CN107325814B

CN107325814B - A kind of fluorescence silicon nano dots and the preparation method and application thereof

Info

Publication number: CN107325814B
Application number: CN201710478865.2A
Authority: CN
Inventors: 吴富根; 陈晓凯; 张晓东
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2017-06-22
Filing date: 2017-06-22
Publication date: 2018-11-09
Anticipated expiration: 2037-06-22
Also published as: CN107325814A

Abstract

The invention discloses a kind of fluorescence silicon nano dots (SiNDs), it is prepared by silane and Bengal rose red with one step of hydro-thermal method.Compared with prior art, the SiNDs prepared by the present invention has the fluorescence quantum yield (100%) of superelevation, and can realize the long-time specific imaging to mammalian cell lysosome.In addition, the lysosome imaging effect of the SiNDs is not influenced by cell cleaning, fixed and permeabilization etc., have the advantages that resistance to cleaning, resistance to fixation and resistance to permeabilization.Meanwhile the SiNDs also has the advantages that manufacturing cost is low, synthetic method is simple, water dispersible is good, fluorescence emission peak width, good light stability, cell compatibility is good, cell phototoxicity is low etc., is expected to become novel lysosome fluorescence probe.

Description

Fluorescent silicon nanodot and preparation method and application thereof

Technical Field

The invention belongs to the field of nano materials and biotechnology, and particularly relates to a fluorescent silicon nanodot as well as a preparation method and application thereof.

Background

Fluorescent materials have a wide range of applications in biomedicine. Compared with the traditional organic fluorescent small molecules, the fluorescent nano material has the advantages of better light stability, adjustable excitation emission wavelength and the like, and is widely concerned in recent years. There are many kinds of existing fluorescent nano materials, but they all have certain disadvantages, such as: traditional semiconductor quantum dots (such as cadmium selenide, lead sulfide and the like) generally contain heavy metal elements with high toxicity, so the dosage of the traditional semiconductor quantum dots in biomedicine is greatly limited; although the biocompatibility of the noble metal nano-cluster (such as gold nano-cluster and silver nano-cluster) and the up-conversion nano-particles is better than that of semiconductor quantum dots, the quantum yield is generally lower, and the imaging effect is poor; the synthesis method of the polymer quantum dots is complex, and the surface modification of the polymer quantum dots can greatly influence the fluorescence property of the polymer quantum dots; the carbon dots and the graphene quantum dots have wide fluorescence emission peaks, have multicolor luminescence property and are not beneficial to being used with other probes; other novel nano-dots (such as black phosphorus quantum dots) have the problems of difficult surface modification, harsh preparation conditions and the like. Therefore, the development of the super-bright fluorescent nano material with excellent performance and meeting the biomedical significance is of great significance.

Lysosomes, on the other hand, are key organelles in eukaryotic cells, are single-layer membrane-coated vesicular structures, contain a variety of acid hydrolases, and can decompose and digest a variety of exogenous and endogenous molecules and substances in cells, and are the digestion centers of the cells. Lysosomes also play an important role in a range of physiological processes including apoptosis, autophagy, intracellular cholesterol homeostasis, plasma membrane repair, cytoskeletal and tissue remodeling, pathogen defense, and intracellular signal transduction. Therefore, the observation and tracking of the number, size, morphology, etc. of lysosomes is very important for understanding the behavior and fate of cells. Lysosomes are acidic in vivo (pH 4.5-5.0) compared to other organelles, and therefore lysosomal imaging is often achieved using molecules capable of targeting an acidic environment. For example, 3- (2, 4-dinitroaniline) -3' -amino-N-methyldipropylamine (DAMP) is a commonly used lysosomal targeting molecule, but because the molecule is not fluorescent by itself, grafting a fluorescent molecule onto the primary amine group of the molecule is required to achieve lysosomal imaging. Some fluorescent molecules (e.g., neutral red and acridine orange), while capable of imaging lysosomes, also image nucleic acids that are acidic in the nucleus. Currently, commercial lysosomal fluorescent probes (such as LysoTracker series dyes from Thermo Fisher) have a good lysosomal imaging effect, but have the disadvantages of high synthesis cost, poor light stability and short lysosomal imaging time. Meanwhile, the surface of the fluorescent probe has no modifying group, so that the fluorescent probe cannot be further modified and applied. Emerging fluorescent nanomaterials are considered to be effective approaches to solve the above problems. For example, organic fluorescent nanoparticles (PVP and BPVP) prepared by Lin et al target lysosomes of cancer cells, but do not enable lysosomal imaging of normal cells (org.biomol.chem.,2009,7,2036.). After the surface of gold nanoparticles is modified by a fluorescent molecule FITC and a polypeptide capable of targeting lysosomes, Dekiwadia et al realize imaging of lysosomes (J.Pept.Sci.,2012,18,527.). None of the above methods, however, evaluated the effect of nanoparticles on imaging lysosomes in cells after immobilization and permeabilization. In addition, long-term real-time observation of lysosomal behavior has an important role in studying physiological changes of cells, and none of the above methods can achieve long-term imaging of lysosomes of living cells. Therefore, the development of a fluorescent nanoparticle with good performance as a fluorescent probe for long-time lysosome observation is urgently needed to realize long-time imaging of the lysosome of living cells and imaging of the lysosome of fixed and permeabilized cells.

Disclosure of Invention

The invention aims to provide fluorescent silicon nanodots (SiNDs) to realize lysosome imaging with ultra-long time, washing resistance, fixation resistance and permeabilization resistance, and solve the problems of high cost, poor stability, short imaging time and the like in the prior art.

The invention also aims to solve the technical problem of providing a preparation method and application of the fluorescent silicon nanodot.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a fluorescent silicon nanodot comprises the following components in parts by weight:

100 parts of water-soluble silane;

1-10 parts of rose bengal.

Wherein, the following components in parts by weight are preferred:

100 parts of water-soluble silane;

rose bengal 3 parts.

Wherein the water-soluble silane is Aminopropyltrimethoxysilane (APTMS), Aminopropyltriethoxysilane (APTES), 3- (2-aminoethylamino) propyltrimethoxysilane (DAMO), 3- [2- (2-aminoethylamino) ethylamino ] propyltrimethoxysilane (AEEA) or gamma-Glycidoxypropyltrimethoxysilane (GPTMS), preferably 3- [2- (2-aminoethylamino) ethylamino ] propyltrimethoxysilane (AEEA).

The preparation method of the fluorescent silicon nanodot comprises the following steps:

dissolving rose bengal in the formula amount in ultrapure water, adding water-soluble silane in the formula amount, and reacting; and after the reaction is finished, dialyzing the reaction solution cooled to room temperature to obtain the fluorescent silicon nanodot aqueous solution.

Wherein,

the reaction temperature is 120-250 ℃, and preferably 160 ℃;

the reaction time is 2-24 h, preferably 3 h;

the reaction device is preferably a hydrothermal reaction kettle.

Wherein the cut-off molecular weight of the dialysis bag used for dialysis is 500-1000.

The application of the fluorescent silicon nanodots as the lysosome fluorescent probe is also within the protection scope of the invention.

The application of the rose bengal in preparing the fluorescent silicon nanodots is also within the protection scope of the invention; the preparation of the fluorescent silicon nanodots is performed by the method described above with reference to a water-soluble silane (preferably 3- [2- (2-aminoethylamino) ethylamino ] propyltrimethoxysilane).

In the invention, the rose bengal is not only used as a coloring agent, but also participates in the synthesis of the fluorescent silicon dots, has an important effect on the synthesis of the fluorescent silicon dots, and cannot be replaced by other coloring agents.

Has the advantages that:

compared with the prior art, the invention has the following advantages:

(1) ultra-high fluorescence quantum yield: the quantum yield of the fluorescent silicon nano-dot can reach 100%, and an excellent lysosome imaging effect can be obtained only with the sample concentration of 20 mu g/mL;

(2) excellent lysosome specific targeting: the co-localization rate with the commercial lysosomal probe is more than 98 percent, and the probe is only present in lysosomes even if the probe is incubated with cells for a long time;

(3) can realize the imaging of the lysosome for a very long time: after the probe is incubated with cells and washed by phosphate buffer solution, the probe still has a good lysosome imaging effect within 48 hours, and the commercialized lysosome probe only has a good lysosome imaging effect within 2 hours;

(4) has excellent photobleaching resistance: the silicon nanodots are not easily photobleached under laser irradiation, and the light stability of the silicon nanodots is better than that of commercial lysosomal dyes, so that long-time continuous imaging observation can be realized;

(5) has good washing resistance, fixation resistance and permeabilization resistance: after cells are washed, fixed and permeabilized for many times, the silicon nanodots can still have a good imaging effect on lysosomes;

(6) has better biocompatibility: the silicon nanodots have low toxicity to cells at a concentration of 500 [ mu ] g/mL or less through cytotoxicity evaluation experiments; after long-time irradiation by laser, the cytotoxicity is still very low, which proves that the material has good biocompatibility and lower phototoxicity;

(7) the preparation method is simple, the raw materials are cheap and easy to obtain, and the mass preparation can be realized.

Drawings

FIG. 1 is a schematic representation of the preparation of ultra-bright fluorescent silicon nanodots (SiNDs) using silane reagents AEEA and rose bengal in example 1;

FIG. 2 is a transmission electron microscope photograph of SiNDs obtained in example 1;

FIG. 3 is a graph showing the statistical results of particle sizes of SiNDs produced in example 1;

FIG. 4 is a graph showing UV-VIS absorption spectra of SiNDs obtained in example 1;

FIG. 5 is a graph showing fluorescence excitation and emission spectra of SiNDs obtained in example 1;

FIG. 6 is a graph showing extinction at 650nm in various pH environments for SiNDs obtained in example 1;

FIG. 7 is a graph showing the imaging effect of lysosomes by SiNDs prepared in example 1 after incubation in A549 cells for various periods of time;

FIG. 8 is a graph of the imaging effect of lysosome after incubation of commercial Green lysosomal fluorescent dye LysoTracker Green (LT-Green) with A549 cells for various periods of time;

FIG. 9 is a graph of the effect of imaging lysosomes with the commercial Red lysosomal fluorescent dye LysoTracker Red (LT-Red) after incubation with A549 cells for various periods of time;

FIG. 10 is a graph comparing imaging effects of SiNDs prepared in example 1 with lysosomes from LT-Green and LT-Red after rinsing for various periods of time;

FIG. 11 is a graph comparing the fluorescence stability of SiNDs and LT-Red obtained in example 1 after being irradiated with laser light for various periods of time;

FIG. 12 is a graph comparing the effects of immobilization and permeabilization resistance of the SiNDs prepared in example 1 with LT-Green and LT-Red in A549 cells;

FIG. 13 is a graph showing imaging effects of lysosomes from SiNDs obtained in example 1 in different cell lines;

FIG. 14 is a graph showing cytotoxicity of SiNDs prepared in example 1 in A549 cells under light and normal conditions.

Detailed Description

Example 1

Taking AEEA as an example, the preparation of the fluorescent silicon nanodots comprises the following steps:

(1) rose bengal is weighed and dissolved well in ultrapure water, and the appropriate volume of AEEA is added so that the mass ratio of AEEA to rose bengal is 100: 3. fully and uniformly mixing the two and transferring the mixture into a hydrothermal reaction kettle;

(2) reaction: reacting for 3 hours at 160 ℃ in a hydrothermal reaction kettle to form a silicon nano-dot solution;

(3) and (3) purification: dialyzing to obtain the target ultra-bright fluorescent silicon nano-dot solution.

The reaction is schematically shown in FIG. 1, and the transmission electron microscopy results of the prepared SiNDs are shown in FIG. 2, the statistical results of the particle size distribution are shown in FIG. 3, the ultraviolet-visible absorption spectrum is shown in FIG. 4, the fluorescence excitation emission spectrum is shown in FIG. 5, and the extinction degrees at 650nm in different pH environments are shown in FIG. 6. As can be seen from the above figures, the prepared SiNDs are in the shape of uniform spheres, the particle size is approximately 2.5nm, the maximum ultraviolet absorption peak is located at 511nm, and the maximum excitation wavelength and the maximum emission wavelength are 511nm and 525nm respectively. The fluorescence quantum yield of the SiNDs can reach 100 percent through calculation. In particular, as can be seen from fig. 6, SiNDs are easy to aggregate and precipitate under the condition of weak acid environment, and the characteristic causes particle aggregation when entering acid lysosomes in cells, thereby realizing long-time lysosome imaging.

Example 2

The imaging effect of SiNDs prepared in example 1 on lysosomes of a549 cells was tested by the following method:

(1) cell culture: a549 cells at 5X 10³Density of cells/well 96-well plates were seeded at 37 ℃ with 5% CO₂Culturing for 24h in the environment;

(2) cell staining: the culture medium in each well was replaced with fresh culture medium containing 20 g/mL SiNDs at 37 deg.C with 5% CO₂After incubation for 2,4, 18 and 24h in ambient, respectively, washing was performed 2 times with Phosphate Buffered Saline (PBS). Then, staining was performed with a commercial Red lysosomal fluorescent dye (LT-Red) and a nuclear staining reagent (Hoechst 33342) for 30 and 10 minutes, respectively. Finally, the cells were washed 2 times with PBS;

(3) and (3) confocal fluorescence microscope imaging observation: laser with the wavelength of 405, 488 and 552nm is used as exciting light, Hoechst 33342 dye emits blue fluorescence under the excitation of 405nm exciting light, SiNDs emits green fluorescence under the excitation of 488nm exciting light, and LT-Red dye emits Red fluorescence under the excitation of 552nm laser.

Lysosome imaging and co-localization results are shown in figure 7. As can be seen, the SiNDs are mainly distributed at some punctate positions after entering cells, and after the SiNDs are subjected to colocalization analysis with Red commercial lysosome fluorescent dye (LT-Red), the lighted areas of the SiNDs are proved to be lysosomes, and the colocalization rate with the lysosome dye is up to 98% after calculation. In addition, even if the dosing time is extended to 24h, the SiNDs stay in lysosomes. Therefore, the capability of the synthesized SiNDs in the invention to target lysosomes is not different along with the change of dosing time, and the stability of lysosome imaging is reflected.

Examples 3 and 4

Commercial lysosomal Green fluorescent dye (LT-Green) and Red fluorescent dye (LT-Red) were tested for their lysosomal imaging effects on A549 cells.

The procedures of examples 3 and 4 are substantially the same as those of example 1, except that SiNDs in step (2) are replaced by 1LT-Green and LT-Red, and the SiNDs are cultured for 30min, 4h and 8h, respectively.

Lysosomal imaging effects are shown in figures 8 and 9. As can be seen from the observation of FIGS. 8 and 9, the change in the dosing time had little effect on the Green lysosomal fluorescent dye LT-Green, but the LT-Red site was greatly changed. With the addition time extended to 4h, LT-Red was transferred to the cytoplasmic and nuclear membrane regions other than lysosomes, and even at 8h some of the dye had entered the nucleus. Therefore, compared with LT-Red, the synthesized SiNDs are more suitable for long-time tracking lysosome imaging.

Example 5

The imaging stability of the SiNDs prepared in example 1 and commercial LT-Green and LT-Red on washed lysosomes was tested by the following method:

(1) cell culture: the procedure was in accordance with step (1) in example 2;

(2) cell staining and washing: the culture solution in the cell culture wells was changed to fresh culture solution containing SiNDs (20. mu.g/mL), LT-Green (1. mu.M) and LT-Red (1. mu.M), respectively, and the wells were stained for 2h, 30min and 30 min. After washing twice with PBS, the mixture was washed at 37 ℃ with 5% CO₂Culturing in dark environment for 0, 1, 2,4, 12, 24, 36 and 48h, and observing under confocal fluorescence microscope;

(3) lysosome imaging effect observation: 488nm and 552nm lasers were used as excitation light. Wherein, SiNDs and LT-Green emit Green fluorescence under 488nm laser, and LT-Red emits Red fluorescence under 552nm laser.

The fluorescence imaging results are shown in FIG. 10. Comparing the imaging results of the three dyes, the fluorescence of the commercial lysosomal dyes LT-Green and LT-Red is obviously weakened within 2h after the cells are washed. Obviously, the SiNDs prepared by the method still have strong fluorescence within 48 hours after the cells are washed, so that the long-time sharp imaging of lysosomes can be realized.

Example 6

The SiNDs prepared in example 1 were tested for their stability in imaging lysosome fluorescence with commercially available LT-Red after exposure to laser light for various periods of time. The method comprises the following steps:

(1) cell culture: the procedure was in accordance with step (1) in example 2;

(2) cell staining: respectively changing the culture solution in the cell culture hole into fresh culture solution containing SiNDs (20 mu g/mL) and LT-Red (1 mu M), staining for 2h and 30min, and washing the cells twice with PBS;

(3) laser irradiation: irradiating the stained cells under 488nm laser for 0, 0.5, 1, 3, 5, 10, 15, 20 and 30min respectively, and observing by using a confocal fluorescence microscope;

(4) and (3) observing fluorescence stability: 488nm and 552nm lasers were used as excitation light. Wherein, SiNDs emit green fluorescence under 488nm laser, and LT-Red emits Red fluorescence under 552nm laser.

As can be seen from FIG. 11, the LT-Red fluorescence is significantly reduced within 3min of laser irradiation, while the SiNDs prepared by the invention still maintain clear lysosome imaging effect after 20min of irradiation. The experiment proves the remarkable advantages of SiNDs in the aspects of photobleaching resistance and stable imaging.

Example 7

SiNDs prepared in example 1 were tested for their ability to withstand immobilization and strike-through with commercially available LT-Green and LT-Red. The method comprises the following steps:

(1) cell culture: the procedure was in accordance with step (1) in example 2;

(2) cell staining: the procedure was identical to that of step (2) in example 5, and fresh culture solutions containing SiNDs (20. mu.g/mL), LT-Green (1. mu.M) and LT-Red (1. mu.M) were stained for 2h, 30min and 30min, respectively. Observing the stained cells under a confocal fluorescence microscope;

(3) cell fixation: placing the stained cells in a phosphate buffer solution containing 4% glutaraldehyde for 30 minutes, washing the cells for 2 times by using PBS (phosphate buffer solution), and observing a lysosome imaging result under a confocal fluorescence microscope;

(4) cell permeabilization: after the cell fixation step, the cells were treated with 0.1% Triton X-100 in phosphate buffer for 10 min, and finally washed 2 times with PBS and observed under a confocal fluorescence microscope. The setup conditions of the confocal fluorescence microscope were the same as in step (3) of example 5.

As can be seen from FIG. 12, both LT-Green and LT-Red are not suitable for imaging lysosomes of immobilized and permeabilized cells, and the lysosome imaging probe prepared by the invention has the capabilities of immobilization resistance and permeabilization resistance.

Example 8

The imaging effect of the SiNDs in different cell lines is tested, the operation steps of the imaging method are approximately the same as those of the imaging method in example 2, only the A549 cells in example 2 need to be replaced by normal lung cells AT II, breast cancer cells MCF-7 and macrophages Raw 264.7 respectively, and the staining time is changed to 2 hours.

FIG. 13 shows that the synthesized SiNDs of the present invention have excellent lysosome imaging ability in different cell lines.

Example 9

The cytotoxicity of SiNDs prepared in example 1 to a549 cells under dark and light conditions was tested by the following procedure:

(1) cell culture: the procedure was in accordance with step (1) in example 2;

(2) SiNDs treatment: respectively changing the culture solution in the cell culture hole into fresh culture solution containing SiNDs with different concentrations, wherein the concentration gradient of the SiNDs is as follows: 0, 50, 100, 200, 500 and 1000. mu.g/mL, 5% CO at 37 ℃₂After 24h incubation in the environment. The cells were divided into two groups, one group was exposed to white light for 30min, and the other group was exposed to dark. Subsequently, both groups of cells were plated at 37 ℃ with 5% CO₂Culturing for 4h in the environment;

(3) and (3) detecting the activity of the cells: cytotoxicity was detected by MTT method.

The cell viability results are shown in figure 14. As can be seen from the figure, even if the concentration of the SiNDs is as high as 200 mug/mL, the cells in the illuminated group and the non-illuminated group both keep more than 90% of activity, and the low toxicity of the SiNDs synthesized by the invention is reflected, so that the SiNDs become a safe lysosome imaging fluorescent probe.

Claims

1. The fluorescent silicon nanodot is characterized by being prepared from the following raw materials in parts by mass:

100 parts of water-soluble silane;

1-10 parts of rose bengal.

2. The fluorescent silicon nanodot of claim 1, wherein the water soluble silane is aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 3- (2-aminoethylamino) propyltrimethoxysilane, 3- [2- (2-aminoethylamino) ethylamino ] propyltrimethoxysilane or gamma-glycidoxypropyltrimethoxysilane.

3. The method for preparing fluorescent silicon nanodots according to claim 1, comprising the steps of:

dissolving rose bengal with the formula amount in water, and adding water-soluble silane with the formula amount for reaction; and after the reaction is finished, dialyzing the reaction solution cooled to room temperature to obtain the fluorescent silicon nanodot aqueous solution.

4. The method according to claim 3, wherein the reaction temperature is 120 to 250 ℃ and the reaction time is 2 to 24 hours.

5. The method according to claim 3, wherein the cut-off molecular weight of the dialysis bag used in dialysis is 500 to 1000.

6. Use of the fluorescent silicon nanodots of claim 1 as lysosomal fluorescent probes.

7. Application of rose bengal in preparing fluorescent silicon nanodots.