruthenium compound, tin compound, thallium compound , Bismuth compound, tungstic acid compound, molybdate compound, sulfur compound, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadolinium compound, thulium compound, ytterbium compound, lutetium compound, DY630, DY631, DY633, DY635, DY636, DY650, Y651 (above, manufactured by Dyomics GmbH), Oyster643, Oyster656 (above, made by Denovo Biolabels) 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ', 4 , 4 ', 5', 7,7'-hexachlorofluorescein, 6-carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7' -Dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alex Fluor 568 , Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 530/550 BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (above, manufactured by Invitrogen), methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7 (or more Or a dye containing at least one substance selected from the group of GE Healthcare).

In the monodispersed state maintaining method, the fluorescent dye-containing particles may be embedded or polymerized in at least one of a polymer, a gel, a hydrogel, a glass carrier, and a substrate.
In the above-mentioned monodispersed state maintaining method, the surface of the monodispersed particle is physically combined with at least one of antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, protein, peptide, and nucleic acid molecule, A label having chemical or biological affinity may be provided.

Another aspect of the present invention is a method for detecting an analyte, comprising reacting monodisperse particles obtained using the above-described method for maintaining a monodispersed state of particles with an analyte.
In the detection method, the label provided on the surface of the monodisperse particles is reacted with the analyte having physical, chemical, or biological affinity with the label. Also good.

In the detection method, the analyte may be at least one of an antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, protein, peptide, and nucleic acid molecule.
In the above detection method, the amount of the analyte reacted with the monodisperse particles is determined by enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), cell immunostaining method. , Tissue immunostaining, immunoprecipitation, flow cytometry, fluorescence activated cell sorting (FACS), immunochromatography, quartz crystal microbalance (QCM), surface plasmon resonance (SPR), two-sided polarization It is good also as measuring using 1 or more methods chosen from the group of a wave type | formula interferometry (DPI), an ellipsometry method, an ELISPOT method, and a western blotting method.

In the above detection method, the labeled substance provided on the surface of the monodisperse particle is a nucleic acid molecule, and the analyte to be reacted with the nucleic acid molecule is amplified, transcribed, or labeled with one or more sequences. Measurement may be performed using one or more methods selected from the group consisting of a method in which hybridization, translation, and ligation are performed as an enzymatic reaction, and a pull-down method in detecting transcription factors.
In the detection method, the nucleic acid molecule is at least one of oligonucleotide, DNA corresponding to the target gene, DNA binding to the protein, RNA corresponding to the target gene, mRNA lacking a stop codon, and RNAi double-stranded RNA. It may be one.

  According to one embodiment of the present invention, monodisperse particles can be supported on a support. And when using monodisperse particles, the particle | grains which maintained the monodispersed state can be obtained by eluting the support body in which the monodisperse particles were supported to the 2nd dispersion medium. For this reason, if it is one aspect | mode of this invention, the monodispersed state of particle | grains can be maintained for a long period of time compared with a prior art.

It is a figure which shows typically the process of the monodispersed state maintenance method of the particle | grains which concern on embodiment of this invention. It is a figure which shows typically the process of the detection method of the analysis target which concerns on embodiment of this invention. It is a figure which shows the result of the particle size distribution measurement of the particle | grains when not using the monodispersed state maintenance method of the particle | grains concerning embodiment of this invention. It is a figure which shows the result of having ultrasonically processed the particle | grains at the time of not using the monodispersed state maintenance method of the particle | grains concerning embodiment of this invention, and measuring the particle size distribution. It is a figure which shows the result of the particle size distribution measurement of the particle | grains at the time of using the monodispersed state maintenance method of the particle | grains concerning embodiment of this invention. It is a figure which shows the result of the fluorescence intensity measurement of the measurement using the monodisperse particle before and behind drying.

Hereinafter, a method for maintaining a monodispersed state of particles according to the present embodiment and a method for detecting an analyte using monodispersed particles obtained by maintaining the monodispersed state by the method will be described. First, a method for maintaining a monodispersed state of particles will be described, and then a method for detecting an analyte will be described.
<Method for maintaining monodispersed state of particles>
FIG. 1 is a diagram schematically showing the steps of the method for maintaining a monodispersed state of particles according to the present embodiment. As shown in FIG. 1, the method for maintaining a monodispersed state of particles according to the present embodiment supports a dispersion 10 in which monodispersed particles 14 are dispersed in a dispersion medium (first dispersion medium) 12 and supports monodispersed particles 14. A step of applying and infiltrating to the possible support 20 (infiltration step), a step of drying the support 20 to remove the water of the dispersion medium 12 infiltrated into the support 20 (drying step), and monodisperse particles The step of immersing the support 20 that has been dried and stored during use in the dispersion medium (second dispersion medium) 32 to elute the monodisperse particles 14 supported by the support 20 into the dispersion medium 32. (Elution step). Details of these steps will be described below. In the present embodiment, “monodisperse particles” mean particles having a uniform size (particle diameter) and good dispersibility.

(Infiltration process)
In the particle monodispersed state maintaining method according to the present embodiment, first, as shown in FIG. 1A, the dispersion 10 in which the monodispersed particles 14 are dispersed in the dispersion medium 12 can be supported by the monodispersed particles 14. It is applied to the support 20 and infiltrated. After the dispersion 10 is infiltrated, the support 20 is left on a sponge-like base. FIG. 1B shows a state in which the dispersion 10 is infiltrated into the entire support 20.
The support 20 may be any support that can support the monodisperse particles 14. For example, the support 20 is fibrous or porous, and is formed of an inorganic material or an organic material. Hereinafter, a specific example will be described.

  Examples of the fibrous material (fiber material) used for the support 20 include synthetic fibers (polyester fiber, polyethylene terephthalate fiber, polyamide fiber, polycarbonate fiber, polyimide fiber, polyether ether ketone fiber, polyether sulfone fiber, polyphenylene). Sulfide fiber, polyacetal fiber, phenol resin fiber, polyarylate fiber, liquid crystal polymer fiber, etc., semi-synthetic fiber (cellulose ester fiber such as cellulose acetate), natural fiber (cellulose fiber, cotton, hemp, rock wool, wool fiber, etc.) ) And inorganic fibers (carbon fiber, silicon carbide fiber, glass fiber, quartz fiber, silica-alumina fiber, etc.). In addition, you may use these materials individually or in combination of 2 or more types. In addition, among the above materials, in order to further increase the elution efficiency of the monodisperse particles 14, it is more preferable to form the support 20 with glass fibers or quartz fibers.

  Examples of the porous material (porous material) used for the support 20 include kaolin, calcined clay, aluminum hydroxide, magnesium hydroxide, light calcium carbonate, heavy calcium carbonate, barium sulfate, alumina, titanium oxide, Examples include talc, mica powder, and silica. In addition, about the material of the support body 20, if it is a well-known thing, it will not be limited to these examples. Among the above materials, in order to further increase the elution efficiency of the monodisperse particles 14, it is preferable to form the support 20 with silica gel or zeolite.

The shape of the support 20 is not particularly limited, and is, for example, a pellet shape, a granular shape, a honeycomb shape, or a flat plate shape. Among these shapes, a flat plate shape that can uniformly dry the support 20 is most preferable.
The dispersion 10 infiltrated into the support 20 includes monodispersed particles 14 and a dispersion medium 12 that disperses the monodispersed particles 14 as shown in FIG.

  The monodisperse particles 14 are formed of any one of a simple substance, a compound, and a mixture, and can be dried and solidified. More specifically, the monodisperse particles 14 are composed of at least one of metal colloid particles, fluorescent dye-containing particles, and visible dye-containing particles. The metal colloid particles described above are composed of at least one of gold colloid particles, silver colloid particles, iron colloid particles, aluminum colloid particles, and platinum colloid particles.

  The above-mentioned fluorescent dye-containing particles contain the fluorescent dye and the magnetic substance. The fluorescent dyes encapsulated in the fluorescent dye-containing particles are divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, trivalent or tetravalent titanium. , Copper, silver, divalent samarium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, ruthenium compound, tin compound, thallium compound, Bismuth compound, tungstic acid compound, molybdate compound, sulfur compound, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadolinium compound, thulium compound, ytterbium compound, lutetium compound, DY630, DY631, DY633, DY635, DY636 , DY650, DY651 Above, manufactured by Dynamics GmbH), Oyster 643, Oyster 656 (manufactured by Denovo Biolabels) 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ', 4,4 ', 5', 7,7'-hexachlorofluorescein, 6-carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxy Fluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 568, Alexa Fluor 568 Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY5 Y 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (above, manufactured by Invitrogen), methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7 (or more A pigment containing at least one substance selected from the group of GE Healthcare. The fluorescent dye-containing particles are embedded or polymerized in at least one of a polymer, a gel, a hydrogel, a glass carrier, and a substrate.

The surface of the monodisperse particle 14 has physical, chemical, or biological affinity with at least one of antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, protein, peptide, and nucleic acid molecule. A sign 16 having the following is provided.
The particle diameter of the monodisperse particles 14 is preferably in the range of 10 nm to 1,000 nm.

In addition to the monodispersed particles described above, the monodispersed particles 14 may be, for example, non-pigmented particles or colored particles. Further, the material of the monodisperse particles 14 may be, for example, silica particles, latex particles, semiconductor nanoparticles, gold particles, metal nanoparticles, inorganic crystal particles, and the like other than the materials described above.
The dispersion medium 12 contains at least one of a saccharide, a surfactant, and a polymer as a buffer. By including these buffering agents in the dispersion medium 12, aggregation of the monodispersed particles 14 is prevented when the support 20 is dried, and at the same time, when the support 20 is immersed in the dispersion medium 32, the dispersion 20 is monodispersed. The particles 14 can be easily eluted.

  Examples of the saccharide contained in the dispersion medium 12 include trehalose, glucose, fructose, galactose, xylose, mannose, maltose, sucrose, lactose, maltosyl trehalose, dextrin, starch, and pullulan. In addition, saccharides include monosaccharides, oligosaccharides, and polysaccharides. Monosaccharides and oligosaccharides are particularly preferable as the saccharides according to this embodiment. Moreover, it is desirable that the final concentration of saccharides in the dispersion 10 is in the range of 0.1% (w / v) to 5% (w / v).

  The polymer contained in the dispersion medium 12 may be hydrophilic or amphiphilic, for example, polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyethyleneimine, polyacrylic acid, polymethacrylic acid, poly-1 1,3-dioxolane, 2-methacryloyloxyethyl phosphorylcholine polymer, poly-1,3,6-trioxane, polyamino acid, protein, particularly polyethylene glycol are preferred. The molecular weight of polyethylene glycol is preferably in the range of 300 or more and 20,000 or less. The final concentration of the polymer in the dispersion 10 is desirably in the range of 0.01% to 0.25%.

  Examples of the surfactant contained in the dispersion medium 12 include sodium cholate, sodium deoxycholate, sodium lauryl sulfate, sucrose monocholate, β-D-fructopyranosyl-α-D-glucopyranoside monododecanoate, β-D-fructopyranosyl-α-D-glucopyranoside monodecanoate, n-octanoyl-N-methylglucamide, n-nonanoyl-N-methylglucamide, n-decanoyl-N-methylglucamide, n -Octyl-β-D-thioglucopyranoside, n-octyl-β-D-maltopyranoside, n-octyl-β-D-glucopyranoside, n-nonyl-β-D-thiomaltopyranoside, n-dodecyl-β- D-maltopyranoside, n-decyl-β-D-maltopyranoside, N, N-bis (3-D-glu N-amidopropyl) colamide, N, N-bis (3-D-gluconamidopropyl) deoxycolamide, 3-[(3-colamidopropyl) dimethylammonio] propanesulfonic acid, 3-[(3-colamide Propyl) dimethylammonio] -2-hydroxypropanesulfonic acid and the like. These surfactants may be used alone or in combination of two or more.

  Examples of a method for applying and infiltrating the dispersion 10 to the support 20 include, for example, an impregnation method, a spray method, a Mayer bar method, a gravure method, a micro gravure method, a die method, a blade method, a micro rod method, and an air knife method. And publicly known coating methods such as a curtain method, a slide method, and a roll method.

(Drying process)
After the above infiltration process, the support 20 infiltrated with the dispersion 10 is dried. More specifically, as shown in FIG. 1C, the water of the dispersion medium 12 infiltrated into the support 20 is removed (evaporated) from the support 20. As a method for drying the support 20, a known technique can be used. More specifically, the stage on which the support body 20 infiltrated with the dispersion 10 is placed is left in a vacuum desiccator equipped with a desiccant, and the inside of the vacuum desiccator is depressurized with an oil rotary vacuum pump. The body 20 is dried under reduced pressure. At this time, if a sponge-like platform is used instead of a flat plate made of plastic or glass as a platform on which the support 20 is placed, the air permeability of the surrounding environment of the infiltrated support 20 is increased and drying is performed more uniformly. . By doing so, the particles can be present (maintained) in a monodispersed state inside the support 20 that has been dried under reduced pressure. In other words, the monodisperse particles 14 can be supported on the support 20 without aggregating.
As described above, the monodisperse particles 14 are stored.

(Elution process)
After the above-described drying step, the support 20 containing the monodisperse particles 14 is immersed in the dispersion medium 32 when the monodisperse particles 14 are used. More specifically, as shown in FIG. 1 (d), when using the monodisperse particles 14, the dried support 20 is immersed in the dispersion medium 32 and stirred to be supported by the support 20. The monodispersed particles 14 are eluted in the dispersion medium 32. Finally, as shown in FIG. 1 (e), the support 20 is taken out from the dispersion medium 32. In this way, a dispersion 30 in which the monodisperse particles 14 are dispersed in the dispersion medium 32 is obtained.
The dispersion medium 12 and the dispersion medium 32 may be the same type of dispersion medium or different types of dispersion medium.
Through the above steps, the method for maintaining a monodispersed state of particles according to this embodiment is completed.

(Example of use)
A reagent or sample using the monodispersed particles 14 obtained by using the above-described method for maintaining a monodispersed state of particles is manufactured. The reagent or sample thus produced is used for, for example, chemical analysis, experiment, test research, examination, and treatment.
(effect)
(1) As described above, in the above monodispersed state maintaining method, the dispersion 10 in which the monodisperse particles 14 are dispersed in the dispersion medium 12 is infiltrated into the support 20 that can support the monodisperse particles 14 and then the support. 20 is dried and the support 20 is stored. When the monodisperse particles 14 are used, the stored support 20 is immersed in the dispersion medium 32, and the monodisperse particles 14 supported by the support 20 are used as the dispersion medium 32. It is eluted.

  With this configuration, since the monodispersed particles 14 are supported by the support 20, the monodispersed state can be maintained without agglomerating the monodispersed particles 14. Then, when using the monodisperse particles 14, the monodisperse particles 14 can be obtained by eluting the monodisperse particles 14 from the support 20 into the dispersion medium 32. For this reason, if it is this embodiment, the monodispersed state of particle | grains can be maintained for a long period of time compared with a prior art.

  Furthermore, according to the present embodiment, the form can be used immediately and the surface activity of the monodisperse particles 14 can be maintained, so that the monodisperse particles 14 can be used in various measurements using the particles. Further, according to the present embodiment, measurement errors due to particle aggregation can be prevented, so that measurement with improved stability and reproducibility is possible. In addition, according to the present embodiment, distributed processing is not necessary, so that the operation can be simplified, the work time can be shortened, and the work efficiency can be improved.

(2) In the monodispersed state maintaining method, the monodispersed particles 14 are formed of any one of a simple substance, a compound, and a mixture, and can be dried and solidified.
With this configuration, since the physical strength and chemical strength of the monodisperse particles 14 are increased, the characteristics of the monodisperse particles 14 are not easily changed even if the support 20 infiltrated with the dispersion 10 is sufficiently dried. For this reason, the maintenance period of the monodisperse particles 14 that can be used for analysis or the like can be made longer.

(3) In the monodispersed state maintaining method, the monodispersed particles 14 are composed of at least one of metal colloid particles, fluorescent dye-containing particles, and visible dye-containing particles.
With this configuration, since the monodisperse particles 14 can be optically identified, the analyte can be easily detected when the analyte is detected using the monodisperse particles 14.

(4) In the monodispersed state maintaining method, the metal colloid particles are composed of at least one of gold colloid particles, silver colloid particles, iron colloid particles, aluminum colloid particles, and platinum colloid particles.
If it is this structure, since the acquisition of the substance used as the material of the monodisperse particle 14 becomes easy, the manufacturing cost of the monodisperse particle 14 can be reduced.

(5) In the monodispersed state maintaining method, the support 20 is made of an inorganic material or an organic material while being fibrous or porous.
With this configuration, since there is an appropriate space inside the support 20, the monodisperse particles 14 can be efficiently dispersed in the support 20.
(6) In the monodispersed state maintaining method, the support 20 is formed of any one of silica gel, glass fiber, quartz fiber, and zeolite.
If it is this structure, since the acquisition of the substance used as the material of the support body 20 will become easy, the manufacturing cost of the support body 20 can be reduced.

(7) In the monodispersed state maintaining method, the dispersion medium 12 contains at least one of saccharides, a surfactant, and a polymer.
With this configuration, the monodisperse particles 14 dispersed in the support 20 can be efficiently eluted into the dispersion medium 32.
(8) Moreover, in the monodispersed state maintaining method, the fluorescent dye-encapsulated particles encapsulate the fluorescent dye and the magnetic substance with an organic polymer.
With this configuration, the fluorescence intensity of the fluorescent dye-encapsulated particles can be increased. Therefore, when the analyte is detected using the fluorescent dye-encapsulated particles as the monodisperse particles 14, the analysis object can be easily detected. .

(9) In the above monodispersed state maintaining method, the fluorescent dye included in the fluorescent dye-containing particles is divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent. Valent iron, trivalent or tetravalent titanium, copper, silver, divalent samarium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl Compound, ruthenium compound, tin compound, thallium compound, bismuth compound, tungstic acid compound, molybdate compound, sulfur compound, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadolinium compound, thulium compound, ytterbium compound, lutetium Compound, DY630, DY631, DY633, DY635 DY636, DY650, DY651 (manufactured by Dynamics GmbH), Oyster643, Oyster656 (manufactured by Denovo Biolabels) 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy- 2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein, 6-carboxy-2', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4 ', 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluo 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 555, Alexa Fluor 555, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 546 Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 493/503, BODIPY 493/503 DIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (above, manufactured by Invitrogen), methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7 (or more A pigment containing at least one substance selected from the group of GE Healthcare.
With this configuration, the fluorescence intensity of the fluorescent dye-encapsulated particles can be further increased. Therefore, when the analyte is detected using the fluorescent dye-encapsulated particles as the monodisperse particles 14, the analyte can be detected more easily. It becomes.

(10) In the monodispersed state maintaining method, the fluorescent dye-containing particles are embedded or polymerized in at least one of a polymer, a gel, a hydrogel, a glass carrier, and a substrate.
With this configuration, the physical strength and chemical strength of the fluorescent dye-encapsulated particles can be increased. Therefore, when the analyte is detected using the fluorescent dye-encapsulated particles as the monodisperse particles 14, the fluorescent dye-encapsulated particles are used. Is easy to handle.

(11) In the above-described monodispersed state maintaining method, the surface of the monodispersed particle 14 has at least one of antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, protein, peptide, and nucleic acid molecule. A label having physical, chemical or biological affinity is provided.
With this configuration, since the label 16 is provided on the surface of the monodisperse particles 14, when the analyte is detected using the monodisperse particles 14, the number of types of objects that can be analyzed is increased. it can.

<Detection method of the analyte>
FIG. 2 is a diagram schematically showing the steps of the method for detecting an analyte according to the embodiment of the present invention. As shown in FIG. 2, the method for detecting an analyte according to the present embodiment is performed by reacting monodisperse particles obtained using the above-described monodispersed state maintaining method for particles with the analyte. This is a method for detecting an object. Hereinafter, an example of a method for detecting an analyte according to an embodiment of the present invention will be briefly described with reference to FIG.

FIG. 2A is a diagram illustrating a state where the marker 42 is fixed to the plate 40.
First, as shown in FIG. 2B, the labeled object 42 and the analysis object 50 are reacted. The analyte 50 has physical, chemical, or biological affinity with the label 42.
Next, as shown in FIG. 2 (c), the label 16 provided on the surface of the monodisperse particle 14 is reacted with the analysis target 50. The monodispersed particles 14 are monodispersed particles 14 obtained by the above-described method for maintaining a monodispersed state of particles. Thus, the analyte 50 is indirectly detected by detecting the monodisperse particles 14 fixed to the plate 40. The label 16 has physical, chemical, or biological affinity with the analyte 50.

  The analyte 50 according to this embodiment is, for example, at least one of an antigen, an antibody, DNA, RNA, a sugar, a sugar chain, a ligand, a receptor, a protein, a peptide, and a nucleic acid molecule. The nucleic acid molecule is at least one of oligonucleotide, DNA corresponding to the target gene, DNA binding to the protein, RNA corresponding to the target gene, mRNA lacking a stop codon, and RNAi double-stranded RNA.

In addition to the above, the analyte 50 can be selected from lipids, carbohydrates, and proteins, and is particularly preferably selected from metabolites, hormones, viruses, microorganisms, and cells.
The amount of the analyte 50 that has reacted with the monodisperse particles 14 is, for example, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay (FIA), a cell immunostaining method, or a tissue immunostaining method. , Immunoprecipitation, flow cytometry, fluorescence labeled cell sorting (FACS), immunochromatography, quartz crystal microbalance (QCM), surface plasmon resonance (SPR), two-plane polarization interferometry ( It can be measured using one or more methods selected from the group of DPI), ellipsometry, ELISPOT, and Western blotting. In addition, as a biological sample used for the said measurement, a liquid thing is used. For example, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostate fluid, cowper's fluid or ejaculation Prefluid, sweat, feces, hair, tears, cystic fluid, pleural and ascites, pericardial fluid, lymph, sputum, milk, bile, interstitial fluid, menstrual discharge, pus, sebum, vomiting, vaginal discharge It is particularly preferred to use secretions from mucous membranes, stool, pancreatic juice, lavage fluid, bronchopulmonary aspirate, blastocyst fluid, or umbilical cord blood.

(Example of use)
The above-described method for detecting an analyte is used for, for example, chemical analysis, experiment, test research, examination, and treatment.
(effect)
(1) As described above, in the detection method, the monodisperse particles obtained by using the above-described monodispersed state maintaining method for particles are reacted with the analysis object.
With this configuration, it is possible to easily and quickly detect an analyte with improved stability and reproducibility while using monodispersed particles stored for a long period of time.

(2) Further, in the case of the above detection method, the label 16 provided on the surface of the monodisperse particle 14 and the analyte 50 having physical, chemical, or biological affinity with the label 16 Is reacting.
With this configuration, the analysis object 50 can be specifically measured.
(3) In the above detection method, the analyte 50 is at least one of an antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, protein, peptide, and nucleic acid molecule.
With this configuration, a wide variety of substances can be analyzed.

(4) In the above detection method, the amount of the analyte 50 that has reacted with the monodisperse particles 14 is determined using enzyme immunoassay (EIA), radioimmunoassay (RIA), or fluorescence immunoassay (FIA). , Cell immunostaining, tissue immunostaining, immunoprecipitation, flow cytometry, fluorescence activated cell sorting (FACS), immunochromatography, quartz crystal microbalance (QCM), surface plasmon resonance (SPR) ), Two-plane polarization interferometry (DPI), ellipsometry, ELISPOT, and western blotting.
If it is this structure, since a general purpose measuring method can be used, the cost concerning the detection of the analysis target object 50 can be reduced.

(5) In the above detection method, the label 16 provided on the surface of the monodisperse particle 14 is used as a nucleic acid molecule, and the analyte 50 reacted with the nucleic acid molecule is amplified, transcribed, or labeled. Measurement is performed using one or more methods selected from the group consisting of a method in which hybridization, translation, and ligation with the probe nucleic acid thus performed are performed as an enzyme reaction, and a pull-down method in transcription factor detection.
This configuration can be used not only for detection and measurement of the analyte 50 but also for multi-step processes such as gene expression analysis and transcription factor recovery work.
Hereinafter, a method for maintaining a monodispersed state of particles according to the present invention and a method for detecting an analyte using monodispersed particles obtained by using the method will be described in more detail based on examples.

Example 1
(1) Monodispersed particles In this example, particles having a fluorescent dye compound-containing magnetic material disclosed in International Publication No. 2010-029739 pamphlet (hereinafter also simply referred to as “fluorescent dye-containing particles”) are monodispersed. Used as particles 14. The monodisperse particles 14 have a polymer layer having an affinity for a fluorescent substance and a magnetic material disposed in the polymer layer. Examples of the polymer layer include polystyrene, polyglycidyl methacrylate (polyGMA), and copolymers thereof. By using such a polymer layer, for example, it can be bonded to other substances through functional groups (particle surface functional groups) such as an epoxy group of poly-GMA, so that a physiologically active substance is selectively bonded to the polymer layer. It becomes possible.

  In this example, a particle whose surface functional group is a carboxyl group is prepared, and an antibody, which is a protein, is produced using N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. Bonded to the surface. More specifically, mouse monoclonal antibody anti-PSA antibody 5A6 (trade name, HyTest) was used. Further, antibody-labeled particles were prepared by adding 100 μg of the antibody to the reaction solution containing 1 mg of the above particles and reacting and binding them. Thus, antibody-labeled particles were produced as the monodisperse particles 14 according to this example. The monodispersed particles 14 had a particle diameter (diameter) of about 200 nm.

(2) Method for producing support holding dry particles First, 20 mM Tris-HCl (pH 8.2), 0.05% PEG (molecular weight: 20,000), 3.5 in a 1.5 ml Eppendorf tube. The dispersion medium 12 containing% sucrose was mixed with the monodispersed particles 14 prepared in (1) above, and sonicated at 28 kHz for 30 seconds. Thus, a dispersion 10 in which the above particles were monodispersed was obtained.

Next, a total of 900 μl of the dispersion 10 was dropped into a 10 mm × 150 mm glass fiber pad (glass fiber pad, Millipore) as the support 20 to infiltrate.
Finally, the glass fiber pad infiltrated with the dispersion 10 was dried for 18 hours in an environment reduced in pressure using a vacuum vacuum pump. In this manner, a dry particle holding support (a support on which dry particles were held) according to this example was produced.

(3) Confirmation of Particle Aggregation Dispersion 10 obtained in (2) above was placed in a 1.5 ml Eppendorf tube and stored in a 4 ° C. environment. The particle size of particles that had passed for 28 days after standing was measured with a particle size distribution analyzer (SALD-2100, manufactured by Shimadzu Corporation). The particle size distribution measured in this way is shown in FIG. Moreover, the particle size distribution of the particle | grains measured by ultrasonically treating the particle | grains which passed for 28 days is shown in FIG. 3 and 4, the horizontal axis represents the particle diameter (μm), and the vertical axis represents the frequency (%).

(4) Elution of particles from the support on which the dry particles are held and confirmation of the particle size of the eluted particles From the dry particle holding support stored for 28 days, in order to grasp the particle size of the particles held dry on the support 20 The particles were eluted and the particle size was measured. More specifically, after storing the dried particle holding support prepared in (2) above for 28 days, 8 pieces obtained by cutting the support into 10 mm × 5 mm were placed in a 1.5 ml Eppendorf tube. It was immersed in 5 ml of ultrapure water and then stirred for 30 seconds with a vortex mixer. The particle diameter of the thus eluted particles in the eluate was measured with a particle size distribution analyzer (SALD-2100, manufactured by Shimadzu Corporation). The measured particle size distribution is shown in FIG. In FIG. 5, the horizontal axis represents the particle diameter (μm), and the vertical axis represents the frequency (%).
In addition, the measurement range of the above-mentioned particle size distribution measurement was limited to a range of 30 nm to 1000 μm in terms of measurement performance.

(5) Comparison of particle diameters When the measurement results of (3) and (4) above are compared, the particles stored in the liquid have about 40% of particles having a diameter of 300 to 450 nm as shown in FIG. However, the remaining 60% are particles of 1 μm or more, and it is considered that various particle sizes are mixed. FIG. 4 shows the result of measuring the particle size by sonicating the particles stored in the liquid. In consideration of the fact that the particle size is distributed within the range of 160 to 365 nm and is essentially 200 nm, the particle size distribution of 160 to 365 nm shown in FIG. This is considered to be caused by the particle size distribution of the particle having a diameter of 200 nm. As shown in FIG. 5, the particles eluted from the support 20 on which the dried particles are held have a particle size distribution within a range of 200 to 500 nm, and are maintained in a monodispersed state from storage in liquid. it is conceivable that. From the above, it has been found that the use of the support 20 is effective for preserving the monodisperse particles 14.

(Example 2)
The reactivity of the antibody (labeled substance 16) bound on the surface of the fluorescent dye-encapsulated particles as the monodisperse particles 14 eluted from the support 20 and the antigen (analytical object 50) was confirmed. Fluorescent dye-encapsulated particles eluted from the dry particle holding support (hereinafter also referred to as “post-drying particles”) and fluorescent dye-encapsulated particles stored in the dispersion medium 12 (hereinafter also referred to as “pre-drying particles”). The reaction efficiency of the fluorescent dye-encapsulated particles was compared with the antigen having affinity with the antibody. The measurement is performed by immobilizing an antibody on a 96-well plate (hereinafter also simply referred to as “plate”), adding and reacting an antigen with a known concentration, and then binding antibody-bound particles (fluorescent dye-containing particles with antibodies on the surface). Added and reacted. The reactivity of the antibody on the fluorescent dye-encapsulated particles and the antigen was measured by detecting the fluorescent dye of the fluorescent dye-encapsulated particles captured on the plate. Details are as follows.

  First, 1 μg / ml of anti-PSA antibody 1H12 (HyTest), a mouse monoclonal antibody that recognizes an epitope different from the anti-PSA antibody (5A6) bound to the particle surface, was added to 96 well Black Plate Maxisoap (trade name, Nunc) 1 well. 50 μl was added and solidified for 18 hours in a 4 ° C. environment. Next, the above-described antibody-immobilized plate was washed with a surfactant-containing solution and blocked with a Skim Milk solution at room temperature. After blocking, it was washed with a solution containing a surfactant. 50 μl of PSA solution prepared at an antigen concentration of 0, 0.16, 0.8, 4, 20 ng / ml was added per well, and reacted for 1 hour in an environment at 25 ° C. Next, the plate was washed with a surfactant-containing solution, and 50 μl of a dispersion liquid having a concentration of 1 μg / ml containing pre-drying particles and post-drying particles was added to each well, and each was in an environment of 25 ° C. For 1 hour.

  After the reaction, the plate was washed with a surfactant-containing solution, and the fluorescence intensity of the particles before drying and the particles after drying was measured with a fluorescence measurement reader Envision (PerkinElmer). The measurement results are shown in FIG. In addition, the particle | grains before drying used what carried out the ultrasonic treatment to the particle | grains preserve | saved in the liquid, and carried out the monodispersion. In addition, after drying (elution particles), 8 pieces obtained by cutting a support on which dry particles were supported into 10 mm × 5 mm were placed in 0.5 ml of ultrapure water placed in a 1.5 ml Eppendorf tube. It was obtained by dipping and stirring for 30 seconds with a vortex mixer.

As shown in FIG. 6, the fluorescence intensity with respect to each antigen concentration was substantially the same between the particles before drying and the particles after drying. From this result, the particles eluted from the support on which the dry particles are supported (post-dry particles) have the same level of antigen recognition ability as the particles stored in the liquid (pre-dry particles), and the same level. It was confirmed to have antigen concentration dependency.
In addition, the above-mentioned Example is an example of this invention, This invention is not limited to these Examples at all.

DESCRIPTION OF SYMBOLS 10 Dispersion liquid 12 Dispersion medium 14 Monodisperse particle 16 Labeled object 20 Support body 30 Dispersion liquid 32 Dispersion medium 40 Plate 42 Labeled object 50 Analyte

Claims (17)

  A dispersion in which monodisperse particles are dispersed in a first dispersion medium is infiltrated into a support that can support the monodisperse particles, and then the support is dried to store the support, Maintaining a monodispersed state of particles, wherein the preserved support is immersed in a second dispersion medium during use, and the monodispersed particles supported on the support are eluted into the second dispersion medium. Method.   The method for maintaining a monodispersed state of particles according to claim 1, wherein the monodispersed particles are formed of any one of a simple substance, a compound, and a mixture, and can be dried and solidified. .   The method for maintaining a monodispersed state of particles according to claim 1 or 2, wherein the monodispersed particles are composed of at least one of metal colloid particles, fluorescent dye-containing particles, and visible dye-containing particles. .   4. The monodispersed state of particles according to claim 3, wherein the metal colloid particles are composed of at least one of gold colloid particles, silver colloid particles, iron colloid particles, aluminum colloid particles, and platinum colloid particles. Maintenance method.   5. The monodisperse particles according to claim 1, wherein the support is fibrous or porous and is formed of an inorganic material or an organic material. State maintenance method.   The said support body is formed with any one of a silica gel, glass fiber, quartz fiber, and a zeolite, The monodispersed state maintenance of the particle | grains as described in any one of Claims 1-5 characterized by the above-mentioned. Method.   The method for maintaining a monodispersed state of particles according to any one of claims 1 to 6, wherein the first dispersion medium contains at least one of a saccharide, a surfactant, and a polymer. .   The method for maintaining a monodispersed state of particles according to claim 3, wherein the fluorescent dye-containing particles include a fluorescent dye and a magnetic substance.   The fluorescent dye is divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, trivalent or tetravalent titanium, copper, silver, divalent samarium. , Divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, ruthenium compound, tin compound, thallium compound, bismuth compound, tungstate compound, molybdic acid Compound, sulfur compound, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadolinium compound, thulium compound, ytterbium compound, lutetium compound, DY630, DY631, DY633, DY635, DY636, DY650, DY651 (above, Domicics G H), Oyster 643, Oyster 656 (above, Denovo Biolabels) 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein, 6-carboxy-2', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein, naphtho Fluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluo 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 594, Alexa Fluor 568, Alex Fluor 568 Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 56 / OD4 PY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (above, manufactured by Invitrogen), methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7 (above, GE Healthcare) The method for maintaining a monodispersed state of particles according to claim 8, wherein the pigment contains at least one substance selected from the group of (manufactured).   10. The fluorescent dye-containing particle is embedded or polymerized in at least one of a polymer, a gel, a hydrogel, a glass carrier, and a substrate, according to any one of claims 3, 8, and 9. A method for maintaining a monodispersed state of the described particles.   The surface of the monodisperse particle has physical, chemical, or biological affinity with at least one of antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, protein, peptide, and nucleic acid molecule. The method for maintaining a monodispersed state of particles according to any one of claims 1 to 10, wherein a labeling substance having a particle size is provided.   A method for detecting an analyte, comprising reacting monodispersed particles obtained by using the method for maintaining a monodispersed state of particles according to any one of claims 1 to 11 and the analyte. .   13. The labeling object provided on the surface of the monodisperse particles, and the labeling substance and the analyte having physical, chemical, or biological affinity are reacted with each other. The detection method of the analysis target object of description.   14. The analysis object according to claim 12, wherein the analyte is at least one of an antigen, an antibody, DNA, RNA, a sugar, a sugar chain, a ligand, a receptor, a protein, a peptide, and a nucleic acid molecule. Method for detecting an analyte in   The amount of the analyte reacted with the monodisperse particles is determined by enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), cell immunostaining, tissue immunostaining, Sedimentation method, flow cytometry method, fluorescence-labeled cell sorting method (FACS), immunochromatography method, quartz crystal microbalance method (QCM), surface plasmon resonance method (SPR), two-plane polarization interference method (DPI) The measurement of the analyte according to any one of claims 12 to 14, wherein the measurement is performed using one or more methods selected from the group of ellipsometry, ELISPOT, and Western blotting. Detection method.   The labeled substance provided on the surface of the monodisperse particles is a nucleic acid molecule, and the analyte reacted with the nucleic acid molecule is amplified, transcribed, hybridized with a labeled probe nucleic acid, translated, The method for detecting an analyte according to claim 13, wherein the measurement is performed using one or more methods selected from the group consisting of a method in which ligation is carried out as an enzyme reaction and a pull-down method in detecting transcription factors.   The nucleic acid molecule is at least one of an oligonucleotide, DNA corresponding to a target gene, DNA binding to a protein, RNA corresponding to a target gene, mRNA lacking a stop codon, and double-stranded RNA for RNAi, The method for detecting an analysis object according to claim 16.

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