Method for producing fluorescent silica nanoparticles, method for adjusting the particle size of fluorescent silica nanoparticles, and method for adjusting the fluorescence intensity of fluorescent silica nanoparticles.
The method for producing fluorescent silica nanoparticles adjusts particle size and maintains fluorescence intensity by using alkoxysilane and ammonia, addressing the need for control in manufacturing these nanoparticles.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- TOSOH CORP
- Filing Date
- 2024-11-14
- Publication Date
- 2026-05-26
AI Technical Summary
There is a need for novel methods to manufacture fluorescent silica nanoparticles and to control their particle size and fluorescence intensity.
The method involves producing fluorescent silica nanoparticles by adding alkoxysilane and ammonia to an aqueous solution containing a rare earth fluorescent complex, hexadecyltrimethylammonium bromide, and ethyl alcohol, allowing for adjustment of particle size and fluorescence intensity by varying the amounts of alkoxysilane and ammonia.
This method enables the production of fluorescent silica nanoparticles with adjustable particle sizes ranging from 20 nm to 250 nm and maintains fluorescence intensity even with smaller particle sizes.
Smart Images
Figure 2026086271000001 
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Abstract
Description
Technical Field
[0001] The present invention relates to a method for producing fluorescent silica nanoparticles, a method for adjusting the particle size of fluorescent silica nanoparticles, and a method for adjusting the fluorescence intensity of fluorescent silica nanoparticles.
Background Art
[0002] In recent years, it has been proposed to use fluorescent nanoparticles, particularly fluorescent silica nanoparticles, as detection reagents. For example, fluorescent nanoparticles are used in the field of biochemistry, particularly for cell staining and protein labeling.
[0003] Regarding such fluorescent nanoparticles, various studies have been conducted, and particularly, fluorescent silica nanoparticles encapsulating europium(III) chelate compounds have been studied (Non-Patent Documents 1 and 2).
Prior Art Documents
Non-Patent Documents
[0004]
Non-Patent Document 1
Non-Patent Document 2
Summary of the Invention
Problems to be Solved by the Invention
[0005] As mentioned above, various studies have been conducted on fluorescent nanoparticles. However, there is a need for novel methods to manufacture fluorescent silica nanoparticles. Furthermore, there is a need for methods to control the particle size and fluorescence intensity of fluorescent silica nanoparticles. [Means for solving the problem]
[0006] The inventors of this invention, after diligent research, discovered that fluorescent silica nanoparticles can be manufactured, the particle size of the fluorescent silica nanoparticles can be adjusted, and the fluorescence intensity of the fluorescent silica nanoparticles can be adjusted by the following means, thereby completing the present invention.
[0007] <Aspect 1> To provide an aqueous solution containing a rare earth fluorescent complex, hexadecyltrimethylammonium bromide, ethyl alcohol, and water, and Adding alkoxysilane and ammonia to the aqueous solution, A method for producing fluorescent silica nanoparticles, including [the specified ingredient]. <Aspect 2> The method according to embodiment 1, wherein the particle size of the fluorescent silica nanoparticles is 20 nm to 250 nm. <Aspect 3> The method according to embodiment 1, wherein the particle size of the fluorescent silica nanoparticles is 20 nm to 100 nm. <Aspect 4> The fluorescent silica nanoparticle according to embodiment 1, wherein the rare earth fluorescent complex is a europium(III) chelate compound. <Aspect 5> A method for adjusting the particle size of fluorescent silica nanoparticles, comprising adjusting the amount of alkoxysilane and ammonia added, according to the method described in any one of embodiments 1 to 4. <Pattern 6> A method for adjusting the fluorescence intensity of fluorescent silica nanoparticles, comprising adjusting the amount of alkoxysilane and ammonia added, according to the method described in any one of embodiments 1 to 4. [Effects of the Invention]
[0008] According to the present invention, it is possible to produce fluorescent silica nanoparticles, adjust the particle size of the fluorescent silica nanoparticles, and adjust the fluorescence intensity of the fluorescent silica nanoparticles. [Brief explanation of the drawing]
[0009] [Figure 1] Figure 1 shows the particle sizes of silica nanoparticles A1-A3 (Example A) and silica nanoparticles B1-B3 (Example B). [Figure 2] Figure 2 shows the relationship between the particle size of silica nanoparticles A1-A3 (Example A) and B1-B3 (Example B), and the fluorescence intensity due to DTBTA-Eu(III) encapsulated within these silica nanoparticles. [Modes for carrying out the invention]
[0010] Method for producing fluorescent silica nanoparticles The present invention's method for producing fluorescent silica nanoparticles includes the following steps: To provide an aqueous solution containing a rare earth fluorescent complex, hexadecyltrimethylammonium bromide, ethyl alcohol, and water, and Add alkoxysilane and ammonia to the aqueous solution.
[0011] According to the method of the present invention for producing fluorescent silica nanoparticles, fluorescent silica nanoparticles can be produced. Here, the particle size of these fluorescent silica nanoparticles may be 20 nm or more, 30 nm or more, 40 nm or more, or 45 nm or more, and may also be 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 80 nm or less, or 250 nm or less.
[0012] The particle size of the fluorescent silica nanoparticles can be measured by the dynamic light scattering method. Specifically, for example, the particle size of the fluorescent silica nanoparticles can be measured using a Zetasizer Ultra (manufactured by Malvern Panalytical) and the attached software. Here, a 12 mm square polystyrene cell (DTS0012 manufactured by Malvern Panalytical) can be used, and the equilibration time can be set to 120 seconds.
[0013] The fluorescent silica nanoparticles produced by the method of the present invention have a rare earth fluorescent complex encapsulated in the silica. Therefore, these fluorescent silica nanoparticles can be used, for example, as a diagnostic reagent using time-resolved fluorescence immunoassay (TR-FIA). Further, the fluorescent silica nanoparticles produced by the method of the present invention can be used as a fluorescent detection material that requires durability.
[0014] <Provision of an aqueous solution> In the method of the present invention for producing fluorescent silica nanoparticles, first, an aqueous solution containing a rare earth fluorescent complex, hexadecyltrimethylammonium bromide, ethyl alcohol, and water is provided.
[0015] Examples of the rare earth fluorescent complex that can be used in the method of the present invention include complexes formed by complexation of rare earth metals such as europium (Eu), samarium (Sm), terbium (Tb), and dysprosium (Dy) with organic ligands, particularly europium (III) chelate compounds. More particularly, examples of the rare earth fluorescent complex that can be used include DTBTA-Eu(III) (2,2′,2″,2″-{4′-{[(4,6-dichloro-1,3,5-triazin-2-yl)amino]biphenyl-4-yl}-2,2′-{2,2′,2″,2″-{4′-{[(4,6-dichloro-1,3,5-triazin-2-yl)amino]biphenyl-4-yl}-2,2′:6′,2″-terpyridine-6,6″-diyl}bis(methylenenitrilo)}tetrakis(acetate)-Eu 3+ ) can be mentioned.
[0016] The fluorescence of the rare earth complex is due to the energy transfer from the ligand in the complex to the central ion, which is excited by the absorption of light by the ligand in the near-ultraviolet region and emits fluorescence characteristic of rare earth ions in the visible light region. Examples of the organic ligand include DTBTA, β-diketone ligands (such as 2-naphthoyltrifluoroacetone (NTFA), 2-thenoyltrifluoroacetone (TTA), benzoyltrifluoroacetone (BFA), trifluoroacetylacetone, etc.). The rare earth fluorescent complex preferably has a silanol group and / or a silicon alkoxide group, and can form a silica bond (Si—O—Si) together with a difunctional, trifunctional, or tetrafunctional silicon alkoxide.
[0017] The concentration of the rare earth in the rare earth fluorescent complex in the fluorescent silica nanoparticles produced by the method of the present invention may be 0.1 μM or more, 0.5 μM or more, 1.0 μM or more, 1.5 μM or more, and may also be 30.0 μM or less, 20.0 μM or less, 10.0 μM or less, 5.0 μM or less, or 3.0 μM or less.
[0018] Here, the concentration of the rare earth in the rare earth fluorescent complex can be determined by using an ultraviolet-visible spectrophotometer (for example, UV-1700 manufactured by Shimadzu Corporation) to measure the absorption spectrum in a quartz cuvette and using the absorption peak and its molar absorption coefficient. For example, for the measurement of the concentration of europium as a rare earth, the absorption spectrum in a quartz cuvette with an optical path length of 1 cm can be measured and determined using the absorption peak at 340 nm and its molar absorption coefficient (ε = 31,000).
[0019] As hexadecyltrimethylammonium bromide, commercially available ones can be used, for example, those available from Fujifilm Wako Pure Chemical Corporation.
[0020] As ethyl alcohol, commercially available ones can be used.
[0021] As water, ultrapure water obtained by reverse osmosis can be used.
[0022] <Addition of alkoxysilanes, etc.> In the present invention's method for producing fluorescent silica nanoparticles, alkoxysilane and ammonia are added to an aqueous solution. This allows for the hydrolysis and dehydration condensation of silicon alkoxide to produce fluorescent silica nanoparticles, i.e., silica nanoparticles containing rare earth fluorescent complexes.
[0023] As the silicon alkoxide, any silicon alkoxide that can form silica by hydrolysis and dehydration condensation in an aqueous solution can be used, and in particular, bifunctional, trifunctional, or tetrafunctional silicon alkoxides, and more particularly tetraethyl orthosilicate (TEOS), can be used.
[0024] Commercially available ammonia can be used, and it can be added in the form of aqueous ammonia.
[0025] In the method of the present invention, the particle size and fluorescence intensity of the fluorescent silica nanoparticles produced can be adjusted by adjusting the amount of alkoxysilane and ammonia added. Specifically, for example, in the method of the present invention, the particle size of the resulting fluorescent silica nanoparticles can be reduced by increasing the amount of alkoxysilane and ammonia added. Furthermore, while generally, when the particle size of fluorescent silica nanoparticles is small, the fluorescence intensity decreases, in the method of the present invention, by increasing the amount of alkoxysilane and ammonia added, the fluorescence intensity can be maintained even when the particle size of the resulting fluorescent silica nanoparticles is small.
[0026] Methods for adjusting the particle size of fluorescent silica nanoparticles and methods for adjusting the fluorescence intensity of fluorescent silica nanoparticles. As described above, in the method for producing fluorescent silica nanoparticles, the particle size of the resulting fluorescent silica nanoparticles can be reduced by increasing the amount of alkoxysilane and ammonia added, and the fluorescence intensity can be maintained even when the particle size of the resulting fluorescent silica nanoparticles is small.
[0027] Therefore, the method of the present invention for adjusting the particle size of fluorescent silica nanoparticles includes adjusting the amount of alkoxysilane and ammonia added in the method of the present invention for producing fluorescent silica nanoparticles. Furthermore, the method of the present invention for adjusting the fluorescence intensity of fluorescent silica nanoparticles also includes adjusting the amount of alkoxysilane and ammonia added in the method of the present invention for producing fluorescent silica nanoparticles. For details on the components of the method of the present invention for adjusting the particle size of fluorescent silica nanoparticles and the method of the present invention for adjusting the fluorescence intensity of fluorescent silica nanoparticles, refer to the description of the method of the present invention for producing fluorescent silica nanoparticles. [Examples]
[0028] <Example A> Purified water (25°C, resistivity 18 MΩcm) was prepared using a reverse osmosis (thermoscientific RO) system and used in all experiments.
[0029] (1) Preparation of DTBTA-Eu(III) Europium(III) chelate compound (DTBTA-Eu(III)) was prepared from ATBTA-Eu(III)(4′-(4′-amino-4-biphenylyl)-2,2′:6′,2″-terpyridine-6,6″-diyrbis(methyliminodiaacetate)) sodium europium(III) (manufactured by Tokyo Chemical Industry Co., Ltd.) according to the method disclosed by Tokyo Chemical Industry Co., Ltd.
[0030] Specifically, 10 mg of ATBTA-Eu(III) was dissolved in 0.3 mL of 0.1 mol acetate buffer (pH 4.9). Next, 2.15 mg of cyanuric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 1 mL of ultrapure water in a water bath at 50-60°C and added to the ATBTA-Eu(III) solution while stirring for 30 minutes. Furthermore, 5 mL of acetone was added to the resulting solution, and the solution was centrifuged at 14,000 rpm and 16°C for 10 minutes to obtain a precipitate of DTBTA-Eu(III).
[0031] The DTBTA-Eu(III) precipitate was further washed with 5 mL of acetone, recovered by centrifugation, and dried overnight to obtain DTBTA-Eu(III) powder.
[0032] (2) Preparation of DTBTA-Eu(III)-encapsulated silica nanoparticles in the presence of CTAB The preparation of DTBTA-Eu(III)-encapsulated silica nanoparticles was carried out according to the method described in Non-Patent Document 2.
[0033] Specifically, 10 mg of DTBTA-Eu(III) was dissolved in 4 mL of ultrapure water, and then 10 mg of hexadecyltrimethylammonium bromide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "CTAB") was added and the mixture was vigorously stirred.
[0034] Next, 400 mL of ethyl alcohol and 100 mL of ultrapure water were added to the obtained solution and mixed. To the solution thus obtained, 0.2 mL of tetraethyl orthosilicate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "TEOS") and 0.2 mL of 28-30% ammonia solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter simply referred to as "ammonia solution") were added and the mixture was stirred for two days to obtain preparation A.
[0035] (3) Growth of silica nanoparticles To further promote particle growth, 0.6 mL of TEOS and 2 mL of ammonia solution were added to the prepared solution A obtained above, and the mixture was stirred at room temperature for 12 hours.
[0036] (4) Filtration of silica nanoparticles Using an Amicon Ultracell (MILLIPORE) equipped with a 10kDa membrane filter, 500 mL of preparation solution A, in which silica nanoparticles were grown as described above, was ultrafiltered until the amount of preparation solution A at the top of the membrane filter was 50 mL.
[0037] The first 200 mL of the filtrate was collected as the first filtrate, and the following 200 mL was collected as the second filtrate. The collected first and second filtrates were each added to beakers and concentrated on a hot plate at 80-125°C to obtain 30 mL each of first concentrated filtrate A1 and second concentrated filtrate A2. The silica nanoparticles in first concentrated filtrate A1 were designated as silica nanoparticles A1, and the silica nanoparticles in second concentrated filtrate A2 were designated as silica nanoparticles A2.
[0038] (5) Introduction of carboxyl group One μL of a 35% aqueous solution of N-[(3-trimethoxysilyl)propyl]-ethylenediamine triacetate trisodium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter simply referred to as "carboxyl group introducing agent") was added to the second concentrated filtrate A2 to introduce carboxyl groups into silica nanoparticles. These silica nanoparticles, into which carboxyl groups were introduced, were designated as silica nanoparticles A3.
[0039] <Example B> Example B was carried out in the same manner as Example A, except for the following points. (i) In the above "(2) Preparation of DTBTA-Eu(III)-encapsulated silica nanoparticles in the presence of CTAB", instead of adding 0.2 mL of TEOS and 0.2 mL of ammonia solution to the solution obtained by adding ethyl alcohol and ultrapure water, 4 mL of TEOS and 4 mL of ammonia solution were added to obtain preparation solution B, and (ii) In the above "(3) Growth of silica nanoparticles," instead of adding 0.6 mL of TEOS and 2 mL of ammonia solution to preparation solution A in order to further grow the particles, 0.8 mL of TEOS and 0.8 mL of ammonia solution were added to preparation solution B. (iii) In the above "(4) Filtration of silica nanoparticles", the first concentrated filtrate and the second concentrated filtrate were referred to as the first concentrated filtrate B1 and the second concentrated filtrate B2, respectively, and the silica nanoparticles in the first concentrated filtrate B1 were referred to as silica nanoparticles B1, and the silica nanoparticles in the second concentrated filtrate B2 were referred to as silica nanoparticles B2, and (iv) In the above "(5) Introduction of carboxyl groups", instead of adding 1 μL of a 35% aqueous solution of the carboxyl group introducing agent to the second concentrated filtrate A2, 0.1 μL of this aqueous solution was added to the second concentrated filtrate B2 to introduce carboxyl groups into the silica nanoparticles, and the silica nanoparticles to which carboxyl groups were introduced in this manner were designated as silica nanoparticles B3.
[0040] <Rating> Figure 1 shows the particle sizes of silica nanoparticles A1-A3 obtained in Example A, and the particle sizes of silica nanoparticles B1-B3 obtained in Example B. Figure 2 shows the relationship between the particle size of these silica nanoparticles and the fluorescence intensity (wavelength 614 nm) due to DTBTA-Eu(III) encapsulated within them. As shown in Figures 1 and 2, the particle size and fluorescence intensity of DTBTA-Eu(III)-encapsulated silica nanoparticles could be adjusted by adjusting the content of TEOS and ammonia solution in the presence of CTAB.
Claims
1. To provide an aqueous solution containing a rare earth fluorescent complex, hexadecyltrimethylammonium bromide, ethyl alcohol, and water, and Adding alkoxysilane and ammonia to the aqueous solution, A method for producing fluorescent silica nanoparticles, including [the specified ingredient].
2. The method according to claim 1, wherein the particle size of the fluorescent silica nanoparticles is 20 nm to 250 nm.
3. The method according to claim 1, wherein the particle size of the fluorescent silica nanoparticles is 20 nm to 100 nm.
4. The fluorescent silica nanoparticle according to claim 1, wherein the rare earth fluorescent complex is a europium(III) chelate compound.
5. A method for adjusting the particle size of fluorescent silica nanoparticles, comprising adjusting the amount of alkoxysilane and ammonia added, according to any one of claims 1 to 4.
6. A method for adjusting the fluorescence intensity of fluorescent silica nanoparticles, comprising adjusting the amount of alkoxysilane and ammonia added, according to any one of claims 1 to 4.