JP2003270154A - Fluorescent dye molecule-containing silica ball - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent dye molecule-containing silica ball with superior performance and stability in manufacturing. <P>SOLUTION: The fluorescent dye molecule-containing silica ball is prepared by using a fluorescent dye molecule represented by structural formula 1 or R-CO-NH-(CH<SB>2</SB>)<SB>3</SB>-Si-(C<SB>2</SB>H<SB>5</SB>O)<SB>3</SB>(in the formula, R is a substance capable of having NHS in a side chain). <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a labeled molecule-containing silica sphere useful as a detection system reagent.

[0002]

2. Description of the Related Art In recent years,
Various studies on biochemical examination methods using fine particles containing a fluorescent dye have been conducted. For example, the alpha screen technique has been commercialized by Wackard. This is diameter 2
A 50 nm latex donor and Accector beads (registered trademark) are used. After the donor beads and Accector beads are bound, when the donor beads are excited by a laser, the fluorescence from the internal fluorescent molecules causes singlet oxygen molecules to be emitted. Is generated, which chemically reacts with the fluorescent substance in the axe sector beads to generate chemiluminescence, which is observed.

On the other hand, various methods for producing silica spheres containing fluorescent dye molecules inside have been proposed, and as typical ones,
There is one prepared by directly binding FITC (fluorocein isothiocyanate) directly to APS (γ-aminopropyl orthosilicate) (J. Phys. Chem. B 199).
9,103, 1408-1415), which holds the highest concentration of fluorescent dye molecules in silica spheres in this type of manufacturing method. But,
It has also been reported that quenching occurs inside the silica sphere at its maximum concentration. Thus, by surrounding the fluorescent dye molecule with silica, there is no quenching by an external factor (absorption of excitation energy by a biochemical polymer), and it is expected to be applied to various tests.

However, as a result of further investigations by the present inventors, in the production of the above silica spheres, the binding property of FITC and APS is not high, the production efficiency is poor, and the obtained particle size is uniform. There were drawbacks such as several hundred nanometers.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems of the prior art, and an object thereof is to provide a fluorescent dye molecule-containing silica sphere which is excellent in manufacturing stability and performance. To do.

The inventors of the present invention have conducted extensive studies to achieve such an object, and as a result, have found that it is extremely effective to introduce a specific compound into silica spheres containing fluorescent dye molecules, and have completed the present invention. It was

[0007] That is, the present invention is a fluorescent dye molecule-containing silica sphere prepared by using a fluorescent dye molecule represented by the following structural formula (1).

R—CO—NH— (CH ₂ ) ₃ —Si— (C ₂ H ₅ O) ₃ (1) (In the formula, R is a substance capable of having NHS in the side chain.) Preferably , R is a substance selected from FITC (fluorocein isothiocyanate), a dye, a magnetic substance and a free radical.

In particular, FITC-CO-NH- (CH ₂ ) ₃ -Si
_{- (C 2 H 5 O)} 3 (1 ') is preferable. In formula (1 '), FITC
Represents fluoroscein isothiocyanate.

FITC is an example, and instead, other dyes such as rhodamine, magnetic substances such as iron oxide, stable free radicals such as spin labeling agents, R is NHS.
Include all substances that can have a.

Further, the present invention provides a silica sphere having various functions provided by surface-modifying the surface of the above-mentioned fluorescent dye molecule-containing silica sphere by chemical treatment.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below.
The present invention is a process for producing “FITC-APS-silica” which is a conventionally proposed fluorescent sphere-containing silica sphere,
FITC-NHS having NHS (N-hydroxysuccinimide ester) as a side chain is used for FITC,
FITC-CO-APS was prepared by utilizing the strong binding ability between the amine group of APS and the CO group of FITC, and the production stability thereof was remarkably improved.

The method for producing the fluorescent sphere-containing silica spheres of the present invention may be the same as that of the above-mentioned prior art. That is, N-
Fluorescein isothiocyanate or biotin molecule having hydroxysuccinimide ester as a side chain is used as a raw material and is a silane coupling agent.
-With the compound formed by the binding of aminopropyl orthosilicate as the nucleus, according to the method of Stober, that is, by hydrolysis and polycondensation of tetraorthosilicate with ammonia in aqueous ethanol solution at room temperature in the atmosphere of 4: 1 ethanol. Tetraorthosilicate can be used in aqueous solution to produce silica spheres containing fluorescent dye molecules.

More specifically, FITC-NHS-APS
DMSO solution (5 ml) was added to water (6 ml), the ratio of ethanol (20 ml) to the aqueous solution was 4: 1, and tetraorthosilicate (0.3 ml) and about 30% ammonia water (2 ml) were added to the mixture. To do.

In this case, by changing the concentration of tetra-orthosilicate and the number of times of this aging, the size of the silica spheres obtained is changed from several nm in diameter to several hundreds nm, and further, μ.
Can be freely adjusted to m-order.

Further, instead of tetraorthosilicate,
γ-Mercaptopropyltriethoxysilane (MPS)
It is also possible to manufacture using. In the above method, instead of 0.3 ml of tetra-orthosilicate, 5M MPS
Silica spheres are similarly formed by adding 0.3 ml, and the efficiency of containing the fluorescent dye and the efficiency of modifying the surface layer are very high. As described above, even when an effective silica sphere is formed by using a silane coupling agent other than APS, it is expected to contain a high fluorescent dye, and its surface modification ability and property change are expected.

The silica spheres thus obtained are purified by removing coexisting ions by a well-known ultrafiltration method or adjusted to a desired particle size distribution.

As described above, the silica sphere containing a fluorescent dye molecule of the present invention can have a size of several nanometers and several tens of nanometers, and when it is bound to a protein by preparing NHS on its surface, the silica spheres are removed. It is useful as a detection system reagent that is difficult to undergo and is not easily quenched. In addition, the conventional "F
The "ITC-NHS" molecule has poor stability, so the FITC that binds to the protein of the antibody is only about 4 molecules, and the sensitivity is low, and it can be applied only to the fixed shape on the dish, that is, it has poor practical performance. On the other hand, in the fluorescent dye molecule-containing silica spheres of the present invention, since thousands of FITCs are densely packed in the silica spheres without concentration quenching,
It has an excellent effect that it emits strong light when irradiated with laser and has high sensitivity.

Further, in the case of the fluorescent sphere-containing silica spheres of the present invention, silica is chemically inactive and easily modified, so that, for example, its surface is made mesoporous or newly specified. It is possible to immobilize on the surface an acceptor molecule for binding to the protein.

That is, the functional addition of nanoparticles is important for controlling substances at the nano level and improving functional characteristics.
Modification of the surface of the nanoparticles is mentioned as an important issue for the functional addition of the nanoparticles. In the case of the fluorescent dye molecule-containing silica sphere of the present invention, various substances are modified on the surface layer by chemically treating the surface. It is possible.

The substances used for such purpose include γ-aminopropyl orthosilicate (APS),
γ-Mercaptopropyltriethoxysilane (MPS)
Examples include various silane coupling agents such as silane coupling agents and their various cross-linking agents, various proteins, various DNAs, and the like. Specific chemical treatments (modification treatments) include fluorescent dye molecule-containing silica spheres. A method such as stirring in an aqueous solution of the above substance may be used.

By such a modification treatment of silica spheres, a wide variety of functional additions can be carried out, and such functionalized silica spheres are more versatile for various application techniques due to their shape, inclusion and surface modification characteristics. Various applications are possible.

First of all, from its shape, biologically,
It may be possible to reconstitute microorganisms such as bacteria and animal cells. For example, a virus surface protein binds to the surface of a sphere,
For example, a "pseudovirus" whose outline is similar to the virus but does not have the gene of interest is proposed, which may be applied to immunization or vaccine of animals that do not require adjuvant. It is also possible to measure the antibody titer using the "pseudovirus" containing fluorescence inside and the technique of "Measuring method of agglutination reaction" described in PCT / JP / 11573. Furthermore, it is also possible to use the "pseudovirus" as a means of drug delivery by utilizing the organ-specific infectivity of the virus.

Also, by binding and presenting antibodies and T cell receptors on the surface, "immunized silica spheres" similar to B cells and T cells can be prepared, and not only for research,
It can be expected to be applied to diagnosis and treatment in cancer and immune diseases. Furthermore, if it becomes possible to reconstitute the receptor, signal molecule, and gene expression system in the silica sphere, it is considered that it can be used for “silica (β) cells” such as cells artificially reproducing pancreatic β cells that secrete insulin. To be

The surface-modified property can give the silica sphere the property of binding substances such as arbitrary proteins and genes and presenting its function as described above. However, this is not limited to what develops solely from the nature of binding. That is, the isolated unit of a sphere also has the property that it can efficiently bind to a substance and concentrate the substance on the surface of the sphere, as will be shown in Examples below. This can be achieved, for example, by binding various enzymes that catalyze multi-step reactions onto silica spheres and concentrating them to improve reaction efficiency (reaction-enhanced silica spheres), by multiplexing antibodies on silica spheres. It is considered that it can be exerted by the usage (bond-reinforced silica spheres) for improving the bonding property.

Alcohol dehydrogenase (AD
H) After attaching the silane coupling reagent (APS) to the surface, an activated silica solution (containing 27% SiO ₂ ) was used to
By covering DH with silica, ADH can be fixed inside the silica sphere (modification of functional protein).
The contained silica spheres show enzyme activity in the presence of ethanol and coenzyme (NAD), and production of NADH can be confirmed from its fluorescence peak.

Further, a method of adsorbing a substance diffusing in a liquid to silica spheres and concentrating it (substance-concentrating (recovering) silica spheres) can be mentioned, and the examples described later can be interpreted as the result of this characteristic.

These effects are not limited to the direct characteristics of silica spheres, such as concentration of antibodies using silica spheres surface-modified with antigen, concentration of biotinylated proteins using silica spheres surface-modified with avidin, etc. It is also effective in indirect and multi-stage usage.

Further, the property as a usage (adhesive silica sphere) of conjugating two or more kinds of substances which are originally difficult to conjugate through silica spheres is also conceivable.

As another application technique, for example, a bar code labeling method can be considered. In silica spheres to which various functions have been added (for example, various antibodies and peptides are displayed on the surface as a library, etc.), it is extremely convenient for the application of functional silica spheres to quickly distinguish them. Is one of the methods, and a bar code label is one of the methods. Specifically, by mixing two or more types of fluorescently labeled fluorescent dye molecule-containing silica spheres of the present invention at various mixing ratios, barcode-labeled silica spheres having versatile fluorescent characteristics can be prepared and flow It can be distinguished by cytometry or fluorescence microscopy. Further, the surface can be labeled with a barcode by modifying the surface layer with a dye, a protein, a gene or the like at various mixing ratios using the surface modification technique. In particular, in recent years, gene sequencing technology has been simplified, and it is possible to identify a specific gene sequence by binding it to the surface of silica spheres and, if necessary, amplifying it by PCR and reading the base sequence. Become.

[0031]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Fluorescein isothiocyanate or biotin molecule having N-hydroxysuccinimide ester as a side chain was used as a raw material, and a silane coupling agent, 3-
Using the compound formed by the binding of aminopropyl orthosilicate as a nucleus, according to the method of Stober, that is, by hydrolysis and polycondensation of tetraorthosilicate with ammonia water in an ethanol aqueous solution at room temperature in the atmosphere in a 4: 1 ethanol aqueous solution. Then, silica spheres containing fluorescent dye molecules were prepared using tetraorthosilicate.

When the biotin / silica particles thus prepared were mixed with a protein having an avidin chain, avidin-protein aggregation could be observed by the biotin of the same sample.

Further, similarly, when biotin-silica particles containing a fluorescein isothiocyanate molecule were prepared and the same observation was carried out, the same agglutination was observed, and the protein labeling by the fluorescein isothiocyanate-silica particles was confirmed. It could be confirmed. Example 2 Silica spheres containing fluorescent dye molecules prepared in Example 1 (A: 1
FIG. 1 shows an absorption spectrum of an aqueous solution of (st Growth, B: 2nd Growth). 1st Growth
The color of the solution was deep yellow and was stable even after 1 month. This indicates that the solution pH at the time of sample preparation is strongly alkaline and remains in that state inside the silica spheres. In both cases, a typical absorption peak can be observed at 490 nm, and in the 2nd Growth absorption spectrum, a scattering effect based on large particles in which the absorption increases toward the short wavelength side was observed.

The concentration of fluoroscein isothiocyanate estimated from the molecular extinction coefficient was 0.075 mM in the case of 1st Growth. Furthermore, when the fluorescence spectra were measured, both had a typical fluorescence peak.

Further, according to the result of the fluorescence lifetime measurement (FIG. 2), a free state in an aqueous solution (3.8 nse
Since it was almost the same as that in c), it was found that self-quenching did not occur in this sample.

When the same sample was observed with a transmission electron microscope, particles of several nm to several hundred nm were observed, and 1st G
The person with low ghth (Fig. 3) has unevenness on the surface and has a mesoporous appearance, and the person with 2 nd growh (Fig. 4)
Had a smooth surface. Example 3 (Preparation of amine surface-modified silica spheres and surface-modified properties) 5 mM of the fluorescent dye molecule-containing silica spheres prepared in Example 1 was used.
Of APS (γ-aminopropyl orthosilicate) aqueous solution to attach APS to the surface layer and N
It was possible to make silica spheres that present H ₂ on the surface. This amine surface-modified silica sphere does not require any other special reagent and can be mixed with a protein solution to form a protein or DN on the surface.
A was adsorbed. a) Protein-modified silica spheres are attached to Green fluorescein pr
Immediately after mixing with the protein (GFP), it was possible to confirm that the silica spheres emitted GFP fluorescence immediately after that under a fluorescence microscope. b) DNA obtained by fluorescently modifying gene-modified silica spheres (PCR primer)
FITC-GST-AS (FITC-5'-GGCAG
When mixed with ATCGTCAGTTCAGTCAC-3 ')), it was confirmed immediately after that that the silica spheres emit FITC fluorescence under a fluorescence microscope. Example 4 (Preparation of SH surface-modified silica sphere and surface modification property) 5 mM of the silica dye containing a fluorescent dye molecule prepared in Example 1 was used.
By agitating in the MPS (γ-mercaptopropyltriethoxysilane) aqueous solution of, it was possible to fabricate silica spheres in which MPS was attached to the surface layer and SH was presented on the surface. When this SH surface layer-modified silica sphere was mixed with GFP, it was possible to confirm that the silica sphere emits GFP fluorescence immediately after that, under a fluorescence microscope. Example 5 (Preparation of protein surface layer-modified silica spheres) a) Immobilization of carboxyl group of protein The amine surface layer-modified silica spheres prepared in Example 3 were cross-coupled with 1-ethyl-3- (3-dimethylaminopropyl). ) When carbodiimide was added, the amino group on the surface of the silica sphere and the carboxyl group of the protein could be firmly bound. b) Immobilization of amino group of protein On the amine surface-modified silica spheres prepared in Example 3, succinomidyl trans-4- which is a cross-coupling reagent.
When (maleimidylmethyl) cyclohexane-1-carboxylate was added, the amino groups on the surface of the silica sphere and the amino groups of the protein could be firmly bound. Example 6 (Application to detection reaction) a) Detection on silica spheres 5 ml of DMSO solution of Rhodamine-NHS-APS,
In addition to 6 ml of water, the ratio of 20 ml of ethanol to the aqueous solution was 4: 1, and 0.3 ml of 5M γ-mercaptopropyltriethoxysilane (MPS) and 2 ml of about 30% ammonia water were added to this, and stirred for one day. While standing still, silica spheres containing fluorescent dye molecules were prepared. A transmission electron micrograph and a fluorescence microscopic image of this product are shown in FIGS. 5a and 5b.
Shown in.

Next, silica spheres containing fluorescent dye molecules were prepared by the above-mentioned method, and Rhodamine-labeled glutathione-labeled on the surface.
S-transferase (GST) protein was attached (Fig. 6a). Rhodamine fluorescence was confirmed on the silica spheres by a fluorescence microscope (Fig. 6b). Next, silica spheres containing no fluorescent dye were prepared by a method using MPS, and Rhodamine labeled G
The ST protein was bound (Fig. 7a). After blocking with bovine serum albumin, it was washed using a centrifuge. The washed GST surface-modified silica spheres were reacted with an anti-GST antibody labeled with FITC and observed with a fluorescence microscope. As a result, as shown in FIG.
Fluorescence of TC was observed, and it was confirmed that the anti-GST antibody was bound to the antigen GST on the silica sphere.

[Brief description of drawings]

FIG. 1 is a silica sphere containing a fluorescent dye molecule prepared in Example 1 (A: 1st Growth, B: 2nd Growth).
It is a figure which shows the absorption spectrum in the aqueous solution of h).

FIG. 2 Silica spheres containing fluorescent dye molecules prepared in Example 1 (A: 1st Growth, B: 2nd Growth)
It is a figure which shows the fluorescence decay curve of h).

3 is a TEM photograph (× 90,000) of the fluorescent dye molecule-containing silica spheres (1st Growth) prepared in Example 1. FIG.

FIG. 4 is a TEM photograph (× 15,000) of the fluorescent dye molecule-containing silica spheres (2st Growth) prepared in Example 1.

5 is a TEM photograph (× 10,000) of the fluorescent dye molecule-containing silica spheres prepared in Example 6 (FIG. 5a) and a finding under a fluorescence microscope in which the SH surface-modified silica spheres and GFP were mixed (×). 400) (Fig. 5b).

FIG. 6 is a finding (b) in which Rhodamine glutathione-S-transferase (GST) protein was attached onto the fluorescent dye molecule-containing silica spheres (a) prepared in Example 6 (× 40).
0).

FIG. 7: On a silica sphere containing no fluorophore
Rhodamine glutathione-S-transferase (G
(ST) Protein is attached (a) and then FITC-labeled anti-GST antibody is bound (b) (x400).

Claims (3)

[Claims] 1. A fluorescent dye molecule-containing silica sphere prepared using a fluorescent dye molecule represented by the following structural formula (1). _{R-CO-NH- (CH 2} ) 3 -Si- (C 2 H 5 O) 3 (1) ( wherein, R is a substance that may have an NHS in a side chain.) 2. The fluorescent dye molecule-containing silica sphere according to claim 1, wherein R is a substance selected from FITC (fluorocein isothiocyanate), a dye, a magnetic substance and a free radical. 3. A silica sphere whose surface layer is modified by chemically treating the surface of the fluorescent sphere-containing silica sphere according to claim 1 or 2.