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WO2006070582A1 - Procede de preparation d’une sphere de silice contenant une molecule marquee - Google Patents

Procede de preparation d’une sphere de silice contenant une molecule marquee Download PDF

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Publication number
WO2006070582A1
WO2006070582A1 PCT/JP2005/022628 JP2005022628W WO2006070582A1 WO 2006070582 A1 WO2006070582 A1 WO 2006070582A1 JP 2005022628 W JP2005022628 W JP 2005022628W WO 2006070582 A1 WO2006070582 A1 WO 2006070582A1
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Prior art keywords
silica
labeled molecule
labeled
molecule
sphere
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English (en)
Japanese (ja)
Inventor
Hirokazu Miyoshi
Michihiro Nakamura
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Techno Network Shikoku Co Ltd
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Techno Network Shikoku Co Ltd
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Priority to JP2006550652A priority Critical patent/JP4982687B2/ja
Publication of WO2006070582A1 publication Critical patent/WO2006070582A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica

Definitions

  • the present invention relates to a method for producing a labeled molecule-containing silica sphere useful as a detection reagent. More specifically, the present invention relates to a method for preparing a labeled molecule-containing silica sphere capable of stably and efficiently fixing a labeled molecule to a silica sphere under mild conditions. Furthermore, the present invention relates to a labeled molecule-containing silica sphere obtained by a powerful method.
  • Non-Patent Document 1 Patent documents 1 to 7
  • This is made using latex donor beads (registered trademark) and axector bead (registered trademark) made of latex with a diameter of 250 mm.
  • the donor beads and axe beads are bonded, the donor beads are excited with a laser.
  • the singlet oxygen molecule is generated by the fluorescence from the internal fluorescent molecule, and this chemically reacts with the fluorescent substance in the first sector bead to produce chemiluminescence, and this is observed.
  • silica spheres as the above beads (fine particles) have been studied, and various methods for producing silica spheres containing fluorescent dye molecules have been proposed.
  • Such silica spheres have a form in which fluorescent dye molecules held inside are surrounded by silica, and as a result, quenching by external factors (for example, absorption of excitation energy by biochemical polymers) can be suppressed. Therefore, it is expected to be applied to various tests as a highly sensitive detection reagent.
  • Non-patent Document 2 Concentration of fluorescent dye molecules (FITC) is retained inside the silica sphere On the other hand, it has also been reported that quenching occurs inside the silica sphere at its maximum concentration (Non-patent Document 2). In addition, as a result of investigations by the present inventors, the above-described method has a too high bondability between FITC and APS, resulting in poor production efficiency, and the resulting silica sphere has a single particle size and several forces. Compared to a hundred nanometers, it was powerful. Patent Document 1: US 4918200 A
  • Patent Document 2 WO 917087 A1
  • Patent Document 3 EP 502060 A1
  • Patent Document 4 Japanese Patent Laid-Open No. 5-501611
  • Patent Document 5 US 5252743 A
  • Patent Document 6 US 5451683 A
  • Patent Document 7 US 5482867 A
  • Non-patent literature l Analytica Chimica Acta 1998, 367, 159
  • the present invention has been made in view of the above-mentioned problems of the prior art, and a novel method for efficiently and stably preparing a labeled molecule-containing silica sphere containing a desired labeled molecule.
  • the purpose is to provide.
  • Another object of the present invention is to provide a method for producing the above-described labeled molecule-containing silica sphere by adjusting it to a desired size.
  • an object of the present invention is to provide a labeled molecule-containing silica sphere obtained by the above method and to provide a use as a detection reagent thereof.
  • succinimidyl ester ester formed by binding a labeled molecule and succinimide via an ester bond (-CO-0-) has been studied.
  • silica compound (1) When the compound (1) is reacted with an amino group-containing silica compound (2), the carbonyl group of the succinimidyl ester compound (1) and the amino group of the silica compound (2) are bonded with an amide bond (-NH-CO -) To obtain a silica compound containing a labeled molecule (silica compound containing a labeled molecule) (3) And by further reacting the labeled molecule-containing silica compound (3) with the silica compound (4), silica spheres stably containing the labeled molecule can be efficiently prepared, and the size ( It was found that the particle size can be adjusted freely.
  • silica compound (4) used for the reaction with the labeled molecule-containing silica compound (3), various desired groups (for example, OH group, SH group, amine group, SCN group, epoxy group, CNO group, etc.) can be introduced, and such silica spheres can bind various functional molecules using these groups as acceptor groups, and can be applied to various reactions. It was confirmed that it can be used effectively as a possible reaction reagent.
  • desired groups for example, OH group, SH group, amine group, SCN group, epoxy group, CNO group, etc.
  • the present invention has been completed on the basis of strong knowledge.
  • the present invention includes the following modes:
  • Item 2 As the silica compound having an amino group (2), 3- (aminopropyl) triethoxysilane or 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane should be used.
  • Item 2. A method for preparing a labeled molecule-containing silica sphere according to Item 1.
  • the silica compound (4) includes tetraethoxysilane, ⁇ -mercaptopropyltriethoxysilane, aminopropyltriethoxysilane, 3-thiocyanatopropyltriethoxysilane, and 3-glycidyloxypropyltriethoxysilane.
  • a group power consisting of 3-isocyanatopropyltriethoxysilane and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, wherein at least one silica compound selected from the group forces is used.
  • Item 3. The method for preparing a labeled molecule-containing silica sphere according to Item 1 or 2.
  • Item 4 The method for preparing a labeled molecule-containing silica sphere according to any one of Items 1 to 3, wherein the step (b) is performed in the presence of water, alcohol, and ammonia.
  • Item 5. The method for preparing a labeled molecule-containing silica sphere according to Item 4, wherein the volume ratio of water to alcohol is 1: 0.5 to 1: 8.
  • Item 6 A labeled molecule-containing silica sphere obtained by the method according to any one of Items 1 to 5.
  • the labeled molecule-containing silica sphere obtained by the method according to Item 1 of Item 1 to Item 5 is further converted to a silica compound (4) different from the silica compound (4) used in step (b).
  • Item 8 A labeled molecule-containing silica sphere obtained by the method according to Item 8.
  • Item 9 A silica sphere obtained by binding a peptide, protein, gene, microorganism, coupling agent, piotin, avidin, or a labeled molecule to the surface of the labeled molecule-containing silica sphere described in Item 9.
  • Item 10 A labeled molecule-containing silica sphere obtained by the method according to any one of Items 1 to 5, and
  • step (c) optionally having a step of treating with a silica compound (4) different from the silica compound (4) used in step (b);
  • a method for preparing a multiple bond of labeled spheres containing labeled molecules is provided.
  • Item 11 A multi-bond product of labeled spheres containing labeled molecules obtained by the method according to item 10. The present invention will be described in detail below.
  • the method for preparing a labeled molecule-containing silica sphere of the present invention is characterized by having the following steps (a) and (b).
  • succinimidyl ester compound (1) used in the step (a) examples include compounds represented by the following general formula.
  • R means a labeled molecule. More specifically, R can be bonded to succinimide via an ester bond (-CO-0-) as shown in the following formula.
  • R represents a labeled molecule
  • R ′ represents a hydrogen atom or an arbitrary group.
  • R forms a carboxylic acid or a derivative thereof by bonding a -COOR 'group (R' means a hydrogen atom or an arbitrary group) as a side chain.
  • R' means a hydrogen atom or an arbitrary group
  • Examples of the carboxylic acid or its derivative shown as the compound (0) in the above include 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5 (6) -carboxy-fluorescein, 6-carboxy-2 ′, 4 , 4 ', 5', 7,7, -Hexaclo oral fluorescein, 6-carboxy-2,4,7,7, -Tetrachloro oral fluorescein, 6-carboxy-4,5, -dichloro-2, 7, -dimethoxyfluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5 (6) -carboxy-rhodamine, Alexa Fluor 350 carboxylic acid, Alexa Fluor 4
  • the succinimidyl ester compound (1) used in the step (a) of the present invention is a carboxylic acid or a derivative thereof [compound (0)] as shown in the above formula.
  • N-hydroxysuccinimide can be prepared by esterification according to a conventional method. However, it can also be obtained commercially.
  • succinimidyl ester compound (1) 5-succinimidyl ester-fluorescein, corresponding to the carboxylic acid or derivative thereof [compound (1)], 6 -Succinimidyl ester-fluorescein, 5 (6) -succinimidyl ester-fluorescein, 6-succinimidyl ester-2 ', 4,4', 5 ', 7,7' Fluorescein, 6-succinimidyl ester-2 ', 4,7,7'-Tetrachrome mouth fluorescein, 6-succinimidyl ester-4,5,5-dichloro-2,7, -dimethoxyfur Olethein, 5-succinimidyl ester-rhodamine, 6-succinimidyl ester-rhodamine, 5 (6) -succinimidyl ester-rhodamine, succinimidyl ester-Alexa Fluor 350, succinimidyl ester-Alexa
  • the silica compound (2) having an amino group is not particularly limited, and examples thereof include 3- (aminobutylpyr) triethoxysilane, 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxy.
  • Examples include silane, N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, and 3-aminopropyltrimethoxysilane.
  • the reaction between the succinimidyl ester compound (1) and the silica compound having an amino group (2) is performed by dissolving in a solvent such as DMSO or water and stirring at room temperature. It can be done by doing.
  • the carbo group of the succinimidyl ester compound (1) and the amino group of the silica compound (2) having an amino group are bonded to an amide bond (-NH-CO -)
  • the labeled molecule-containing silica compound (3) has an embodiment in which the labeled molecule and the silica compound are bonded via an amide bond.
  • step (b) the labeled molecule-containing silica compound (3) is reacted with the silica compound (4).
  • the silica compound (4) used here is not particularly limited, but includes tetraethoxysilane, ⁇ -mercaptopropyltriethoxysilane, aminopropyltriethoxysilane, 3-thiocyanatopropyltriethoxysilane, 3-glycidyl. Mention may be made of oxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane.
  • the ratio of the labeled molecule-containing silica compound (3) and the silica compound (4) is not particularly limited, but the molar ratio of the silica compound (4) to 1 mole of the labeled molecule-containing silica compound (3) is 100 to 40000. Preferable ⁇ is 300 to 20000, more preferable ⁇ is 500 to 10000, and more preferable ⁇ is 600 to 7000. [0021] This reaction is carried out in the presence of alcohol, water and ammonia. Examples of the alcohol include lower alcohols having 1 to 3 carbon atoms such as methanol, ethanol and propanol.
  • the ratio of water and alcohol in the powerful reaction system is not particularly limited, but preferably 0.5 to 8 parts by volume of alcohol, preferably 1 to 5 parts by volume, more preferably 1 part by volume of water.
  • the range of 1-2 volume parts can be mentioned.
  • the amount of ammonia is not particularly limited, for example, a molar ratio of 200 to 250000, preferably 400 to 150,000, more preferably 2500 to 25000 can be mentioned with respect to 1 mol of the labeled molecule-containing silane compound to be reacted. it can.
  • This reaction can be performed at room temperature, and is preferably performed with stirring.
  • the silica sphere (5) containing the target labeled molecule can be prepared by reaction for several tens of minutes to several tens of hours.
  • the size (diameter) of the silica spheres to be prepared can be appropriately adjusted by adjusting the concentration of the silica compound (4) used or adjusting the reaction time. it can. Larger silica spheres can be prepared by increasing the concentration of the silica compound (4) used or increasing the reaction time (eg Blaaderen et al., “Synthesis an d Characyerization of Monodisperse Collidal Organo -silica Spheres ", J. Colloid and Interface Science 156, 1-18.1993). Also, larger silica spheres can be prepared by repeating step (b) a plurality of times.
  • the size (diameter) of the obtained labeled molecule-containing silica sphere can be freely adjusted to a desired size, for example, from the nm order to the m order.
  • the labeled molecule-containing silica sphere having a size of several to several tens of nm, specifically 3 to 3 Onm. It is also possible to prepare. Further, if necessary, it can be adjusted to a desired particle size distribution by subsequent treatment, and thus silica spheres in a desired particle size distribution range can be obtained.
  • the labeled molecule-containing silica spheres thus obtained may be purified by removing conventional coexisting ions and unnecessary coexisting substances using a conventional method such as ultrafiltration membrane, if necessary.
  • the labeled molecule is immobilized in the silica sphere by using the method of the present invention.
  • the sensitivity can be increased more than that of a free labeled molecule.
  • many labeling molecules fluorescent dye molecules
  • Silica is generally known to be chemically inert and easy to modify.
  • the labeled molecule-containing silica sphere of the present invention prepared by the method described in (1) above can also easily bind a desired molecule to the surface, and the surface can be mesoporous or smooth. It can also be.
  • a labeled molecule-containing silica sphere having a group on the surface can be provided.
  • Table 1 shows the relationship between the silica compound (4) used for the reaction and the acceptor group formed on the surface of the labeled molecule-containing silica sphere obtained thereby.
  • the labeled molecule-containing silica sphere (5) obtained by the method (1) an acceptor group different from the acceptor group introduced onto the surface by the silica compound (4) used in the reaction was introduced. If so, the labeled molecule-containing silica sphere (5) is further treated with a silica compound different from the silica compound (4) used in step (b). This treatment can be performed by performing the same operation as in the above step (b) using a silica compound different from the silica compound (4) used in the step (b).
  • the labeled molecule-containing silica sphere of the present invention (surface-modified labeled molecule-containing silica sphere) prepared by such a method has a desired molecule [eg, peptide, protein, depending on the type of acceptor group on the surface. , Genes (poly or oligonucleotides such as RNA and DNA), microorganisms, coupling agents, piotin, avidin, and labeled molecules) can be bound to the surface.
  • a desired molecule eg, peptide, protein, depending on the type of acceptor group on the surface.
  • Genes poly or oligonucleotides such as RNA and DNA
  • microorganisms microorganisms
  • coupling agents piotin, avidin, and labeled molecules
  • a surface-modified-labeled-molecule-containing silica sphere having an OH group is formed on the surface of various silica compounds [for example, the silica described above via a silane bond (-Si-0-Si-). Compound (4), etc.]
  • Surface modification with SH groups Labeled molecule-containing silica spheres can be converted to peptides, proteins on their surface via bonds via disulfide bonds (-SS-), thioester bonds, or thiol substitution reactions. , Genes, etc .: surfaces with NH groups
  • Modified Labeled molecule-containing silica spheres have peptides, proteins, etc. on their surfaces via amide bonds or cheaure bonds; surface-modified silica molecules containing SCN groups have peptide molecules on their surfaces via chearea bonds Surface modification with epoxy group Labeled molecule-containing silica spheres have peptide or protein on the surface via amide bond; Surface modification with CNO group Labeled molecule-containing silica sphere has amide bond Thus, peptides, proteins, and the like can be respectively bound to the surface.
  • Molecules such as peptides, proteins, or genes bound to the modified labeled molecule-containing silica spheres as such become further acceptor molecules such as antigen-antibody reaction, piotin-avidin reaction, base sequence, and the like. Further, a desired molecule can be bound by utilizing a specific reaction such as hybridization utilizing the homology of each other.
  • the labeled molecule-containing silica sphere of the present invention (surface-modified labeled molecule-containing silica sphere) prepared by such a method can be applied to various applied technologies due to its fine shape, inclusion, and surface modification characteristics. More diverse applications are possible.
  • a mimic of a virus, a bacterium, a living microorganism, or an animal cell from its minute shape.
  • a surface protein such as a virus is labeled with the labeled molecule of the present invention.
  • the outer shell resembles the virus, but without the gene, a “pseudovirus” can be created. This could be applied to animal immunization and vaccine preparation without the need for adjuvants.
  • pseudovirus can be used as a means for drug delivery using the organ-specific infectivity of viruses.
  • the labeled molecule-containing silica sphere of the present invention binds a substance such as an arbitrary protein or gene based on its surface modification property (acceptor group) and presents its function on the surface. It can have the property that Such binding characteristics are useful not only in terms of adding functions by simply binding desired molecules, but also in terms of being able to concentrate substances such as arbitrary proteins and genes on the surface of silica spheres. For example, it is possible to improve reaction efficiency by binding and concentrating various enzymes that catalyze multistep reactions on silica spheres (reaction-enhanced silica spheres), and antibodies on silica spheres.
  • a bar code labeling method is conceivable.
  • silica spheres with various functions added for example, a library with various antibodies and peptides displayed on the surface
  • functional silica spheres to quickly distinguish them.
  • One of the methods is bar code sign.
  • a fluorescent dye molecule-containing silica compound labeled with two or more fluorescent dye molecules is mixed at various blending ratios to produce fluorescent dye molecule-containing silica spheres, thereby having various fluorescent properties.
  • Barcode labeled silica spheres can be made and can be distinguished by flow cytometry and fluorescence microscopy. Furthermore, using the above surface modification technology,
  • barcode labeling can also be performed by modifying proteins or genes labeled with dyes at various compounding ratios.
  • gene sequencing technology since gene sequencing technology is being simplified in recent years, it is possible to identify specific gene sequences by binding them to the surface of silica spheres, amplifying them with PCR as necessary, and reading the base sequences. It becomes.
  • the present invention provides a method for preparing a multiple bond product of labeled molecule-containing silica spheres as a method for increasing the strength of the labeled molecules possessed by the labeled molecule-containing silica spheres.
  • This method is carried out by further coupling the labeled molecule-containing silica spheres to the labeled molecule-containing silica spheres obtained by the above method using a coupling agent corresponding to the acceptor group of the labeled molecule-containing silica spheres. (Step (d)).
  • step (b) when the acceptor group of the labeled molecule-containing silica sphere is changed to a desired one different from the original acceptor group, the silica compound (4) used in step (b) described above is used before step (d).
  • a treatment with a silica compound (4) having a desired acceptor group, which is different from) may be carried out [step (c)].
  • Examples of the coupling agent that can be used here include those listed in Table 2 depending on the acceptor group on the surface of the labeled molecule-containing silica sphere.
  • the reaction with the coupling agent can be performed by reacting the labeled molecule-containing sirisphere of the present invention in the presence of the coupling agent.
  • a method in which a stirring reaction is performed at room temperature for several tens of minutes to several tens of hours can be used.
  • the ratio of the coupling agent to be used is 300 to 6000 parts, preferably 600 to 5400 parts, more preferably 2100 to 3000 parts by mole with respect to 1 mole of the labeled molecule-containing silica sphere.
  • the labeled molecule-containing silica spheres of the present invention are multiply bonded through the coupling agent.
  • Such a method can be effectively used as a means for increasing the strength of a labeled molecule (for example, a fluorescent pixel or a radical) derived from the labeled molecule-containing silica sphere.
  • the granular material formed by multiple bonding is not particularly limited, but can have a particle size in the range of 60 to 150 nm.
  • FIG. 1 is a graph showing a fluorescence decay curve of fluorescein (labeled molecule) -containing silica sphere A prepared in Example 1.
  • FIG. 2 is a schematic diagram showing the surface treatment of silica spheres described in Example 2.
  • FIG. 3 Spectrum A: Absorption spectrum of fluorescein (labeled molecule) -containing silica sphere A (Ist Growth) (diameter: 20 nm) prepared in Example 1 in an aqueous solution, spectrum B: adjusted in Example 2 (1)
  • FIG. 3 is a graph showing absorption spectra of the produced fluorescein (labeled molecule) -containing silica sphere (2nd Growth) (diameter: 230 nm) in an aqueous solution.
  • FIG. 4 is a diagram showing an image of a TEM photograph (X 90,000) of a fluorescein (labeled molecule) -containing silica sphere (diameter: 20 nm) (1st Growth) prepared in Example 1.
  • FIG. 5 Fluorescein (labeled molecule) -containing silica sphere prepared in Example 2 (1) (diameter: 230 nm) It is a figure which shows the image of the TEM photograph (X 15,000) of (2nd Growth).
  • FIG. 7 Fluorescence microscope findings (Fig. A) of silica spheres (SH base surface modified silica spheres) containing fluorescent dye molecules (fluorescein) prepared in Example 5, and silica with rhodamine-labeled GST attached to the silica spheres Shows the sphere's findings ( Figure b) (X 400) (Original is a color chart).
  • FIG. 8 Transmission electron microscope (T) of silica spheres containing rhodamine (labeled molecules) prepared in Example 7.
  • FIG. 9 A diagram showing the findings (a) in which rhodamine-labeled GST was attached to silica spheres that did not contain fluorescent dye molecules (a) and then the findings (b) in which FITC-labeled anti-GST antibody was bound (X) (X) 400)
  • a silica compound (3) containing fluorescein as a labeled molecule was prepared according to the following formula.
  • succinimidyl ester compound fluorescein (labeled molecule: represented by R in the formula) and succinimide are bonded through an ester bond.
  • FLUoS Carboxyfluorescein— N—hydroxysuccinimide ester
  • Aminopropyl) triethoxysilane [3- (aminopropyl) triethoxysilane: hereinafter also referred to as “APS”! /, U)] (2) is added to equimolarity with the above FLUOS, and stirred for about 1 hour using a stirrer piece.
  • Fluorescein (labeled molecule) -containing silica formed by reacting with stirring to form an amide bond between the carbo group of the succinimidyl ester compound [FLUOS (l)] and the amino group of the silica compound [APS (2)] Compound (3) was prepared.
  • FLUOS (1) which had a yellow color, was added with DMSO solution strength APS (2), it turned orange.
  • fluorescein (labeled molecule) -containing silica spheres (5) were prepared from the fluorescein (labeled molecule) -containing silica compound (3) according to the following formula.
  • reaction solution was subjected to ultrafiltration (Amicon (registered trademark) stirring cell) (filter 1; UF disk YM100 Ultracell RC100K NMWL) (sales company: MILLIPORE ⁇ Nomin al Molecular Weight Limit (NMWL ): 100 kDa] and repeated filtration and washing with distilled water several times to obtain 2 ml of sample solution A (containing fluorescein (labeled molecule) -containing silica spheres (5).
  • ultrafiltration Analogened trademark
  • filter 1 UF disk YM100 Ultracell RC100K NMWL
  • MILLIPORE Nomin al Molecular Weight Limit
  • Sphere is called “silica sphere A.”
  • the filtrate obtained here is further filtered with an ultrafiltration device (Amicon (registered trademark) stirring cell) (filter; UF disk YM-3 Ultracell RC100K NMWL) Company: MILLIPORE ⁇ Nominal Molecular Weight Limit (NMWL): 3 kDa], and repeated filtration and washing with distilled water several times to obtain 3 ml of sample solution B (fluorescein (labeled molecule)) Contains silica sphere (5) . This silica sphere Is called “silica sphere B”).
  • the reaction completed solution (yellow green) after reacting the reaction solution for 24 hours was diluted 10-fold in the same manner as the reaction solution (all component mixture solution before reaction), and the absorption spectrum was measured. However, the absorbance of the peak was 0.607.
  • the molecular extinction coefficient of FLUOS (7.5xl0 4), when calculating the concentration of Furuoresein molecules contained in the reaction-terminated liquid is 80.9 / z mol / l, the concentration of Furuoresein molecules contained in mixed Goeki before the reaction It was about 1.2 times larger than that.
  • the excitation wavelength was determined for the peak power of the emission spectrum of each emission peak.
  • the fluorescence intensity (excitation wavelength: 496 nm, emission wavelength: 520 nm) was measured for the pre-reaction mixture and the post-reaction reaction end solution. The results were 17.06 (pre-reaction mixture) and 33.95 (reaction end solution), respectively. Yes, it was obvious that the fluorescence intensity increased by about 2 times due to the reaction.
  • the silica sphere A and the silica sphere B prepared by the above-described method have 18.4% and 46.7% of the labeled molecules (fluorescein) bound (labeled), respectively.
  • the utilization rate of FLUOS fluorescein molecule used in the reaction was found to be 65.1%.
  • the labeling rates of silica sphere A and silica sphere B are both Imhofe's paper (A. Imhof, et al., “Spectroscopy or fluorescein (riT) Dyea Colloidal bilica spheres, J. Phys. Chem. B 1999, 103, 1408. -1415), which exceeded the maximum label rate of 13%.
  • the silica sphere A in the sample solution A and the silica sphere B in the sample solution B prepared in (1) were transferred to a transmission electron microscope (Tokushima University School of Medicine) and an ultra high voltage electron microscope (Osaka University Ultra High Voltage Electron Microscope Center). When observed with, particle images with diameters of about 20 and 4 were observed. From this result, it was judged that the diameter of silica sphere A was about 20 mm, and the diameter of silica sphere B was about 4 nm.
  • the diameter of the silica sphere obtained by Imhofb's method is 18 4-305 nm (A. Imhof, et al., Spectroscopy of Fluorescein (FITC) Dyea Colloida 1 Silica Spheres ", J. Phys. Chem. B 1999, 103, 1408-1415).
  • Silica spheres fluorescence intensity of A1 particles 2.0x10- 11 (the number of silica spheres A contained in the sample solution A 2 ml is 4.72X10 "number / 2 ml: fluorescence intensity of the sample solution A 0.03 ml is 143.38), and silica spheres B1 the number of silica spheres B fluorescence intensity of the particles contained in 2.2x10- 13 (sample solution B 2 ml 1.16x10 "/ 2ml: The fluorescence intensity of 0.03ml of sample solution B is 255.10).
  • the fluorescence intensity of one molecule of free fluorescein is 1.8 ⁇ 10-13 (the fluorescence intensity is 957.562 in 10 ml sample of 0.0029 mmol / l).
  • the number of Furuorese Inn molecules contained in the sample because it is 15 5.25Xl0, fluorescence intensity per molecule becomes 1.8X10- 13].
  • the fluorescence intensity of silica sphere A1 particles is 11% of the fluorescence intensity of one molecule of fluorescein.
  • the fluorescence intensity of the silica sphere B1 particle is 1.2 times the fluorescence intensity of one molecule of fluorescein.
  • the volume of the silica spheres A1 particles (particle size 20 nm) are 4.2x10- 18 cm 3, since the number of Furuoresein molecules are Ru contained per one particle is 97 molecules / SiO particles in silica spheres A1 particles
  • silica sphere A has a fluorescence intensity equivalent to 111 molecular weight fluorescein molecule, although it contains 97 molecules of fluorescein molecule (concentration of fluorescein molecule: 38.4 mmol / l) in one particle. It can be said that.
  • the volume of the silica spheres B1 particles (particle size 4 nm) is 3.4x10- 2 ° cm 3, 1 molecule / SiO particles and the possible forces et number of Furuoresein component element included in one particle, silica spheres B1 Of the fluorescein molecule in the particle
  • silica sphere B contains one fluorescein molecule (concentration of fluorescein molecule: 48.9 mmol / l) in one particle, but has a fluorescence intensity corresponding to a 1.2 molecular weight fluorescein molecule. You can hear it.
  • the concentration of fluorescein molecules in one particle of silica sphere obtained by Imhofe's method is 3 ⁇ mmol / 1 (A. Imhof, et al., "Spectroscopy of Fluorescein (FIT) Dyed Colloida 1 Silica Spheres ", J. Phys. Chem. B 1999, 103, 1408-1415). From this, the concentration of fluorescein molecules contained in one particle of silica sphere B (48.9 mmol / l) is 1.58 times that amount.
  • Fluorescence lifetime was measured for the silica sphere A (diameter 20 °) obtained above. Specifically, a sample (an aqueous solution of silica sphere A) is irradiated using pulsed light (nanosecond (nsec) order) that is not stationary light when excited at an excitation wavelength (494 nm). Excited by the pulsed light The intensity of the emitted fluorescent peak was measured. Figure 1 shows the results (fluorescence decay curve) with time on the horizontal axis and emission peak intensity on the vertical axis.
  • the label rate (labeled molecule content) is 18.4% and 46.7%, respectively, 20 nm and 4 nm fluorescein (labeled molecule) -containing silica nanoparticles (particle size: several Several tens of thousands) can be prepared.
  • the number of fluorescein molecules per particle was 1 and 97, respectively, and the concentration of fluorescein molecule per particle calculated was 38.4 mmol / l and 48.9 mmol / l.
  • the fluorescence intensity of the fluorescein molecule per particle was 111 times and 1.2 times that of one free fluorescein molecule, respectively. This is because one molecule of fluoresce in one particle of SiO molecule.
  • the surface of the fluorescein (labeled molecule) -containing silica sphere formed by the above reaction has an OH group as an acceptor group based on the silica compound (tetraethoxysilane) (4) used in the reaction. (OH-based surface modified silica sphere).
  • Example 1 [Fluorescein (labeled molecule) -containing silica particles (diameter 20 nm)] (1st Growth) in an aqueous solution of 1 ml, ethanol 4 ml, tetraethoxysilane (TEOS) 50 ⁇ 1, 27 wt% aqueous ammonia 50 ⁇ 1 were mixed, and then magnetic stirring was performed at room temperature for 24 hours. Then, ultrafiltration (filter: UF disk ⁇ 100 Ultracel RC 100 K NMWL) was performed, washed several times with distilled water, and taken out as silica spheres containing 2nd Growth fluorescein (labeled molecules).
  • TEOS tetraethoxysilane
  • the diameter of the silica sphere was 230 nm, which was about 10 times larger than the diameter of silica sphere A (20 nm).
  • R means fluorescein (labeled molecule)
  • the aqueous solution of silica sphere A (diameter 20 nm) (1st Growth) had a deep yellow color and was stable even after one month.
  • fluorescein molecules are known to emit stable and strong fluorescence under alkaline conditions. Since the solution used for the preparation of silica spheres is strongly alkaline, the results obtained above (strong and stable fluorescence) are attributed to the alkali inside the prepared silica spheres. Conceivable.
  • silica sphere A (diameter: 20 nm) prepared in Example 1 and the silica sphere (diameter: 230 nm) prepared in (1) above were observed with a transmission electron microscope.
  • a transmission electron microscope image of silica sphere A is shown in FIG. 4, and a transmission electron microscope image of silica sphere prepared in (1) is shown in FIG.
  • Silica sphere A prepared in Example 1 (diameter: 20 nm) (1st Growth) (Fig. 4) has a mesoporous shape with irregularities on the surface, whereas the silica sphere prepared in (1) (diameter : 230nm) (2nd Growth) ( Figure 5) had a smooth surface.
  • the sample of the present invention had a fluorescence intensity of 9.8 (excitation wavelength of 484 nm and an emission wavelength of 515 nm), which was clearly about 1/15 of the fluorescence intensity of the control sample.
  • the fluorescence intensity of the sample of the present invention treated with crab dartal aldehyde was decreased. From this decrease in fluorescence intensity (quenching phenomenon), by adding dartal aldehyde, NH
  • FIG. 6 schematically shows the above reaction.
  • the silica spheres are bonded to each other to form large particles by blending the sample solution of the present invention to a predetermined amount (in this case, 0.2 ml), that is, by adjusting the amount of the mixture solution to be blended. It is possible to make a lump (multiple bond).
  • silica spheres bind to each other and It was obvious that a mass of forceballs (multiple bonds) could be formed. That is, it can be said that larger fluorescein (labeled molecule) -containing silica particles can be prepared by the above reaction.
  • the obtained solution was subjected to ultrafiltration (Amicon (registered trademark) stirring cell) (filter; UF disk YM100 Ultracell RC100K NMWL) (sales company: MILLIPORE ⁇ Nominal Molecular Weight Limit (NMWL) : 100 kDa], and filtration and washing with distilled water were repeated several times to prepare fluorescein (labeled molecule) -containing silica spheres.
  • the SH group is derived from the silica compound (MPS) used in the above reaction (SH group surface modified silica sphere), and when this silica sphere was observed with a fluorescence microscope, the fluorescence of fluorescein could be observed ( Figure 7a).
  • Piotin (labeled molecule) -containing silica compound (7) was prepared according to the following formula.
  • R means piotin (labeled molecule).
  • * is a bond with an ester group.
  • D-Biotin-N-hyd is formed by binding biotin (labeled molecule) (shown by R in the formula) and succinimide through an ester bond.
  • silica particles (8) containing piotin and fluorescein as labeling molecules were prepared according to the following formula.
  • R represents a fluorescein molecule
  • R represents a piotin molecule
  • the reaction solution obtained above [Piotin (labeled molecule) -containing silica compound] (7) 2.5 ml and the fluorescein (labeled molecule) -containing silica compound (3) obtained in Example 1
  • TEOS tetraethoxysilane
  • the silica sphere (8) containing piotin-fluorescein (labeled molecule) (8) is present in a form in which piotin is exposed on the surface layer portion just by the incorporation of piotin inside. So it was found to react with avidin. Therefore, the silica sphere (8) containing piotin-fluorescein (labeling molecule) is useful as a fluorescent reagent for detection using the piotin-avidin reaction.
  • a rhodamine (labeled molecule) -containing silica compound was prepared according to the following formula.
  • R means rhodamine (labeled molecule).
  • * indicates the bond with the ester group.
  • succinimidyl ester compound 5-carboxyltetramethylrhodamine formed by binding podamine (labeled molecule) and succinimide via an ester bond.
  • succinimidyl ester (9) (Molecular Probes) is dissolved in 1 ml of DMSO solution, and 3- (aminopropyl) triethoxysilane (APSX 2) is used as a silica compound having an amino group.
  • APSX 2 3- (aminopropyl) triethoxysilane
  • a hydramine (labeled molecule) -containing silica compound (10) comprising an amide bond between the group and an amino group of the silica compound (2) was prepared.
  • Rhodamine (labeled molecule) -containing silica compound (10) force Rhodamine (labeled molecule) -containing silica sphere (12) was prepared according to the following formula.
  • R means rhodamine (labeled molecule).
  • TEOS tetraethoxysilane
  • water 4: 1, volume ratio
  • ammonia water 2 ml of about 30% ammonia water was added to this and left at room temperature with stirring for one day.
  • Rhodamine (labeled molecule) -containing silica compound (10) prepared by the method described in Example 7
  • Dispersed aqueous solution (10 ml) In 4 ml of ethanol, tetraethyl orthosilicate (TEOS) 60 1, water 1.2 ml Rhodamine (labeled molecule) -containing silica spheres (11) were prepared by adding 0.4 ml of 27 wt% aqueous ammonia and reacting at room temperature.
  • Silica spheres were prepared by mixing 15 ml and 1 ml of 27% by weight aqueous ammonia and leaving it agitated for one day. This silica sphere has SH groups derived from MPS as acceptor groups on its surface (SH base surface modified silica spheres). Next, the silica spheres were washed with ethanol, water, and physiological saline using a centrifuge separator.
  • Washed rhodamine-labeled GST-bound SH group surface modified silica spheres were mixed with anti-GST antibody 5 ⁇ 1 fluorescently labeled with 0.2 mg / ml fluorescein isothiocyanate (FITC) and fluorescent microscope The observation was performed. As a result, as shown in Fig. 9b, FITC fluorescence was observed in the silica sphere. This confirmed that the anti-GST antibody was bound to the antigen GST on the silica sphere.
  • FITC fluorescein isothiocyanate

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Abstract

L’invention concerne : un nouveau procédé permettant de produire de manière performante et stable une sphère de silice contenant une molécule marquée souhaitée ; une sphère de silice contenant une molécule marquée, préparée par le procédé ; et enfin l’utilisation de la sphère de silice en tant que réactif de détection. La sphère de silice (5) contenant la molécule marquée est préparée via (a) une étape au cours de laquelle un ester succinimidylique (1) comprenant du succinimide et une molécule marquée liée à celui-ci via une liaison ester (-CO-O-) est mis à réagir avec un composé de silice (2) portant un groupe amino, de manière à produire un composé de silice (3) contenant la molécule marquée, et (b) une étape au cours de laquelle un ou plusieurs de ces composés de silice (3) contenant une molécule marquée obtenus à l’étape (a) sont mis à réagir avec un composé de silice (4).
PCT/JP2005/022628 2004-12-09 2005-12-09 Procede de preparation d’une sphere de silice contenant une molecule marquee Ceased WO2006070582A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
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JP2006265306A (ja) * 2005-03-22 2006-10-05 Univ Of Tokushima 色材組成物及びそれを含有する発色または発光性製品
WO2007142316A1 (fr) 2006-06-08 2007-12-13 The University Of Tokushima Méthode de production d'une nouvelle nanoparticule de silice et application de la nanoparticule de silice
JP2008247713A (ja) * 2007-03-30 2008-10-16 Univ Of Tokushima 蛍光色素含有ナノシリカ粒子およびその調製方法
JP2009046330A (ja) * 2007-08-15 2009-03-05 Furukawa Electric Co Ltd:The 逆ミセル分散系を用いてなるシリカナノ粒子の製造方法、該方法により得られたシリカナノ粒子、及びそれを用いた標識試薬
JP2009115822A (ja) * 2009-02-23 2009-05-28 Furukawa Electric Co Ltd:The イムノクロマト法試薬用標識シリカナノ粒子、イムノクロマト法試薬、それを用いたイムノクロマト法用テストストリップ、及びイムノクロマト法用蛍光検出システム
WO2009072657A1 (fr) 2007-12-06 2009-06-11 The University Of Tokushima Particules de silice nanofonctionnelles et leur procédé de fabrication
JP2009281760A (ja) * 2008-05-20 2009-12-03 Konica Minolta Medical & Graphic Inc ナノ粒子内包シリカ、それを用いた生体物質の標識物質および生体物質の標識方法
JP2010105896A (ja) * 2008-10-31 2010-05-13 Furukawa Electric Co Ltd:The カルボキシル基を有する機能性有機分子を含有するシリカナノ粒子の製造方法、前記製造方法により得られたシリカナノ粒子、それを用いた標識試薬
US7955866B2 (en) 2007-06-08 2011-06-07 The Furukawa Electric Co., Ltd. Labelled silica nanoparticles for immunochromatographic assays
WO2014119624A1 (fr) * 2013-02-04 2014-08-07 古河電気工業株式会社 Procédé de production d'un anticorps marqué
WO2015163424A1 (fr) * 2014-04-23 2015-10-29 株式会社ニチレイバイオサイエンス Combinaison pour détection de marqueur cible
WO2016068324A1 (fr) * 2014-10-31 2016-05-06 信一郎 礒部 Pigment el organique contenant des groupes alcoxysilyle et son procédé de production

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WO2007142316A1 (fr) 2006-06-08 2007-12-13 The University Of Tokushima Méthode de production d'une nouvelle nanoparticule de silice et application de la nanoparticule de silice
JP2008247713A (ja) * 2007-03-30 2008-10-16 Univ Of Tokushima 蛍光色素含有ナノシリカ粒子およびその調製方法
US7955866B2 (en) 2007-06-08 2011-06-07 The Furukawa Electric Co., Ltd. Labelled silica nanoparticles for immunochromatographic assays
US10209249B2 (en) 2007-06-08 2019-02-19 The Furukawa Electric Co., Ltd. Labelled silica nanoparticles for immunochromatographic reagent, immunochromatographic test strip using the same, and immunochromomatographic fluorescence-detecting system or radiation-detecting system
JP2009046330A (ja) * 2007-08-15 2009-03-05 Furukawa Electric Co Ltd:The 逆ミセル分散系を用いてなるシリカナノ粒子の製造方法、該方法により得られたシリカナノ粒子、及びそれを用いた標識試薬
WO2009072657A1 (fr) 2007-12-06 2009-06-11 The University Of Tokushima Particules de silice nanofonctionnelles et leur procédé de fabrication
JP2009281760A (ja) * 2008-05-20 2009-12-03 Konica Minolta Medical & Graphic Inc ナノ粒子内包シリカ、それを用いた生体物質の標識物質および生体物質の標識方法
JP2010105896A (ja) * 2008-10-31 2010-05-13 Furukawa Electric Co Ltd:The カルボキシル基を有する機能性有機分子を含有するシリカナノ粒子の製造方法、前記製造方法により得られたシリカナノ粒子、それを用いた標識試薬
JP2009115822A (ja) * 2009-02-23 2009-05-28 Furukawa Electric Co Ltd:The イムノクロマト法試薬用標識シリカナノ粒子、イムノクロマト法試薬、それを用いたイムノクロマト法用テストストリップ、及びイムノクロマト法用蛍光検出システム
WO2014119624A1 (fr) * 2013-02-04 2014-08-07 古河電気工業株式会社 Procédé de production d'un anticorps marqué
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US10466234B2 (en) 2013-02-04 2019-11-05 Furukawa Electric Co., Ltd. Method of producing labeled antibody
WO2015163424A1 (fr) * 2014-04-23 2015-10-29 株式会社ニチレイバイオサイエンス Combinaison pour détection de marqueur cible
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US10324084B2 (en) 2014-04-23 2019-06-18 Nichirei Biosciences Inc. Combination product for detecting target marker
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US11156602B2 (en) 2014-04-23 2021-10-26 Nichirei Biosciences Inc. Combination product for detecting target marker
JPWO2016068324A1 (ja) * 2014-10-31 2017-08-10 信一郎 礒部 アルコキシシリル基含有有機ｅｌ色素およびその製造方法
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