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

Abstract

The invention discloses a fluorescent silicon nano-dot (SiNDs), which is prepared by one-step hydrothermal method from silane and rose bengal. Compared with the prior art, the SiNDs prepared by the present invention have ultra-high fluorescence quantum yield (100%), and can realize long-term specific imaging of mammalian cell lysosomes. In addition, the lysosome imaging effect of the SiNDs is not affected by cell washing, fixation, and permeabilization, and has the advantages of resistance to washing, fixation, and permeabilization. At the same time, the SiNDs also have the advantages of low preparation cost, simple synthesis method, good water dispersibility, narrow fluorescence emission peak width, good photostability, good cytocompatibility, and low cellular phototoxicity. probe.

Description

A fluorescent silicon nano-dot and its preparation method and application

technical field

The invention belongs to the field of nano-materials and biotechnology, and in particular relates to a fluorescent silicon nano-dot and its preparation method and application.

Background technique

Fluorescent materials are widely used in biomedicine. Compared with traditional organic fluorescent small molecules, fluorescent nanomaterials have attracted extensive attention in recent years because of their advantages such as better photostability and tunable excitation and emission wavelengths. At present, there are many types of fluorescent nanomaterials, but they all have certain shortcomings. For example, traditional semiconductor quantum dots (such as cadmium selenide, lead sulfide, etc.) generally contain toxic heavy metal elements, so they are widely used in biomedicine. The use dose is greatly limited; although the biocompatibility of noble metal nanoclusters (such as gold nanoclusters, silver nanoclusters) and upconversion nanoparticles is better than that of semiconductor quantum dots, their quantum yields are generally low, and the imaging effect is not good. Good; the synthesis method of polymer quantum dots is relatively complicated, and the surface modification of polymer quantum dots may have a greater impact on its fluorescence properties; the fluorescence emission peaks of carbon dots and graphene quantum dots are broad, and they also have multicolor The nature of luminescence is not conducive to simultaneous use with other probes; other new nanodots (such as black phosphorus quantum dots) have problems such as difficult surface modification and harsh preparation conditions. Therefore, it is of great significance to develop ultra-bright fluorescent nanomaterials with excellent performance and biomedical significance.

On the other hand, lysosome is a key organelle in eukaryotic cells. It is a sac-like structure covered by a single membrane and contains a variety of acid hydrolases, which can decompose and digest a variety of exogenous and endogenous substances in the cell. Molecules and substances that are the digestive center of the cell. Lysosomes also play an important role in a series of physiological processes such as apoptosis, autophagy, intracellular cholesterol balance, plasma membrane repair, cytoskeleton and tissue remodeling, pathogen defense, and intracellular signal transduction. Therefore, the observation and tracking of the number, size and shape of lysosomes are very important for understanding the behavior and fate of cells. Lysosomes are acidic (pH 4.5–5.0) compared to other organelles, so molecules that target the acidic environment are often used to enable imaging of lysosomes. For example, 3-(2,4-dinitroaniline)-3'-amino-N-methyldipropylamine (DAMP) is a commonly used lysosome-targeting molecule, but since the molecule itself is not fluorescent, it It is necessary to graft fluorescent molecules on the primary amino groups of the molecule in order to realize lysosome imaging. Some fluorescent molecules (such as neutral red and acridine orange) can image lysosomes, but they can also image acidic nucleic acids in the nucleus. Currently commercialized fluorescent probes for lysosomes (such as LysoTracker series dyes from Thermo Fisher) have good lysosome imaging effects, but their synthesis costs are high, photostability is not good, and lysosome imaging time is short. At the same time, there is no modification group on the surface of this type of fluorescent probe, so it cannot be further modified and applied. Emerging fluorescent nanomaterials are considered to be an effective way to solve the above problems. For example, although the organic fluorescent nanoparticles (PVP and BPVP) prepared by Lin et al. can target the lysosomes of cancer cells, they cannot realize the lysosome imaging of normal cells (Org. Biomol. Chem., 2009, 7, 2036. ). Dekiwadia et al. modified the fluorescent molecule FITC and lysosome-targeting polypeptides on the surface of gold nanoparticles, and realized imaging of lysosomes (J. Pept. Sci., 2012, 18, 527.). However, none of the above methods evaluated the imaging effect of nanoparticles on fixed and permeabilized cell lysosomes. In addition, long-term real-time observation of lysosome behavior plays an important role in the study of physiological changes in cells, but none of the above methods can achieve long-term imaging of lysosomes in living cells. Therefore, it is urgent to develop a fluorescent nanoparticle with good performance as a fluorescent probe for long-term observation of lysosomes, so as to realize long-term imaging of lysosomes in living cells and imaging of lysosomes in fixed and permeabilized cells. .

Contents of the invention

The technical problem to be solved in the present invention is to provide a fluorescent silicon nano-dot (SiNDs) to realize ultra-long-time, cleaning-resistant, fix-resistant, and permeabilization-resistant lysosome imaging, which solves the high cost of the existing technology, Problems such as poor stability and short imaging time.

The technical problem to be solved by the invention is to provide the preparation method and application of the fluorescent silicon nano-dot.

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

A fluorescent silicon nano-dot, which includes the following components in parts by mass:

100 parts of water-soluble silane;

Bengal rose red 1 to 10 parts.

Wherein, the component of preferred following mass parts:

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 γ-glycidyl etheroxypropyltrimethoxysilane (GPTMS), preferably 3-[2 - (2-Aminoethylamino)ethylamino]propyltrimethoxysilane (AEEA).

The preparation method of above-mentioned fluorescent silicon nano-dot, it comprises the steps:

The formula amount of rose bengal is dissolved in ultrapure water, and the formula amount of water-soluble silane is added to react; after the reaction is completed, the reaction solution cooled to room temperature is dialyzed to obtain an aqueous solution of fluorescent silicon nano-dots.

in,

The reaction temperature is 120-250°C, preferably 160°C;

The reaction time is 2 to 24 hours, preferably 3 hours;

The reaction device is preferably a hydrothermal reaction kettle.

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

The application of the above-mentioned fluorescent silicon nano-dots as lysosome fluorescent probes is also within the protection scope of the present invention.

The application of Rose Bengal in the preparation of fluorescent silicon nano-dots is also within the protection scope of the present invention; wherein, the preparation of fluorescent silicon nano-dots refers to the use of water-soluble silane (preferably 3-[2-(2-aminoethyl) (amino)ethylamino]propyltrimethoxysilane) was prepared with reference to the aforementioned method.

In the present invention, Rose Bengal is not only used as a dye, but participates in the synthesis of fluorescent silicon dots, which plays an important role in the synthesis of fluorescent silicon dots, and cannot be replaced by other dyes.

Beneficial effect:

Compared with the prior art, the present invention has the following advantages:

(1) Ultra-high fluorescence quantum yield: the quantum yield of the fluorescent silicon nanodots can be as high as 100%, and only a sample concentration of 20 μg/mL can obtain excellent lysosome imaging effect;

(2) Excellent lysosome-specific targeting: the co-localization rate with commercially available lysosome probes is as high as 98%, and the probe still only exists in lysosomes even if it is incubated with cells for a long time ;

(3) Ultra-long time imaging of lysosomes can be realized: after the probe is incubated with cells and washed with phosphate buffer, it still has a good imaging effect of lysosomes within 48 hours, while the commercial lysosome probes The needle has a better lysosome imaging effect within 2 hours;

(4) Excellent anti-photobleaching ability: the silicon nanodot is not easy to be photobleached under laser irradiation, and its photostability is better than commercial lysosomal dyes, so it can realize long-term continuous imaging observation;

(5) Good resistance to cleaning, fixation, and permeabilization: After repeated cleaning, fixation, and permeabilization of cells, the silicon nanodots can still have a good imaging effect on lysosomes;

(6) It has good biocompatibility: through the cytotoxicity evaluation experiment, when the concentration of the silicon nano-dot is 500 μg/mL and below, the toxicity to cells is very low; and after long-term irradiation with laser, its cytotoxicity Still very low, proving that the material has good biocompatibility and low phototoxicity;

(7) The preparation method of the present invention is simple, the raw materials are cheap and easy to obtain, and large-scale preparation can be realized.

Description of drawings

Fig. 1 is the schematic diagram that prepares ultra-bright fluorescent silicon nano-dots (SiNDs) with silane reagent AEEA and Bengal Rose Bengal in embodiment 1;

Fig. 2 is the transmission electron micrograph of the SiNDs that makes in embodiment 1;

Fig. 3 is the particle size statistical result figure of the SiNDs that makes in embodiment 1;

Fig. 4 is the ultraviolet-visible absorption spectrogram of the SiNDs that makes in embodiment 1;

Fig. 5 is the fluorescence excitation and emission spectrogram of the SiNDs that make in embodiment 1;

Fig. 6 is the extinction figure at 650nm of SiNDs prepared in embodiment 1 in different pH environments;

Fig. 7 is the effect diagram of lysosome imaging after the SiNDs prepared in Example 1 were incubated in A549 cells for different time;

Fig. 8 is the effect diagram of lysosome imaging after incubation of commercial green lysosome fluorescent dye LysoTracker Green (LT-Green) with A549 cells for different time;

Figure 9 is the lysosome imaging effect diagram after the commercial red lysosome fluorescent dye LysoTracker Red (LT-Red) was incubated with A549 cells for different time;

Fig. 10 is a comparison diagram of lysosome imaging effects of SiNDs prepared in Example 1 and LT-Green and LT-Red after cleaning for different periods of time;

Fig. 11 is a comparison diagram of the fluorescence stability of SiNDs prepared in Example 1 and LT-Red after irradiation with laser light for different times;

Fig. 12 is a comparison diagram of SiNDs prepared in Example 1 and LT-Green and LT-Red in A549 cells in resistance to fixation and resistance to permeabilization;

Fig. 13 is a lysosome imaging effect diagram of SiNDs prepared in Example 1 in different cell lines;

Fig. 14 is the cytotoxicity graph of SiNDs prepared in Example 1 in A549 cells under light and normal conditions.

Detailed ways

Example 1

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

(1) Weigh Rose Bengal and fully dissolve it in ultrapure water, add an appropriate volume of AEEA so that the mass ratio of AEEA to Rose Bengal is 100:3. The two are fully mixed and transferred to a hydrothermal reaction kettle;

(2) Reaction: React at 160°C for 3 hours in a hydrothermal reactor to form a silicon nanodot solution;

(3) Purification: the target ultra-bright fluorescent silicon nano-dot solution is obtained by dialysis.

The schematic diagram of the reaction is shown in Figure 1, the transmission electron microscope results of the prepared SiNDs are shown in Figure 2, the statistical results of the particle size distribution are shown in Figure 3, the UV-Vis absorption spectrum is shown in Figure 4, and the fluorescence excitation emission spectrum is shown in Figure 5 , its extinction at 650nm in different pH environments is shown in Figure 6. It can be seen from the above figures that the prepared SiNDs are uniform spherical, with a particle size of approximately 2.5nm, its maximum ultraviolet absorption peak is located at 511nm, and its maximum excitation wavelength and maximum emission wavelength are 511nm and 525nm, respectively. It is calculated that the fluorescence quantum yield of the SiNDs can be as high as 100%. In particular, it can be seen from Figure 6 that the SiNDs are easy to aggregate and precipitate under weakly acidic environmental conditions. This characteristic makes it cause particle aggregation when entering the acidic lysosome in the cell, thereby realizing long-term lysosome imaging.

Example 2

The lysosome imaging effect of SiNDs prepared in Example 1 on A549 cells was tested as follows:

(1) Cell culture: A549 cells were seeded in a 96-well plate at a density of 5×10 ³ cells/well, and cultured at 37°C and 5% CO ₂ for 24 hours;

(2) Cell staining: the culture medium in each well was replaced with fresh culture medium containing 20 g/mL SiNDs, cultured at 37°C and 5% CO2 for ₂ , 4, 18 and 24 h, and then treated with phosphoric acid Buffer (PBS) washed 2 times. Then, stain with commercial red lysosomal fluorescent dye (LT-Red) and cell nucleus staining reagent (Hoechst 33342) for 30 and 10 minutes, respectively. Finally, wash the cells twice with PBS;

(3) Confocal fluorescence microscope imaging observation: using lasers with wavelengths of 405, 488 and 552nm as excitation light, Hoechst 33342 dye emits blue fluorescence under excitation of 405nm excitation light, while SiNDs emits green fluorescence under excitation of 488nm excitation light, LT -Red dye exhibits red fluorescence under 552nm laser light.

The results of lysosome imaging and co-localization are shown in Figure 7. It can be seen from the figure that the SiNDs are mainly distributed in some punctate positions after entering the cells. After co-localization analysis with a commercially available red lysosomal fluorescent dye (LT-Red), it is confirmed that the lit area of the SiNDs is the lysosome. And the calculated co-localization rate with lysosome dyes is as high as 98%. In addition, the SiNDs remained in lysosomes even when the dosing time was extended to 24 h. Therefore, the ability of SiNDs synthesized in the present invention to target lysosomes will not vary with the time of drug addition, reflecting the stability of its lysosome imaging.

Examples 3 and 4

The lysosome imaging effect of commercial lysosomal green fluorescent dye (LT-Green) and red fluorescent dye (LT-Red) on A549 cells was tested.

The operation process of Examples 3 and 4 is basically the same as that of Example 1, only need to replace SiNDs in step (2) with 1 LT-Green and LT-Red for 30 min, 4 h and 8 h respectively.

The effects of lysosome imaging are shown in Figures 8 and 9. Observing Figures 8 and 9, it can be seen that the change of dosing time has little effect on the green lysosomal fluorescent dye LT-Green, but greatly changes the staining site of LT-Red. As the dosing time was extended to 4h, LT-Red was transferred to the cytoplasm and nuclear membrane other than the lysosome, and even at 8h, some dyes had entered the nucleus. It can be seen that, compared with LT-Red, the SiNDs synthesized in the present invention are more suitable for long-term tracking of lysosome imaging.

Example 5

The lysosome imaging stability of the SiNDs prepared in Example 1 and commercial LT-Green and LT-Red after cleaning was tested as follows:

(1) Cell culture: the process is consistent with step (1) in Example 2;

(2) Cell staining and washing: replace the culture medium in the cell culture wells with fresh culture medium containing SiNDs (20 μg/mL), LT-Green (1 μM) and LT-Red (1 μM), and stain the corresponding wells for 2 h, 30min and 30min. After being washed twice with PBS, cultured in a dark environment of 37°C and 5% CO ₂ for 0, 1, 2, 4, 12, 24, 36 and 48 hours, observed under a confocal fluorescence microscope;

(3) Observation of lysosome imaging effects: 488nm and 552nm lasers were used as excitation light. Among them, SiNDs and LT-Green emit green fluorescence under 488nm laser, and LT-Red emit red fluorescence under 552nm laser.

The results of fluorescence imaging are shown in Figure 10. Comparing the imaging results of the three dyes, it can be seen that the fluorescence of the commercially available lysosomal dyes LT-Green and LT-Red decreased significantly within 2 hours after the cells were washed. The obvious difference is that the SiNDs prepared by the present invention still have strong fluorescence within 48 hours after the cells are washed, so clear imaging of lysosomes can be achieved for a long time.

Example 6

The stability of lysosome fluorescence imaging of the SiNDs prepared in Example 1 and the commercialized LT-Red after irradiation with laser light for different periods of time was tested. Methods as below:

(1) Cell culture: the process is consistent with step (1) in Example 2;

(2) Cell staining: replace the culture medium in the cell culture wells with fresh culture medium containing SiNDs (20 μg/mL) and LT-Red (1 μM), respectively, and wash the cells twice with PBS after staining for 2 h and 30 min, respectively;

(3) Laser irradiation: After the stained cells were irradiated with a 488nm laser for 0, 0.5, 1, 3, 5, 10, 15, 20 and 30 min respectively, they were observed with a confocal fluorescence microscope;

(4) Observation of fluorescence stability: 488nm and 552nm lasers were used as excitation light. Among them, SiNDs emit green fluorescence under 488nm laser, and LT-Red emit red fluorescence under 552nm laser.

It can be seen from Fig. 11 that the fluorescence of LT-Red is significantly weakened within 3 minutes of laser irradiation, while the SiNDs prepared by the present invention still maintain a clear lysosome imaging effect after 20 minutes of irradiation. This experiment confirmed the remarkable advantages of SiNDs in stable imaging against photobleaching.

Example 7

The ability of SiNDs prepared in Example 1 and commercially available LT-Green and LT-Red to resist fixation and permeabilization were tested. Proceed as follows:

(1) Cell culture: the process is consistent with step (1) in Example 2;

(2) Cell staining: This process is consistent with step (2) in Example 5, staining with fresh culture solution containing SiNDs (20 μg/mL), LT-Green (1 μM) and LT-Red (1 μM) for 2 h respectively, 30min and 30min. The stained cells were observed under a confocal fluorescence microscope;

(3) Cell fixation: place the stained cells in phosphate buffer containing 4% glutaraldehyde for 30 minutes, wash them twice with PBS, and observe the lysosome imaging results under a confocal fluorescence microscope;

(4) Cell permeabilization: After the cells were fixed, they were treated with phosphate buffer containing 0.1% Triton X-100 for 10 minutes, and finally the cells were washed twice with PBS, and observed under a confocal fluorescence microscope. The setting conditions of the confocal fluorescence microscope are consistent with step (3) in Example 5.

It can be seen from Figure 12 that neither LT-Green nor LT-Red is suitable for lysosome imaging of fixed and permeabilized cells, while the lysosome imaging probe prepared by the present invention has the ability to resist fixation and anti-permeabilization .

Example 8

To test the lysosome imaging effect of the SiNDs prepared in Example 1 in different cell lines, the operation steps were roughly the same as in Example 2, only the A549 cells in Example 2 were replaced with normal lung cells AT II, mammary gland For cancer cell MCF-7 and macrophage Raw 264.7, the staining time can be changed to 2h.

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

Example 9

To test the cytotoxicity of the SiNDs prepared in Example 1 to A549 cells under dark and light conditions, the operation steps are as follows:

(1) Cell culture: the process is consistent with step (1) in Example 2;

(2) SiNDs treatment: the culture medium in the cell culture wells was replaced with fresh culture medium containing different concentrations of SiNDs. After culturing for 24 h in a 5% CO ₂ environment. The cells were divided into two groups, one group was irradiated with white light for 30 min, and the other group was placed in dark environment. Subsequently, the two groups of cells were cultured at 37°C and 5% CO ₂ for 4 hours;

(3) Detection of cell activity: the cytotoxicity was detected by MTT method.

The results of cell viability are shown in Figure 14. It can be seen from the figure that even if the concentration of SiNDs is as high as 200 μg/mL, the cells in the light and non-light groups maintain more than 90% of the activity, which reflects the low toxicity of the SiNDs synthesized by the present invention, making it a safe lysosome imaging method. fluorescent probe.

Claims (7)

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.