FLUORESCENT SILICA NANOPARTICLES AND METHOD FOR MANUFACTURING FLUORESCENT SILICA NANOPARTICLES

Abstract

The present invention relates to providing fluorescent silica nanoparticles having high luminance even when many fluorescent dyes are contained in silica particles. Fluorescent silica nanoparticles according to the present invention is fluorescent silica nanoparticles including silica nanoparticles and fluorescent dyes contained in the silica nanoparticles, in which a total volume of the fluorescent dyes is 5% or more with respect to a total volume of the fluorescent silica nanoparticles, and an emission quantum yield of the fluorescent silica nanoparticles is 10% or more.

Claims

1. Fluorescent silica nanoparticles comprising: silica nanoparticles; and fluorescent dyes contained in the silica nanoparticles, wherein a total volume of the fluorescent dyes is 5% or more with respect to a total volume of the fluorescent silica nanoparticles, and an emission quantum yield of the fluorescent silica nanoparticles is 10% or more.

2. The fluorescent silica nanoparticles according to claim 1, wherein an atomic number ratio (C/Si) of carbon to silicon on a surface of the fluorescent silica nanoparticles as measured by X-ray photoelectron spectroscopy is 2 to 10.

3. The fluorescent silica nanoparticles according to claim 1, wherein a coefficient of variation in particle diameter of the fluorescent silica nanoparticles is 20% or less.

4. The fluorescent silica nanoparticles of claim 1, wherein the fluorescent silica nanoparticles have an average particle diameter of 5 to 250 nm.

5. A method for manufacturing the fluorescent silica nanoparticles according to claim 1, the method comprising: continuously adding alkoxysilane to liquid containing fluorescent dyes, ammonia, and water so that a molar ratio of the fluorescent dyes to the alkoxysilane is in a range of 1 to 30.

6. The method for manufacturing fluorescent silica nanoparticles according to claim 5, wherein a continuous addition time of the alkoxysilane is 72 hours or less.

7. The method for manufacturing fluorescent silica nanoparticles according to claim 5, wherein a continuous addition time of the alkoxysilane is 1 hour or less.

8. The fluorescent silica nanoparticles according to claim 2, wherein a coefficient of variation in particle diameter of the fluorescent silica nanoparticles is 20% or less.

9. The fluorescent silica nanoparticles of claim 2, wherein the fluorescent silica nanoparticles have an average particle diameter of 5 to 250 nm.

10. The fluorescent silica nanoparticles of claim 3, wherein the fluorescent silica nanoparticles have an average particle diameter of 5 to 250 nm.

Description

EXAMPLES

[0048] Hereinafter, the invention according to the present embodiment will be described in detail with reference to Examples, but the invention according to the present embodiment is not limited to these Examples.

[0049] [Preparation of Fluorescent Silica Nanoparticles]

Example 1

[0050] <Preparation of Fluorescent Dye>

[0051] A perylene dye derivative was prepared according to a synthesis procedure shown below. Propionic acid was added to 1,6,7,12-tetrachloroperylene tetracarboxylic dianhydride (manufactured by FUJIFILM Wako Pure Chemical Corporation, product number: W01COBQA-7294), and refluxed in a solvent for three hours to react (yield 80%). Next, the obtained compound was dissolved in N-methylpyrrolidone (NMP), added with phenol, and caused to react at 80° C. for six hours in the presence of potassium carbonate (K.sub.2CO.sub.3). Next, 3-bromophenol was added, and the compound was caused to react at 120° C. for 16 hours.

[0052] Next, the obtained compound was dissolved in 1,4-dioxane, added with 4-pinacolborane phenylacetic acid ethyl, and caused to react at 100° C. for two hours in the presence of bis(dibenzylideneacetone)palladium (pd(dba).sub.2) and potassium phosphate (K.sub.3PO.sub.4). (Yield 20%).

[0053] Next, the obtained compound was dissolved in dioxane, added with an aqueous solution of sodium hydroxide, and caused to react at 60° C. for two hours (yield 73%). Next, the obtained compound was dissolved in tetrahydrofuran (THF), added with 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide, and caused to reach at 40° C. for four hours (yield 76%). In this way, the perylene dye derivative was obtained.

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[0054] The perylene dye derivative synthesized as described above was dissolved in dimethylformamide (DMF) at 20 mg/ml. Thereafter, by adding 3-aminopropyltriethoxysilane (APS) (manufactured by Tokyo Chemical Industry Co., Ltd.) so that the molar ratio to the perylene dye derivative was 1, and causing reaction at room temperature for 1 hour, a triethoxysilyl group was added to the perylene dye derivative to obtain perylene-APS.

[0055] <Synthesis of Fluorescent Silica Nanoparticles>

[0056] 747 μl of perylene-APS was added to a solution obtained by mixing 10844 μl of ethanol (99.5, manufactured by FUJIFILM Wako Pure Chemical Corporation), 909 μl of water, and 266 μl of aqueous ammonia (28%, manufactured by FUJIFILM Wako Pure Chemical Corporation), 234 μl of tetraethoxysilane (TEOS, manufactured by Tokyo Chemical Industry Co., Ltd.) diluted to 10% with ethanol placed in a microsyringe pump (SPS-1, manufactured by AS ONE Corporation) in advance was added thereto for 5 seconds, and the solution was stirred at room temperature for 48 hours. At this time, the total amount was 13 ml, and a molar ratio of the perylene dye and the tetraethoxysilane (perylene dye/tetraethoxysilane) was 10.

[0057] The reaction solution described above was centrifuged at 18000 G for 15 minutes in a cooled centrifuge (himac CR21N, manufactured by Koki Holdings Co., Ltd.), added with 13 ml of ethanol after removal of supernatant, and irradiated with ultrasonic waves to be redispersed. Washing by centrifugation, supernatant removal, and redispersion in ethanol was repeated three times. In this way, fluorescent silica nanoparticles of Example 1 were obtained.

Examples 2 to 4

[0058] Fluorescent silica nanoparticles of Examples 2, 3, and 4 were individually obtained similarly to Example 1, except that the molar ratio of the perylene dye and the tetraethoxysilane (perylene dye/tetraethoxysilane) was changed to 15, 25, and 2.

Examples 5 to 7

[0059] Fluorescent silica nanoparticles of Examples 5, 6, and 7 were individually obtained similarly to Example 1, except that the addition time of the tetraethoxysilane was changed to 70 hours, 48 hours, and 0.5 hours.

Comparative Example 1

[0060] 747 μl of perylene-APS was added to a solution obtained by mixing 10844 μl of ethanol (99.5, manufactured by FUJIFILM Wako Pure Chemical Corporation), 909 μl of water, and 266 μl of aqueous ammonia (28%, manufactured by FUJIFILM Wako Pure Chemical Corporation), tetraethoxysilane (164 μl, 70% of the total amount) was added at once, and the solution was stirred at room temperature for 3 hours. Thereafter, tetraethoxysilane (70 μl, 30% of the total amount) was further added at once, and the solution was stirred at room temperature for 48 hours. At this time, the total amount is 13 ml, and the molar ratio of the perylene dye and the tetraethoxysilane (perylene dye/tetraethoxysilane) is 10.

[0061] The reaction solution described above was centrifuged and washed similarly to Example 1, to obtain fluorescent silica nanoparticles of Comparative Example 1.

Comparative Example 2

[0062] 164 μl, which was 70% of the total amount of tetraethoxysilane, was added at once, and the solution was stirred at room temperature for 0.5 hours. Fluorescent silica nanoparticles of Comparative Example 2 were obtained similarly to Comparative Example 1 except that 70 μl, which was 30% of the total amount of tetraethoxysilane, was added at once thereafter.

Comparative Example 3

[0063] Fluorescent silica nanoparticles of Comparative Example 3 were obtained similarly to Example 1 except that tetraethoxysilane was continuously added for 200 hours.

Comparative Example 4

[0064] Comparative Example 4 was made similarly to Example 1 except that the molar ratio of the perylene dye and the tetraethoxysilane was changed to 50, but the amount of the perylene dye was too large to form particles.

Comparative Example 5

[0065] Fluorescent silica nanoparticles of Comparative Example 5 were obtained similarly to Example 1 except that the molar ratio of the perylene dye and the tetraethoxysilane was changed to 0.5.

[0066] [Evaluation]

[0067] The fluorescent silica nanoparticles obtained as described above were evaluated as follows.

[0068] (Contained Amount of Dye)

[0069] For a contained amount of dye, amounts of Si and C elements were measured by the following procedure, an element ratio between Si and C was calculated. Thereafter, the Si ratio was converted into a molecular volume of SiO.sub.2, the C ratio was converted into a molecular volume of the fluorescent dye, and a total volume (%) of the fluorescent dyes contained in the fluorescent silica nanoparticles was calculated. At this time, densities of SiO.sub.2 and the fluorescent dye were 2.2 g/cm.sup.3 and 1.2 g/cm.sup.3, respectively.

[0070] <Quantification of Si>

[0071] Sulfuric acid was added to each fluorescent silica nanoparticle to cause ashing, and then lithium tetraborate was added to produce a bead. Quantification of Si was performed by a calibration curve method with a wavelength differential fluorescent X-ray analyzer (ZSX Primus IV manufactured by Rigaku Corporation).

[0072] <Quantification of C>

[0073] Each fluorescent silica nanoparticle was wrapped with a tin foil, and quantification of C was performed using a CHN elemental analyzer (vario EL cube manufactured by Elementer).

[0074] Measurement results are shown in Table 1 below.

[0075] (Emission Quantum Yield)

[0076] An emission quantum yield was measured using an absolute PL quantum yield measuring apparatus (Quantaurus-QY C11347-01 manufactured by Hamamatsu Photonics K.K.) by dispersing fluorescent nanoparticles in ethanol so that absorbance (abs.) was 0.2 to 0.4 at an excitation wavelength of 567 nm. Measurement results are shown in Table 1 below.

[0077] (Average Particle Diameter and Coefficient of Variation (CV Value))

[0078] For an average particle diameter of each silica nanoparticle obtained as described above, a diameter of each particle (100 pieces or more) shown in an image captured with a scanning electron microscope (manufactured by Hitachi High-Technologies Corporation, S-4800) was measured using image analysis software (A-zou Kun, manufactured by Asahi Kasei Engineering Corporation), and calculated as an average value thereof. A coefficient of variation was also calculated from the measured diameter. Measurement results are shown in Table 1 below.

[0079] (Luminance)

[0080] For luminance of each fluorescent silica nanoparticle obtained as described above, dilution was performed with ethanol using a spectrofluorometer F-7000 (manufactured by Hitachi High-Tech Science Corporation) so that a solid content concentration of the fluorescent nanoparticle dispersion was 0.028 mg/ml, a photomultiplier voltage was set to 400 V, and a fluorescence intensity at 610 nm in excitation light of 567 nm was measured. Measurement results are shown in Table 1 below.

[0081] (C/Si)

[0082] For C/Si, which is an atomic ratio of carbon to silicon on a surface of the fluorescent silica nanoparticles, a solid content concentration of the fluorescent nanoparticle dispersion was adjusted with ethanol to 0.028 mg/ml by using X-ray photoelectron spectroscopy (Quantera SXM manufactured by ULVAC-PHI, Inc.), 8 μl was dropped on a back surface of an aluminum foil previously wiped with ethanol, and a naturally dried film was observed. Measurement results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Evaluation Manufacturing condition Contained Emission Average Addition Addition Fluorescent amount of quantum CV particle method of time of dye/TEOS dye yield value diameter TEOS TEOS (molar ratio) (vol %) (%) (%) (nm) Luminance C/Si Example 1 Continuous 5 seconds 10 45 16 14 72 412 5.6 Example 2 Continuous 5 seconds 15 55 11 19 76 320 8.6 Example 3 Continuous 5 seconds 25 58 11 18 78 380 8.7 Example 4 Continuous 5 seconds 2 10 35 8 77 90 2.1 Example 5 Continuous 70 hours 10 47 22 19 240 610 4.3 Example 6 Continuous 48 hours 10 45 18 18 212 484 4.8 Example 7 Continuous 0.5 hours 10 45 16 16 95 420 5 Comparative Example 1 Divided — 10 46 5 36 89 135 12 Comparative Example 2 Divided — 10 47 4 38 89 135 12 Comparative Example 3 Continuous 200 hours 10 2 25 32 320 43 0.3 Comparative Example 4 Continuous 5 seconds 50 Particle — — — — — cannot be formed Comparative Example 5 Continuous 5 seconds 0.5 2 36 8 80 41 0.5

[0083] When Examples 1 to 7 are compared with Comparative Examples 1 and 2, while TEOS was continuously added to the solution containing the fluorescent dyes in Example 1 to 7, TEOS was added in a divided manner in Comparative Examples 1 and 2. As a result, while the emission quantum yield was as high as 10% or more in Example 1 to 7, the emission quantum yield was as low as 5% and 4% in Comparative Examples 1 and 2. Further, while C/Si was in a range of 2 to 10 in Example 1 to 7, C/Si was as high as 12 in Comparative Examples 1 and 2.

[0084] This is considered to be because, in Example 1 to 7, TEOS, which is more likely to be hydrolyzed and polycondensed, is continuously added to the solution containing the fluorescent dyes, which are less likely to be hydrolyzed and polycondensed, and the fluorescent dye and the TEOS are bonded more, preventing the fluorescent dyes from being bonded to approximate each other. As a result, it is considered that, in the fluorescent silica nanoparticles of Example 1 to 7, concentration quenching is suppressed while the fluorescent dyes are contained at a high concentration, and the emission quantum yield is high. Whereas, in Comparative Examples 1 and 2, since the TEOS was added in a divided manner, it is considered that the bonding between the fluorescent dye and the TEOS was smaller, the fluorescent dyes were bonded to each other more, concentration quenching occurred, and the emission quantum yield becomes low.

[0085] When Examples 1 to 7 are compared with Comparative Example 3, while the addition time of the TEOS was set to 5 seconds to 70 hours in Example 1 to 7, it was set to 200 hours in Comparative Example 3. As a result, while a contained amount of dye was as high as 10 vol % or more in Example 1 to 7, it was as low as 2 in Comparative Example 3. This is considered to be because, in Example 1 to 7, while TEOS, which is more likely to be hydrolyzed and polycondensed, is continuously added to the solution containing the fluorescent dyes, which are less likely to be hydrolyzed and polycondensed, and the fluorescent dye and the TEOS were more bonded to each other, the bonding with the TEOS was insufficient in Comparative Example 3 since the addition time of the TEOS was too long and the bonding between the fluorescent dyes increased.

[0086] When Examples 1 to 7 are compared with Comparative Example 4, the molar ratio of the fluorescent dye/TEOS is as high as 40 in Comparative Example 4. As a result, in Comparative Example 4, the amount of the fluorescent dyes was too large to form particles.

[0087] When Examples 1, 5, 6, and 7 are individually compared, the fluorescent dye/TEOS is 10 in all of these, but the continuous addition time of the TEOS became shorter in the order of Examples 5, 6, 7, and 1. As a result, in this order, the particle diameter of the fluorescent nanoparticles became smaller, and the coefficient of variation (CV value) became lower.

[0088] This application claims priority based on Japanese Patent Application No. 2020 016145 filed on Feb. 3, 2020. The contents described in the specification and drawings of this application are all incorporated herein by

INDUSTRIAL APPLICABILITY

[0089] The fluorescent silica nanoparticles according to the present embodiment have high luminance, and thus are useful for fluorescence imaging and the like.