CN112251225A - Preparation method of green fluorescent whitlockite nanocrystal - Google Patents

Preparation method of green fluorescent whitlockite nanocrystal Download PDF

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CN112251225A
CN112251225A CN202010998598.3A CN202010998598A CN112251225A CN 112251225 A CN112251225 A CN 112251225A CN 202010998598 A CN202010998598 A CN 202010998598A CN 112251225 A CN112251225 A CN 112251225A
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terbium
whitlockite
doped
nanocrystal
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CN112251225B (en
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惠俊峰
姚嘉欣
张敏
杨孜晨
范代娣
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Northwestern University
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    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

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Abstract

The invention discloses a preparation method of terbium-doped whitlockite nanocrystal, which comprises the following steps: adding calcium oleate, magnesium oleate and phosphate solution into an oleic acid and ethanol reaction system, uniformly mixing, fully reacting at the temperature of 100-180 ℃, adding terbium nitrate into the mixture, and continuously reacting to obtain the terbium-doped whitlockite nanocrystal with bright green fluorescence characteristics. The green fluorescent nanocrystalline can be applied to the fluorescent labeling of living cells, can also be used as an inorganic raw material for preparing biomedical bracket materials, and has wide application prospect.

Description

Preparation method of green fluorescent whitlockite nanocrystal
Technical Field
The invention relates to a preparation method of a whitlockite nanocrystal with green fluorescence property.
Background
The development of biological cell and tissue imaging technology is directly related to the depth of human cognitive life in the microscopic world. The development of fluorescent dye, quantum dot and rare earth fluorescent material provides different marking means for people, and promotes the rapid development of the field. For biological tissues, the problem of toxicity and side effects is always a non-negligible problem, and the fluorescent labeling material with biocompatibility has unique advantages.
Whitlockite (Ca)18Mg2(HPO4)2(PO4)12) The calcium ion, the magnesium ion and the phosphate radical ion are formed, exist in the hard tissue of an organism, are elementary materials of the hard tissue of the organism, have excellent biocompatibility, osteoinductivity, nonimmunity, degradability and other properties, and can be widely applied to tissue engineering, targeted drug delivery carriers and the like.
Disclosure of Invention
The invention aims to provide a preparation method of a terbium-doped whitlockite nano fluorescent probe which has bright green fluorescence, controllable appearance and size and good biocompatibility.
The realization process of the invention is as follows:
a preparation method of terbium-doped whitlockite nanocrystal comprises the following steps:
dissolving oleic acid, calcium oleate and magnesium oleate in ethanol, adding sodium hydrogen phosphate or disodium hydrogen phosphate aqueous solution, after fully closed reaction at 100-180 ℃, adding terbium nitrate solution, continuing to fully react, cooling, collecting precipitate, dispersing the precipitate in cyclohexane, and performing assisted precipitation centrifugation by using ethanol to obtain the terbium-doped whitlockite nanocrystal with green fluorescence.
In the above step, the molar ratio of calcium oleate, magnesium oleate and phosphate is 9:1:7, and the phosphate is sodium hydrogen phosphate or disodium hydrogen phosphate.
In the above steps, the volume ratio of the oleic acid, the ethanol and the phosphate aqueous solution is (1-3) to (3-10).
In the above step, the reaction temperature is 100 ℃, 120 ℃, 150 ℃ or 180 ℃.
In the steps, after the closed reaction is performed for 5-20h at 100-180 ℃, a terbium nitrate solution is added, and the reaction is continued for 5-10 h.
In the step, the molar ratio of terbium nitrate to the sum of calcium oleate and magnesium oleate is (0.02-0.2): 1.
In the above steps, the particle size of the prepared terbium-doped whitlockite nanocrystal is 5-35 nm.
The terbium-doped whitlockite nanocrystal prepared by the method is used as a green nano fluorescent probe and applied to the fluorescent labeling of living cells.
In the process of preparing the brushite nanocrystalline, the rare earth ions added into the reaction system are utilized to replace calcium and magnesium cations on the surface of the nanocrystalline, so that the effective doping of the rare earth ions on the surface of the nanocrystalline is realized, and lattice defects are formed on the surface of the nanocrystalline due to the difference of valence and ionic radius between the doped ions and the replaced ions, thereby promoting the target material to have bright fluorescence characteristics. If terbium nitrate solution is added at the beginning of the reaction, a green fluorescent terbium-doped whitlockite nanocrystal cannot be obtained.
The invention has the following positive effects: the preparation method has the advantages of cheap and easily-obtained raw materials, low cost, simple synthesis process, friendly reaction environment, easy process amplification and good reproducibility; the nanocrystalline with the green fluorescence characteristic prepared by the method can be applied to fluorescence labeling of living cells, can also be used as an inorganic raw material for preparing degradable artificial bones, bone cement and other biomedical materials, and has wide application prospect.
Drawings
Fig. 1 is a TEM image of terbium-doped whitlockite nanocrystals prepared in example 1.
Fig. 2 is an XRD pattern of terbium-doped whitlockite nanocrystal prepared in example 1.
Fig. 3 is a fluorescence scan of terbium-doped whitlockite nanocrystals prepared in example 1.
Fig. 4 is a TEM image of terbium-doped whitlockite nanocrystals prepared in example 5.
FIG. 5 is a fluorescent photograph of terbium-doped whitlockite nanocrystals having green fluorescence characteristics prepared in example 6 for cell labeling.
Detailed Description
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1 preparation of Terbium-doped whitlockite nanocrystals with Green fluorescence Properties
Adding 4mL of oleic acid and 16mL of ethanol into a 50mL polytetrafluoroethylene hydrothermal kettle, adding 0.93g of calcium oleate and 0.10g of magnesium oleate, stirring to dissolve, adding 8mL of 0.15M sodium hydrogen phosphate solution, adding 12mL of distilled water, stirring uniformly, sealing, placing in a baking oven for reacting for 15h at 120 ℃, adding 0.57mL of 0.15M terbium nitrate solution (the molar ratio of terbium ions to the sum of calcium ions and magnesium ions is 0.05: 1), continuing to react for 10h at 120 ℃, naturally cooling to room temperature, centrifuging to collect precipitate, dispersing the precipitate in cyclohexane, carrying out precipitation centrifugation with ethanol, and repeating for 2-3 times to obtain a final product.
Detecting the product by transmission electron microscope to be nanoparticles with a particle size of about 15-25nm, as shown in FIG. 1; the product was identified as whitlockite by X-ray powder diffraction, as shown in fig. 2; the sample was fluorescence scanned to show good green fluorescence properties, as shown in fig. 3.
Example 2 preparation of Terbium-doped whitlockite nanocrystals with Green fluorescence Properties
Adding 4mL of oleic acid and 16mL of ethanol into a 50mL polytetrafluoroethylene hydrothermal kettle, adding 0.93g of calcium oleate and 0.10g of magnesium oleate, stirring to dissolve, adding 8mL of 0.15M sodium dihydrogen phosphate solution, adding 12mL of distilled water, stirring uniformly, sealing, placing in a baking oven for 10h at 150 ℃, adding 0.57mL of 0.15M terbium nitrate solution (the molar ratio of the sum of terbium ions to calcium ions and magnesium ions is 0.05: 1), continuing to react for 10h at 150 ℃, naturally cooling to room temperature, centrifuging to collect precipitates, dispersing the precipitates in cyclohexane, using ethanol to assist in precipitation and centrifuging for 2-3 times to obtain a final product, wherein the physicochemical properties of the final product are similar to those of the final product in example 1.
Example 3 preparation of Terbium-doped whitlockite nanocrystals with Green fluorescence Properties
Adding 4mL of oleic acid and 16mL of ethanol into a 50mL polytetrafluoroethylene hydrothermal kettle, adding 0.93g of calcium oleate and 0.10g of magnesium oleate, stirring to dissolve, adding 8mL of 0.15M sodium hydrogen phosphate solution, adding 12mL of distilled water, stirring uniformly, sealing, placing in an oven for 5h at 180 ℃, adding 1.14mL of 0.15M terbium nitrate solution (the molar ratio of terbium ions to the sum of calcium ions and magnesium ions is 0.1: 1), continuing to react for 5h at 180 ℃, naturally cooling to room temperature, centrifuging to collect precipitates, dispersing the precipitates in cyclohexane, centrifuging with ethanol for assisting precipitation, and repeating for 2-3 times to obtain a final product, wherein the physicochemical properties of the final product are similar to those of example 1.
Example 4 preparation of Terbium-doped whitlockite nanocrystals with Green fluorescence Properties
Adding 40mL of oleic acid and 160mL of ethanol into a 500mL polytetrafluoroethylene hydrothermal kettle, adding 9.30g of calcium oleate and 1.00g of magnesium oleate, stirring to dissolve, adding 80mL of 0.15M sodium hydrogen phosphate solution, adding 120mL of distilled water, stirring uniformly, sealing, placing in an oven for reacting for 15h at 120 ℃, adding 5.71mL of 0.15M terbium nitrate solution (the molar ratio of terbium ions to the sum of calcium ions and magnesium ions is 0.05: 1), continuing to react for 10h at 120 ℃, naturally cooling to room temperature, centrifuging to collect precipitates, dispersing the precipitates in cyclohexane, centrifuging with ethanol for assisting precipitation, and repeating for 2-3 times to obtain a final product, wherein the physicochemical properties of the final product are similar to those of example 1.
Example 5 comparative experiment with example 1
Adding 4mL of oleic acid and 16mL of ethanol into a 50mL polytetrafluoroethylene hydrothermal kettle, adding 0.93g of calcium oleate and 0.10g of magnesium oleate, stirring to dissolve, adding 8mL of 0.15M sodium hydrogen phosphate solution and 0.57mL of 0.15M terbium nitrate solution (the molar ratio of terbium ions to the sum of calcium and magnesium ions is 0.05: 1), adding 12mL of distilled water, stirring uniformly, sealing, placing in an oven for reaction at 120 ℃ for 15h, cooling to room temperature, centrifuging to collect precipitate, dispersing the precipitate in cyclohexane, carrying out precipitation centrifugation with ethanol, and repeating for 2-3 times to obtain the final product.
The product is detected to be a nanowire with the particle size of about 1nm by a transmission electron microscope (as shown in figure 4), and a sample is subjected to fluorescence detection to show the non-fluorescence characteristic. The experimental results show that the terbium nitrate solution is added at the beginning of the reaction, and the green fluorescent terbium-doped whitlockite nanocrystal cannot be obtained.
Example 6 cell labeling of Terbium-doped whitlockite nanocrystals with Green fluorescence Properties
In the first step, the hydrophilicity and hydrophobicity of the surface of the nanocrystal is converted. Dispersing 10 mg of terbium-doped whitlockite nanocrystal in 2ml of cyclohexane, adding the terbium-doped whitlockite nanocrystal into 10ml of aqueous solution dissolved with glucan, violently stirring for 2-4h, and volatilizing the cyclohexane to obtain turbid suspension. And then centrifuging and collecting the precipitate, and washing the precipitate by using a mixed solution of ethanol and water to obtain the hydrophilic terbium-doped whitlockite nanocrystal with green fluorescence.
And secondly, marking cells of the terbium-doped whitlockite nanocrystal with green fluorescence characteristic.
CO at 37 deg.C2In a cell culture chamber containing 5%, A549 cells were cultured in a DMEM medium containing 10% fetal bovine serum, 2 mM glutamine, 100U/ml penicillin, and 100. mu.g/ml streptomycin, and maintained in an exponential growth state. A549 cell suspension 1X 10 which has been counted is added to a sterilized laser confocal dish5The number of the cells is cultured for 24 hours, then a fluorescent nanoparticle solution of 150 mug/ml is added to be cultured for 4 hours at 37 ℃, sterilized PBS is used for washing off unabsorbed nanoparticles, a laser confocal dish is taken out and is immediately fixed for 10 minutes at room temperature by 4% paraformaldehyde, the fixed cells are observed by a laser scanning confocal microscope, and the result is shown in figure 5, a green cell image with a dark center and bright periphery can be seen on a black substrate, which indicates that terbium-doped whitlockite nanocrystalline is used for carrying out fluorescence labeling on A549 cells successfully.

Claims (8)

1. A preparation method of terbium-doped whitlockite nanocrystal is characterized by comprising the following steps: dissolving oleic acid, calcium oleate and magnesium oleate in ethanol, adding sodium hydrogen phosphate or disodium hydrogen phosphate aqueous solution, carrying out a fully closed reaction at 100-180 ℃, adding a terbium nitrate solution, continuing to carry out a fully reaction, cooling, collecting precipitate, dispersing the precipitate in cyclohexane, and carrying out assisted precipitation centrifugation by using ethanol to obtain the terbium-doped whitlockite nanocrystal with green fluorescence.
2. The method for preparing a terbium-doped whitlockite nanocrystal according to claim 1, wherein: in the above step, the molar ratio of calcium oleate, magnesium oleate and phosphate is 9:1:7, and the phosphate is sodium hydrogen phosphate or disodium hydrogen phosphate.
3. The method for preparing a terbium-doped whitlockite nanocrystal according to claim 2, wherein: in the above steps, the volume ratio of the oleic acid, the ethanol and the phosphate aqueous solution is (1-3) to (3-10).
4. The method for preparing a terbium-doped whitlockite nanocrystal according to claim 1, wherein: in the above step, the reaction temperature is 100 ℃, 120 ℃, 150 ℃ or 180 ℃.
5. The method for preparing a terbium-doped whitlockite nanocrystal according to claim 1, wherein: in the steps, after the closed reaction is performed for 5-20h at 100-180 ℃, a terbium nitrate solution is added, and the reaction is continued for 5-10 h.
6. The method for preparing a terbium-doped whitlockite nanocrystal according to claim 1, wherein: in the step, the molar ratio of terbium nitrate to the sum of calcium oleate and magnesium oleate is (0.02-0.2): 1.
7. The method for preparing a terbium-doped whitlockite nanocrystal according to claim 1, wherein: in the above steps, the particle size of the prepared terbium-doped whitlockite nanocrystal is 5-35 nm.
8. The terbium-doped whitlockite nanocrystal prepared by the preparation method of claim 1 is applied as a green nano fluorescent probe.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102250615A (en) * 2011-05-06 2011-11-23 清华大学 Fluorine-containing hydroxylapatite single crystal nano shuttle with fluorescence labeling and preparation method thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102250615A (en) * 2011-05-06 2011-11-23 清华大学 Fluorine-containing hydroxylapatite single crystal nano shuttle with fluorescence labeling and preparation method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CAIFENG WANG ET AL.: "Synthesis and formation mechanism of bone mineral, whitlockite nanocrystals in tri-solvent system", 《JOURNAL OF COLLOID AND INTERFACE SCIENCE》 *
LI LI ET AL.: "Simultaneously tuning emission color and realizing optical thermometry via efficient Tb3+/Eu3+ energy transfer in whitlockite-type phosphate multifunctional phosphors", 《JOURNAL OF ALLOYS AND COMPOUNDS》 *
杨志平: "《发光与显示技术》", 31 December 2007, 河北大学出版社 *

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