WO2006070582A1

WO2006070582A1 - Method of preparing silica sphere containing labeled molecule

Info

Publication number: WO2006070582A1
Application number: PCT/JP2005/022628
Authority: WO
Inventors: Hirokazu Miyoshi; Michihiro Nakamura
Original assignee: Techno Network Shikoku. Co., Ltd.
Priority date: 2004-12-09
Filing date: 2005-12-09
Publication date: 2006-07-06
Also published as: JPWO2006070582A1; JP4982687B2

Abstract

A novel method of efficiently and stably preparing a silica sphere containing a desired labeled molecule; a labeled-molecule-containing silica sphere obtained by the method; and a use of the silica sphere as a detection reagent. The labeled-molecule-containing silica sphere (5) is prepared through (a) a step in which a succinimidyl ester compound (1) comprising succinimide and a labeled molecule bonded thereto through an ester bond (-CO-O-) is reacted with a silica compound (2) having an amino group to yield a silica compound (3) containing the labeled molecule and (b) a step in which one of or a combination of two or more of such labeled-molecule-containing silica compounds (3) obtained in the step (a) is reacted with a silica compound (4).

Description

Specification

Method for preparing labeled spheres containing labeled molecules

Technical field

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.

Background art

[0002] In recent years, various biochemical inspection methods using fine particles containing a fluorescent dye as a detection reagent have been studied. For example, the Alpha Screen technique has been commercialized by Nockard (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. When 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.

[0003] In addition, detection reagents using 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.

As a typical method for preparing this reagent, 3- (aminopropyl) triethoxysilane [APS

: 3—, aminopropyl) tnethoxysilane] [this contact with fluorescein isothiocyanate] 7 ^ ~~ to [FITC: flu orescein isothiocyanate] APS— FITC [N- 1- (3-triethoxysilylpropyl)-N ' -fluoresceyl thiourea] is reacted with N-tris (hydroxylmethyl) methyl-2-aminoethanesulfonic acid (TES) in an aqueous ethanol solution containing ammonia (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

Special Reference 2: A. Imhof, et al., "Spectroscopy of Fluorescein (FIT)", J. Phys. Chem. B 1999, 103, 1408-1415

Disclosure of the invention

Problems to be solved by the invention

[0005] 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. Furthermore, 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.

Means for solving the problem

[0006] In order to achieve the above object, the present inventors have intensively studied day and night. As a result, the succinimidyl ester ester formed by binding a labeled molecule and succinimide via an ester bond (-CO-0-) has been studied. 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. In addition, depending on the type of 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.

The present invention has been completed on the basis of strong knowledge. The present invention includes the following modes:

Item 1. (a) Succinimidyl ester compound (1) formed by binding of labeled molecule and succinimide via ester bond (-CO-0-) and silica compound having amino group (2 ) To produce a labeled molecule-containing silica compound (3),

as well as

(b) A step of reacting the silica compound (4) with one or more of the labeled molecule-containing silica compounds (3) obtained in the step (a).

A method for preparing a labeled molecule-containing silica sphere (5).

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.

Item 3. The silica compound (4) includes tetraethoxysilane, γ-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.

Item 7. 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). A method for preparing a labeled molecule-containing silica sphere, comprising a treatment step.

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

(c) optionally having a step of treating with a silica compound (4) different from the silica compound (4) used in step (b); and

(d) A step of bonding the labeled molecule-containing silica spheres together using a coupling agent according to the acceptor group of the labeled molecule-containing silica spheres.

A method for preparing a multiple bond of labeled spheres containing labeled molecules.

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.

(1) Preparation method of labeled spheres containing labeled molecules

The method for preparing a labeled molecule-containing silica sphere of the present invention is characterized by having the following steps (a) and (b).

(a) A succinimidyl ester compound (1) obtained by binding a labeled molecule and succinimide through an ester bond (-CO-0-) and a silica compound (2) having an amino group. A step of reacting to produce a labeling molecule-containing silica compound (3),

as well as

(b) The labeled molecule-containing silica compound (3) obtained in (a) is reacted with the silica compound (4) to produce a standard. Forming a silica sphere (5) containing a sensing molecule.

[0009] Examples of the succinimidyl ester compound (1) used in the step (a) include compounds represented by the following general formula.

[0010] [Chemical 1]

[0011] Here, 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.

[0012] [Chemical 2]

[Wherein R represents a labeled molecule, R ′ represents a hydrogen atom or an arbitrary group.]

Specifically, as shown in the above formula, 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. Can be listed. 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 405 carboxylic acid, Alexa Fluor 430 carboxylic acid, Alexa Fluor 488 carboxylic acid, Alexa Fluor 500 carboxylic acid, Alexa Fluor 514 carboxylic acid, Alexa Fluor 5 32 carboxylic acid, Alexa Fluor 546 carboxylic acid, Alexa Fluor 555 carboxylic acid, Alexa Fluo r 568 carboxylic acid, Alexa Fluor 594 carboxylic acid, Alexa Fluor 610 carboxylic acid, Alexa Fluor 633 carboxylic acid, Alexa Fluor 647 carboxylic acid, Alexa Fluor 660 carboxylic acid, Ale xa Fluor 680 carboxylic acid, Alexa Fluor 700 carboxylic acid, Alexa Fluor 750 Dye such as carboxylic acid and biotin; 3-carboxy TEMPO (4-carboxy-2,2,6,6-tetramethylpiperidine 1 — oxy), 3—carboxy PROXYL [3— (carboxy) — 2,2,5,5 — Tetramethyl-l-piperidinyloxy), etc .; ethylenediaminetetraacetic acid, iron (III) sodium salt hydrate, ethylenediaminetetraacetic acid, iron (II) acetate, etc.

[0014] 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. And N-hydroxysuccinimide can be prepared by esterification according to a conventional method. However, it can also be obtained commercially.

[0015] Specifically, as the 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, succini Midyl ester-Al exa Fluor 405, Succini Dilester-Alexa Fluor 430, Succinimidyl ester -Alexa Fluor 488, Succinimidyl ester-Alexa Fluor 500, Succinimidyl ester-Alexa Fluor 514, Succinimidyl ester-Alexa Fluor 532, Succinimidyl Esters-Alexa Fluor 546, Succinimidyl ester-Alexa Fluor 555, Succinimidyl ester-Alexa Fluor 568, Succinimidyl ester-Alexa Fluor 594, Succinimidyl ester-Alexa Fluor 610, Succinimidyl ester-Alexa Fluor 633 , Succinimidyl ester-Alexa Fluor 647, Succinimidyl ester-Alexa Fluor 660, Succinimidyl ester-Alexa Fluor 680, Succinimidyl ester-Alexa Fluor 70 0, Succinimidyl ester-Alexa Fluor 750, succinimidyl ester-biotin; 3-succinimidyl ester- TEMPO, 3-succinimidyl ester- PROXYL; N- sue cinimidyl ester- ethyleneaiaminetetraacetic acid, lron (III) sodium salt hydrate, N- su ccinimidyl ester- ethylenediaminetetraacetic acid, iron (II) Ability to raise acetate etc.

[0016] 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.

[0017] 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 ratio of the succinimidyl ester compound (1) and the silica compound (2) used in the reaction is not particularly limited, but preferably the succinimidyl ester compound (1 ): Silica compound (2) = 1: 0.5 to 4 (molar ratio), more preferably 1: 1 to 2 (molar ratio).

[0018] Thus, 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 -) To produce the labeled molecule-containing silica compound (3). That is, 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.

Next, in 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, γ-mercaptopropyltriethoxysilane, aminopropyltriethoxysilane, 3-thiocyanatopropyltriethoxysilane, 3-glycidyl. Mention may be made of oxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane.

[0020] 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.

[0022] 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. Although 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.

[0023] This reaction can be performed at room temperature, and is preferably performed with stirring. Usually, the silica sphere (5) containing the target labeled molecule can be prepared by reaction for several tens of minutes to several tens of hours.

[0024] In the step (b), 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. Thus, according to the method of the present invention, 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. Specifically, according to the method of the present invention, as shown in Example 1 described later, 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.

[0025] 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.

[0026] As shown in Example 1 to be described later, the labeled molecule is immobilized in the silica sphere by using the method of the present invention. When fixed or included, the sensitivity can be increased more than that of a free labeled molecule. In addition, according to the method of the present invention, even when a fluorescent dye molecule is used as a labeling molecule, many labeling molecules (fluorescent dye molecules) can be fixed or included in the silica sphere without causing self-quenching. . Therefore, according to the method of the present invention, it is possible to provide a highly sensitive detection reagent that can be used even in a minute region.

[0027] (2) Silica spheres containing labeled 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.

[0028] Specifically, according to the method for preparing a labeled molecule-containing silica sphere of the present invention, an acceptor capable of binding to a desired molecule according to the type of the silica compound (4) used in the step (b). A labeled molecule-containing silica sphere having a group on the surface (surface-modified labeled molecule-containing silica sphere) 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.

[0029] [Table 1] Silica compound (4) Acceptor formed on the surface of Siri-ball

OH group

SH group

₂ NH

S CN group

[0030] For 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). [0031] 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. As an example, for example, 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

2

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.

Therefore, for example, even a molecule that diffuses in a solution and exists at a low concentration is bound to the surface according to the labeled molecule-containing silica sphere (surface modified labeled molecule-containing silica sphere) of the present invention. This makes it possible to detect these molecules on silica spheres with high sensitivity.

[0033] 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.

[0034] 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.

[0035] For example, it is possible to construct a mimic of a virus, a bacterium, a living microorganism, or an animal cell from its minute shape. For example, a surface protein such as a virus is labeled with the labeled molecule of the present invention. By binding to the surface of the containing silica spheres, 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. It is also possible to measure the antibody titer using the “pseudovirus” containing fluorescence inside and the “aggregation measurement method” technique described in International Publication (WO 03/060519). is there.

[0036] Furthermore, "pseudovirus" can be used as a means for drug delivery using the organ-specific infectivity of viruses.

[0037] It is also possible to create "immunosilica spheres" similar to B cells and T cells by binding antibodies and T cell receptors to the surface of the labeled molecule-containing silica spheres of the present invention. . This can be applied not only to research but also to diagnosis and treatment of cancer and immune diseases. Furthermore, if the receptor, signal molecule, and gene expression system can be reconfigured in the labeled molecule-containing silica sphere of the present invention, cells that artificially reproduce spleen j8 cells that secrete insulin such as “silica (β) It is thought that “cells” can also be used.

[0038] 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. It is also possible to improve the bonding properties by combining them (bond-reinforced silica spheres). Further, a method of concentrating an antibody by binding to the labeled molecule-containing silica sphere of the present invention surface-modified with an antigen, a labeled molecule-containing silica sphere of the present invention having a surface modified with avidin, and binding and concentrating a bithione protein. The method of doing can also be illustrated.

[0039] Furthermore, even two or more substances that are inherently difficult to conjugate can be conjugated via the labeled molecule-containing silica sphere of the present invention. For example, when SH-based surface modified silica spheres shown in Example 9 described below are reacted with a mixed solution of rhodamine-labeled dartathione-S-transferase and green fluorescein protein (GFP), rhodamine-labeled It is possible to observe the fluorescence of both glutathione-S-transferase and green fluorescein protein (GFP). In addition, when a labeled molecule-containing silica sphere having avidin bound to its surface is reacted with a mixed solution containing two types of pyotinylated proteins, two types of pyotin チン protein can be bound to the surface of the silica sphere.

[0040] As another applied technique, for example, a bar code labeling method is conceivable. In silica spheres with various functions added (for example, a library with various antibodies and peptides displayed on the surface), it is very convenient to apply functional silica spheres to quickly distinguish them. One of the methods is bar code sign. Specifically, 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,

Alternatively, barcode labeling can also be performed by modifying proteins or genes labeled with dyes at various compounding ratios. In particular, 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.

[0041] (3) Preparation of multiple bonds of labeled spheres containing labeled molecules

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)). In addition, 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)].

[0042] 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.

[0043] [Table 2] 标讀 ^ ¾Acceptor group on the surface of Siri force ball coupling agent

OH group Tetraethyl orthosilicate

SH group Mercaptopropylethyl siligate

NH ₂ group dartal aldehyde

S CN group aminopropylethyl silicate

Epoxy group Aminopropylethylsilique

CNO group Aminopropylethyl silicate

[0044] 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. Usually, 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.

[0045] 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.

[0046] Thus, 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.

Brief Description of Drawings

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. 6] NH group modification of a labeled molecule-containing silica sphere described in Example 4 and a coupling agent

2

The schematic diagram which shows the coupling | bonding of the used silica spheres is shown.

[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.

EM) A photograph (X 10,000) (Figure a) and a fluorescence microscope image (Figure b) are shown (the original is a color diagram).

[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)

(The original is a color diagram).

BEST MODE FOR CARRYING OUT THE INVENTION

[0048] Hereinafter, examples will be described in order to describe the present invention in detail. However, the present invention is not limited to these examples.

Example 1

[0049] Preparation of silica compound containing fluorescein (labeled molecule) and silica sphere (fluorescein-containing silica particles) using the same

(1) Preparation of fluorescein (labeled molecule) -containing silica compound

A silica compound (3) containing fluorescein as a labeled molecule was prepared according to the following formula.

[0050] [Chemical 3]

[0051] [In the formula, represents fluorescein (labeled molecule). In 1 ^, * means the bond with the ester group. ]

Specifically, as a succinimidyl ester compound, fluorescein (labeled molecule: represented by R in the formula) and succinimide are bonded through an ester bond. ) — Carboxyfluorescein— N—hydroxysuccinimide ester (hereinafter “h”, “FLUºS”) (1) (approx. 3.3 mg) is dissolved in 1 ml of DMSO solution. 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. When FLUOS (1), which had a yellow color, was added with DMSO solution strength APS (2), it turned orange.

[0052] (2) Preparation of labeled molecule-containing silica sphere

Next, fluorescein (labeled molecule) -containing silica spheres (5) were prepared from the fluorescein (labeled molecule) -containing silica compound (3) according to the following formula.

[0053] [Chemical 4]

[0054] Specifically, 50 1 was collected from the reaction solution containing the fluorescein (labeled molecule) -containing silica compound (3) obtained above and added to 3.95 ml of ethanol. This further tetraethoxysilane as the silica of compound (Tetraethylorthosilicate:! Hereinafter, both "TEOS" /, the Hare) (4) 50 μ 1, distilled water lml, and 27 weight ^_0/0 to about 100 1 aqueous ammonia In addition, the reaction was allowed to stir at room temperature for about 24 hours using a stirrer piece. At this time, the volume ratio of ethanol and distilled water in the reaction solution was set to 4: 1. The obtained solution had a yellowish green color clearly different from the yellow color of the mixed solution before the reaction, and it was confirmed that the reaction occurred.

[0055] The obtained reaction solution was subjected to ultrafiltration (Amicon (registered trademark) stirring cell) (filter 1; UF disk YM100 Ultracell RC100K NMWL) (sales company: MILLIPORE 丌 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). 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 丌 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”).

[0056] (3) Label rate of the labeled spheres containing the labeled molecules (5) (labeled molecule content)

(3-1) The concentration of fluorescein molecules in the above reaction solution (before 24 hours reaction) is calculated to be 67.7 μmol / 1 [FLUOS (molecular weight 473.4) 0.165 mg (3.3 mg x 50 1 / Use 1000 μΐ for the final reaction (approximately 5.15 ml)]. Actually, 10 times the reaction mixture immediately after mixing all components The absorption spectrum (using a square cell with an optical path length of 1 cm) was measured for the diluted solution, and the peak absorbance was determined from it, which was 0.510. Molecular extinction coefficient of FLUOS from 7.5Xl0 ⁴ der Rukoto of the calculation of the concentration of Furuoresein molecules of the reaction solution, Ri Do a 68 / z mol / l, was consistent with the calculated value mentioned above.

[0057] On the other hand, 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. From this result, the molecular extinction coefficient of Furuoresein in silica particles (nanoparticles) is determined to have changed from the original 7.5Xl0 ⁴ to 8.9xl0 ^4. Therefore, in the following calculation of the label rate (labeled molecules including Yuritsu), it was used latter molecular extinction coefficient values (8.9Xl0 ⁴⁾ as a molecular extinction coefficient

[0058] A fluorescence spectrum (a slit width (Ex / Em) = 1.5 nm / 1.5 nm, Low sensitivity) was measured for each of the mixed solution before the reaction and the reaction completed solution after the reaction. 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.

[0059] (3-2) 0.1 ml of 2 ml sample solution A obtained by ultrafiltration using UF disk YM 100 Ultracel RC100K NMWL as a filter, diluted 10-fold with distilled water, and absorption spectrum Was measured to determine the absorbance (absorbance 0.285). From the calculated value (32 mol / 1) using a molecular extinction coefficient (8.9xl0 ⁴⁾ of FLUOS obtained above, when calculating the amount of Furuoresein molecule, 32 μ mol / 1 x 473.4 x 2/1000 = 0.0303mg It became. The labeling rate of silica sphere A in sample solution A was determined to be 0.0303 / 0.165x100 = 18.4% because the amount of fluorescein molecule added was SO.165 mg (more precisely, the amount of FLUOS). .

[0060] Similarly, 0.1 ml of 3 ml of sample solution B obtained by ultrafiltration using UF disk YM-3 Ultracel RC100K NMWL as a filter was diluted 10-fold with distilled water. Then, the absorption spectrum was measured to determine the absorbance (absorbance 0.482). Molecular extinction coefficient of FLUOS calculated value using ^{(8.9xl0 4) (54.2 μ mol} / 1) force, when the to calculate the amount of Furuoresein molecules became 54.2 mol / lx473.4x3 / 1000 = 0.077mg . Since the amount of fluorescein added initially is S0.165 mg (more precisely, the amount of FLUOS), the labeling rate (labeled molecule content) of Siri-Bogball B in sample solution B is 0.077 / 0.165x100 = 46.7%. It was judged.

[0061] From the above, 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%.

[0062] (4) Number of fluorescent molecules per particle

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).

[0063] Assuming that the number of fluorescein molecules contained in one particle of silica sphere B (diameter 4 nm) is one, fluorescein contained in one particle of silica sphere A (diameter 20 nm) based on the diameter of silica sphere The number of molecules was determined. As a result, the number of fluorescein molecules contained in each silica sphere A (diameter 20 mm) was 97 molecules / SiO particles [silica sphere B (diameter 4 nm) per particle.

2

The number of fluorescein molecules contained in 1 molecule / SiO particles].

2

[0064] (5) Fluorescence intensity per particle

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).

On the other hand, 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].

[0066] From this, 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.

[0067] (6) Concentration of fluorescein molecule in one particle

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

2

Concentration of Furuoresein molecule is 38.4mmol / l (97 / 6.02 x 10 23 /4.2 x 10- 18 χ1000 = 38. 4mmol / l). From this, 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. On the other hand, 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

2

The concentration is 48.9 mmol / l (1 / 6.02 × 10 ²³ /3.4×10— ^2, x 1000 = 48.9 mmol / l). From this, 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.

[0068] The concentration of fluorescein molecules in one particle of silica sphere obtained by Imhofe's method is 3 丄 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.

[0069] (7) Self-quenching

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. This result is almost the same as that of the FLU OS aqueous solution (fluorescein coated with silica) measured in the same way (fluorescence lifetime: 3.8 nsec), and silica sphere A (diameter 20 nm). ) Was found not to cause self-quenching.

[0070] (8) Summary

From the above, by the method of (1), 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. In addition, 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.

2

In molecule (in other words, one fluorescein molecule is covered with SiO molecule)

2), the fluorescence intensity of the fluorescein molecule increases by about 1.2 times.

[0071] 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 2

[0072] (1) Silica sphere A prepared in 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 μ1, 27 wt% aqueous ammonia 50 μ1 were mixed, and then magnetic stirring was performed at room temperature for 24 hours. Then, ultrafiltration (filter: UF disk ΥΜ 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). The diameter of the silica sphere was 230 nm, which was about 10 times larger than the diameter of silica sphere A (20 nm). In order to further increase the thickness of the silica layer, it is possible to prepare silica spheres having a desired size by repeating the above reaction operation in consideration of the ratio of the thickness of the silica layer increased by the above reaction. it can. [0073] FIG. 2 schematically shows the above reaction. In the figure, R means fluorescein (labeled molecule), and R 'means a hydrogen atom.

[0074] (2) Absorption spectra of silica sphere A (diameter 20 °) (1st growth) prepared in Example 1 and silica sphere (2nd growth) (diameter 230 nm) prepared in (1) above in an aqueous solution. Figure 3 shows the spectra as A and B, respectively. As can be seen from these results, the silica sphere A of Example 1 (diameter 20 nm) (1st Growth) and the silica sphere (diameter 230 nm) (2nd Growth) prepared in (1) above were all offset by 490 °. A typical absorption peak could be observed. In the absorption spectrum of the silica sphere (diameter 230 nm) (2nd Growth) prepared in (1) above, a tendency for the absorption to increase toward the shorter wavelength side was observed. This was thought to be due to the scattering effect caused by the larger particles.

[0075] In addition, the results are not shown! /, But V, and the silica sphere of the deviation also showed a fluorescence spectrum based on fluorescein (labeled molecule). The concentration of Furuoresein silica spheres A solution containing (diameter 20 nm) (3 mL) was 0.075mM [molecular extinction coefficient of Furuoresein; 7.5Xl0 calculated as ^4].

[0076] The aqueous solution of silica sphere A (diameter 20 nm) (1st Growth) had a deep yellow color and was stable even after one month. In general, 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.

[0077] (3) The 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.

Example 3

[0078] Preparation and modification properties of silica spheres containing labeled molecules with NH groups on the surface

2

Dispersion of fluorescein (labeled molecule) -containing silica sphere A (particle size 20 nm) prepared in Example 1 0.5 ml of aqueous solution together with 4.5 ml of 4 mM 3- (aminopropyl) triethoxysilane [APS] The reaction was allowed to proceed overnight under heat. As a result, it was possible to produce silica spheres (NH-based surface modified silica spheres) having NH groups as acceptor groups on the surface by attaching APS to the silica spheres.

twenty two

[0079] a) Protein modification

Under the fluorescence microscope, NH base surface modified silica sphere 5 / z g obtained above was replaced with 0.5 / z g / ml Gre

2

It was mixed with en fluorescein protein (GFP) 51. Then, immediately after mixing, NH base surface repair

2 Decorative silica sphere power It was confirmed to emit GFP green fluorescence. From this result, it was proved that the above-mentioned NH-based surface modified silica sphere attaches GFP (protein) to its surface.

2

[0080] b) Genetic modification

Under a fluorescent microscope, 5 g of the NH-based surface modified silica sphere obtained above was converted to 20 mM fluorescence.

2

Labeled DNA [PCR primer: FITC-GST-AS (FITC-5, -GGCAGATCGTCAGTCA GTCAC-3 '): SEQ ID NO: 1] (manufactured by Invitrogen) was mixed with 51. Then, immediately after mixing, it was confirmed that the NH base surface modified silica spherical force FITC fluoresced. this

2

The results show that the NH-based surface modified silica sphere attaches DNA to its surface.

2

It was.

Example 4

[0081] Preparation of NH-based surface-modified silica spheres and fluorescein (labeled molecule) -containing silica spheres

2

Binding (1) 3- (aminopropyl) triethoxy diluted 10-fold with dimethyl sulfoxide (hereinafter also referred to as “DMSO”! /, U) in 2 ml of sample solution B (silica sphere B: particle size 4 nm) prepared in Example 1 Silane [APS] solution 21 was added and stirred at room temperature for 1 hour. As a result, silica spheres (NH group surface modified silica spheres) having NH groups derived from APS as acceptor groups on the surface of silica spheres B (fluorescein (labeled molecule) -containing silica spheres) could be produced. .

twenty two

[0082] Thereafter, 0.1 ml of this mixed solution was added to a 1.5 ml reaction tube, and 40 1 of dartal aldehyde (coupling agent) was added thereto and stirred at room temperature (sample of the present invention). In addition, the use ratio of the dartal aldehyde is 1 mol of the NH base surface modified silica sphere.

2

Is 2190 moles per mole and 329 moles per mole of APS. As a control test, 40 μ1 distilled water was used instead of the 40 μ1 dartal aldehyde, and a similar stirring reaction was performed. (Control sample).

[0083] The fluorescence intensity of the sample of the present invention and that of the control sample were measured.

Whereas the excitation wavelength was 503 nm and the emission wavelength was 520 nm, 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

Two surface layers It was observed that the modified silica spheres gathered to form aggregates (multiple bonds of silica spheres). That is, it was speculated that the decrease in the fluorescence intensity was caused by interference between the light excited by the silica spheres gathered and the light emitted.

[0084] FIG. 6 schematically shows the above reaction.

[0085] (2) When 0.1 ml of a mixed solution of silica sphere B, DMSO and APS is further added to this solution (the sample of the present invention), the fluorescence intensity becomes 25, which is compared with the initial fluorescence intensity (about 10). The fluorescence corresponding to about 15 increased. On the other hand, when 0.1 ml of mixed solution was added to the control sample to make 0.2 ml, the fluorescence intensity that was initially 145 became 72.5 (actual measurement). From this, it is considered that the particles corresponding to the fluorescence intensity of 72.5-15 = 57.5 were used in the above preparation of the silica sphere multiple bond.

[0086] When 0.1 ml (0.3 ml in total) of the mixed solution is further added to the above-mentioned sample of the present invention, the fluorescence intensity becomes 70, which is about 45 (fluorescence intensity: 70-25 = 45) compared to the above fluorescence intensity 25. Fluorescence equivalent to increased. On the other hand, when 0.1 ml of distilled water was further added to the above control sample to make 0.3 ml, it was 48 times (actual measurement) because it was diluted 3 times. Since this value is almost equal to the above fluorescence increase 45, it seems that the 0.1 ml of the mixed solution added to the sample of the present invention is not related to the multiple binding of silica spheres. For these reasons, 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).

[0087] From the above, by treating silica spheres with aminopropyltriethoxysilane (silica compound), NH groups were introduced as acceptor groups on the surface of silica spheres, and then coupling

2

By treating with dartal aldehyde as a ligating agent, 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.

Example 5

[0088] Preparation of labeled molecule-containing silica spheres with SH groups on the surface

(1) Fluorescein (labeled molecule) -containing silica compound prepared in Example 1 (1) (3) 50 μ1 was mixed with 3 ml of water, 10 ml of ethanol, 0.15 ml of γ-mercaptopropyltriethoxysilane (MPS), and 27 wt. The mixture was mixed with 1 ml of an aqueous ammonia solution and stirred at room temperature for 24 hours. The obtained solution (reaction completed solution) was subjected to ultrafiltration (Amicon (registered trademark) stirring cell) (filter; UF disk YM100 Ultracell RC100K NMWL) (sales company: MILLIPORE 丌 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).

[0089] (2) Next, 10 μ 1 of SH base surface modified silica spheres were charged with 10 μ 1 of an aqueous solution of dartathione-S-transferase (rhodamine labeled GST) labeled with 1.0 mg / ml of rhodamine, When observed with a fluorescence microscope, rhodamine fluorescence was observed (FIG. 7b). This confirmed that rhodamine-labeled GST can be attached to the surface of silica spheres (SH-based surface-modified silica spheres).

Example 6

[0090] Preparation of silica compound containing piotin (labeled molecule) and silica spheres using this

Preparation of (labeled molecule-containing silica sphere)

(1) Preparation of silica compound containing piotin (labeled molecule)

Piotin (labeled molecule) -containing silica compound (7) was prepared according to the following formula.

[0091] [Chemical 5]

[In the formula, R means piotin (labeled molecule). In R, * is a bond with an ester group.

twenty two

It means a part. ]

Specifically, as a succinimidyl ester compound, D-Biotin-N-hyd is formed by binding biotin (labeled molecule) (shown by R in the formula) and succinimide through an ester bond.

2

After dissolving roxysuccinimide ester (o (Roshe Molecular Biochemicals) (approx. 50 mg) in DMSO solution (approx. 1 ml) (final concentration: 146 mM), 5.7 3- of 3- (aminopropyl) as a silica compound having 35 μ1 and amino groups. ) Triethoxysilane [APS] (2) 1 1 and DMSO 5 ml were mixed and stirred for about 1 hour to react to prepare a silica compound (7) containing piotin (labeled molecule).

(2) Preparation of Silica Sphere (Silica Sphere Containing Piotin and Fluorescein as Labeling Molecules) Next, silica particles (8) containing piotin and fluorescein as labeling molecules were prepared according to the following formula.

[0094] [Chemical 6]

(In the formula, R represents a fluorescein molecule, R represents a piotin molecule, and in R and R,

1 2 1 2

* Means a bonding site with an ester group. )

Specifically, 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 (1) Mix 2.5 ml of the reaction solution containing it, and add 20 ml of ethanol, 6 ml of water, 0.3 ml of tetraethoxysilane (TEOS), 2 ml of 27% by weight ammonia water and stir at room temperature for 24 hours using a stirrer. did. After completion of the reaction, the obtained reaction solution was subjected to ultrafiltration [Amicon (registered trademark) stirring cell] (filter; UF disk YM 100 Ultracell RC100K NMWL) (sales company: MILLIPORE) [Nominal Molecular Weight Limit (NMWL ): 100 kDa]. Thereby, silica spheres containing piotin and fluorescein as labeling molecules [ Piotin-fluorescein (labeled molecule) -containing silica sphere] (8) was prepared.

[0096] Next, 5 μ1 of the silica sphere (8) containing piotin-fluorescein (labeled molecule) obtained in (5) was mixed with 5 μ1 of a 0.5 mg / ml avidin solution. Was recognized. On the other hand, as a control experiment, a silica ball (5) containing fluorescein (labeled molecule) containing no biotin prepared by the method of Example 1 (1) and (2) was mixed with an avidin solution under the same conditions as described above. As a result, no aggregated image was observed in this case.

[0097] From this, 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.

Example 7

[0098] Preparation of Rhodamine (labeled molecule) -containing silica compound, and preparation of silica sphere [rhodamine (labeled molecule) -containing silica sphere] using the same

(1) Preparation of Rhodamine (labeled molecule) -containing silica compound

A rhodamine (labeled molecule) -containing silica compound was prepared according to the following formula.

[0099] [Chemical 7]

[In the formula, R means rhodamine (labeled molecule). In R, * indicates the bond with the ester group.

3 3

means. ]

Specifically, as a succinimidyl ester compound, 5-carboxyltetramethylrhodamine formed by binding podamine (labeled molecule) and succinimide via an ester bond. About 5 mg of 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. Carry out 51.2 1 in DMSO so that it is equimolar with the midyl ester compound (9) and stir with a stirrer piece for about 1 hour to react with the carbonyl of succinimidylsteri compound (9). 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.

[0101] (2) Preparation of silica sphere

Rhodamine (labeled molecule) -containing silica compound (10) force Rhodamine (labeled molecule) -containing silica sphere (12) was prepared according to the following formula.

[0102] [Chemical 8]

(1 1)

[In the formula, R means rhodamine (labeled molecule). ]

Three

Specifically, tetraethoxysilane (TEOS) 0.3 ml, water 6 ml and ethanol 20 ml were added to DMSO solution 5 ml of the reaction solution [rhodamine (labeled molecule) -containing silica compound (10)] obtained above. (Ethanol: water = 4: 1, volume ratio) 2 ml of about 30% ammonia water was added to this and left at room temperature with stirring for one day. The resulting solution (reaction completed solution) was subjected to ultrafiltration (Amicon (registered trademark) stirring cell) (filter; UF disk YM100 Ultracell RC100K NMWL) (sales company: MILLIPORE 丌 Nominal Molecular Weight Limit (NM WL) : 100 kDa], and filtration and washing with distilled water were repeated several times to prepare silica spheres (11) containing tetradamine (labeled molecule), which was transmitted electron microscope (TEM) A photograph (X 10,000) and a fluorescence microscope image are shown in FIGS. 8a and 8b. Example 8

[0104] Preparation of labeled molecule-containing silica spheres with SH groups on the surface and their properties

(1) Rhodamine (labeled molecule) -containing silica compound (10) prepared by the method described in Example 7 (1) 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. Transfer the reaction solution to an ultrafiltration device [Amicon (registered trademark) stirring cell] (filter; UF disk YM 100 Ultracell RC100K NMWL) (Distributor: MILLIPORE) [Nominal Molecular Weight Limit (NMWL): 100 kDa] And washed.

(2) 0.1 ml of the washed silica sphere-containing solution was added to 1 ml of a 5.8 mM MPS (γ-mercaptoprovir triethoxysilane) aqueous solution and allowed to react at room temperature. As a result, MPS was attached to the surface of rhodamine (labeled molecule) -containing silica sphere (11), and a silica sphere (SH group surface-modified silica sphere) having an SH group as an acceptor group on the surface could be produced. .

(3) Next, washing with distilled water was performed using a centrifuge. Mix the obtained silica sphere (SH-based surface modified silica sphere) -containing solution 2 μ 1 and physiological saline solution 31 containing Green fluorescein protein (G FP) at a rate of 34 μg / ml, and use a fluorescence microscope. As a result, it was confirmed that the SH base surface-modified silica spherical force GFP emitted green fluorescence immediately after mixing. From this result, it was found that the SH-based surface modified silica sphere adsorbs protein on its surface just by mixing with a solution containing protein without the need for other special reagents.

Example 9

[0107] 3 ml of water, 10 ml of ethanol, 5.7 M gamma mercaptopropyltriethoxysilane (MPS) 0.

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.

[0108] To the obtained SH base surface modified silica sphere 10 μ1, 1.0 mg / ml rhodamine-labeled dartathione-S transferase (rhodamine-labeled GST) aqueous solution was added to 10 μ1, and fluorescence microscopy was performed. Observation was performed with a microscope. As a result, rhodamine fluorescence was observed (FIG. 9a). Next, after blocking with l.Omg / ml ushi serum albumin, it was washed with a centrifuge. Washed rhodamine-labeled GST-bound SH group surface modified silica spheres were mixed with anti-GST antibody 5 μ1 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.

As described above, from the results of Example 3 (1), Examples 8 and 9, according to the silica sphere of the present invention, proteins can be attached via the acceptor group on the surface thereof. It was shown that an antigen-antibody reaction can be performed.

Claims

The scope of the claims

[1] (a) Succinimidyl ester compound (1) obtained by binding a labeled molecule and succinimide via an ester bond (-CO-0-) (1) and a silica compound having an amino group (2 ) To produce a labeled molecule-containing silica compound (3),

as well as

A method for preparing a labeled molecule-containing silica sphere (5).

[2] As the silica compound having an amino group (2), 3- (aminopropyl) triethoxysilane or 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane is used. The method for preparing a labeled molecule-containing silica sphere according to claim 1.

[3] As the silica compound (4), tetraethoxysilane, γ-mercaptopropyltriethoxysilane, aminopropyltriethoxysilane, 3-thiocyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, A group power consisting of 3-isocyanatopropyltriethoxysilane and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane is used, and at least one silica compound selected is used. A method for preparing a labeled molecule-containing silica sphere according to claim 1 or 2.

[4] The method for preparing a labeled molecule-containing silica sphere according to any one of claims 1 to 3, wherein the step (b) is performed in the presence of water, alcohol, and ammonia.

5. The method for preparing a labeled molecule-containing silica sphere according to claim 4, wherein the volume ratio of water to alcohol is 1: 0.5 to 1: 8.

[6] A labeled molecule-containing silica sphere obtained by the method according to any one of claims 1 to 5.

[7] The labeled molecule-containing silica sphere obtained by the method according to any one of claims 1 to 5, further comprising a silica compound (4) different from the silica compound (4) used in the step (b) (4) A method for preparing a labeled molecule-containing silica sphere, which comprises a step of treating with (1).

[8] A labeled molecule-containing silica sphere obtained by the method according to claim 7.

[9] On the surface of the labeled molecule-containing silica sphere according to claim 6 or 8, peptides, proteins, residues Silica spheres formed by binding genes, microorganisms, coupling agents, piotin, avidin, or labeled molecules.

[10] The labeled molecule-containing silica sphere obtained by the method according to any one of claims 1 to 5, further comprising (c) a silica compound used in step (b), if necessary (4 And a step of treating with a silica compound (4) different from

[11] A multi-bonded product of labeled molecule-containing silica spheres obtained by the method according to claim 10.