Orange fluorescent silicon nanodot, preparation method thereof and application of orange fluorescent silicon nanodot in imaging of mitochondria in living cells

Abstract

The invention discloses an orange fluorescent silicon nanodot, a preparation method thereof and an application of the orange fluorescent silicon nanodot in mitochondrial imaging in living cells, the method comprises the following steps: S1, dissolving tetramethyl rhodamine methyl ester in ultrapure water, then adding N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, uniformly stirring, and carrying out hydrothermal reaction on the obtained mixture; and S2, cooling after the reaction is finished, purifying the product by using a chromatographic column, eluting by using an eluent, collecting the eluent, and drying to obtain the orange fluorescent silicon nanodot. The silicon nanodot provided by the invention has the advantages of good light stability, high quantum yield and the like, and can be successfully applied to positioning imaging of mitochondria in living cells; compared with common blue-green fluorescence, orange fluorescence has better tissue penetrating ability and background interference resistance; the O-SiNDs prepared by the invention also has the property similar to that of lipophilic cations, and can realize high-specificity targeted marking on mitochondria of living cells.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials, and in particular to an orange fluorescent silicon nanodot, its preparation method, and its application in mitochondrial imaging in living cells. Background Technology

[0002] Mitochondria are important, thread-like organelles in eukaryotic cells, and key executors of aerobic respiration. They not only play a crucial role in providing energy for cellular activities but also participate extensively in various cellular functions, including signal transduction, calcium storage, and regulation of intracellular homeostasis. Furthermore, mitochondria perform substance exchange through close interactions with other organelles, such as regulating unfolded protein responses via mitochondrial-endoplasmic reticulum contact or clearing damaged mitochondria through mitochondrial-lysosomal interactions. Mitochondrial dysfunction often directly leads to cell degeneration and even death, and in severe cases, can cause various diseases such as diabetes and arrhythmias.

[0003] Silicon nanodots, as an emerging fluorescent nanomaterial, have demonstrated broad application potential in the biomedical field due to their low biotoxicity, excellent photostability, and easily modifiable surface properties, effectively overcoming the limitations of traditional fluorescent dyes and semiconductor quantum dots. Therefore, developing silicon nanodot-based fluorescent dyes or imaging materials for mitochondrial imaging is of great significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an orange fluorescent silicon nanodot, its preparation method, and its application in mitochondrial imaging in living cells.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides a method for preparing orange fluorescent silicon nanodots, comprising the following steps: S1. Dissolve tetramethylrhodamine methyl ester in ultrapure water, then add N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, stir until homogeneous, and carry out hydrothermal reaction of the resulting mixture. S2. After the reaction is complete, the product is cooled and purified by chromatography. The product is eluted with eluent, collected, and dried to obtain orange fluorescent silicon nanodots.

[0006] Preferably, step S1 specifically includes: Dissolve tetramethylrhodamine methyl ester in ultrapure water, then add N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, stir until homogeneous, transfer the resulting mixture to a reaction vessel, and react at 150-200℃ for 5-20 hours. Preferably, the hydrothermal reaction temperature in step S1 is 180°C and the reaction time is 10 hours.

[0007] Preferably, step S2 is as follows: after the reaction is completed, the product is cooled to room temperature, centrifuged to discard the precipitate, and then purified by silica gel chromatography column. The product is eluted with a mixture of dichloromethane and methanol. The elution solution is collected, freeze-dried, and orange fluorescent silicon nanodots are obtained.

[0008] Preferably, in the eluent of step S2, the volume ratio of dichloromethane to methanol is 5~20:1.

[0009] Preferably, in the eluent of step S2, the volume ratio of dichloromethane to methanol is 10:1.

[0010] Preferably, the method for preparing the orange fluorescent silicon nanodots includes the following steps: S1. Dissolve 0.2g of tetramethylrhodamine methyl ester in 20mL of ultrapure water, then add 1.0mL of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, stir until homogeneous, transfer the resulting mixture to a reaction vessel, and react at 180℃ for 10 hours. S2. After the reaction is complete, cool to room temperature, centrifuge the product to discard the precipitate, and then purify it using a silica gel chromatography column. The eluent consists of dichloromethane and methanol in a volume ratio of 10:1. Collect the eluent, freeze-dry it, and obtain orange fluorescent silicon nanodots.

[0011] In a second aspect, the present invention provides an orange fluorescent silicon nanodot, characterized in that it is prepared by the method described above.

[0012] A third aspect of the invention provides the application of the orange fluorescent silicon nanodots described above in mitochondrial imaging within living cells. For example, the application of the orange fluorescent silicon nanodots in the preparation of mitochondrial imaging formulations.

[0013] A fourth aspect of the present invention provides the use of the orange fluorescent silicon nanodots described above in the preparation of fluorescent dyes targeting mitochondria.

[0014] The beneficial effects of this invention are: This invention develops a silicon nanodot (O-SiNDs) that emits orange fluorescence, which has advantages such as good photostability and high quantum yield, and can be successfully applied to mitochondrial localization imaging in live cells. Compared with common blue-green fluorescence, orange fluorescence has better tissue penetration and resistance to background interference. The O-SiNDs prepared in this invention not only have strong photostability and high quantum yield, but also have properties similar to lipophilic cations, which can achieve highly specific targeted labeling of mitochondria in live cells, and have broad application prospects in the fields of bioimaging and sensing. Attached Figure Description

[0015] Figure 1The FT-IR and XPS characterization results of the O-SiNDs prepared in Example 1; Figure 2 The optical properties and photostability test results of the O-SiNDs prepared in Example 1; Figure 3 The targeting effect of O-SiNDs prepared in Example 1 on mitochondria and nuclei in PANC-1 cells (scale bar: 20 μm). Detailed Implementation

[0016] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0017] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0019] Example 1 An orange fluorescent silicon nanodot is prepared by the following steps: S1. Take 0.2g of tetramethylrhodamine methyl ester and put it into a beaker. Add 20mL of ultrapure water to dissolve it, then add 1.0mL of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and stir until homogeneous. Transfer the resulting mixture to a reaction vessel and react it in a drying oven at 180℃ for 10 hours. S2. After the reaction is complete, cool to room temperature and centrifuge the product (8000 rpm, 5 minutes) to remove precipitate impurities. The remaining liquid is purified by silica gel chromatography. The eluent consists of dichloromethane and methanol in a volume ratio of 10:1. Collect the eluent, freeze-dry it, and obtain orange fluorescent silicon nanodots, denoted as O-SiNDs. Store them in a refrigerator at 4°C for subsequent experiments.

[0020] Sample characterization and performance testing (1) Structure and fluorescence properties of silicon nanodots The FT-IR and XPS characterization results of O-SiNDs are as follows: Figure 1 As shown in a and 1b, the characteristic peaks of silicon and the appearance of silicon-based functional groups prove the successful synthesis of silicon nanodots.

[0021] Figure 2The optical properties and photostability test results of O-SiNDs are referenced. Figure 2 As can be seen, the maximum emission peak of the silicon nanodots is located at 585 nm. After 10 hours of continuous exposure to natural light, their fluorescence intensity still remains above 95% of the initial value. Figure 2 (b) indicates that the material has excellent photostability and can meet the requirements of intracellular imaging.

[0022] (2) Targeted imaging of mitochondria in living cells by silicon nanodots First, PANC-1 cells were cultured at 37°C in 5% CO2 using DMEM medium containing 1% penicillin / streptomycin and 10% fetal bovine serum. After the cells had fully adhered to the confocal culture dish, 20 μg / mL of O-SiNDs was added and incubated for 30 minutes. Then, 30 nmol / L of MTG commercial dye or 1 mg / mL of Hoechst 33342 was added. After staining, the cells were washed three times with PBS, and finally, the imaging results were observed under a fluorescence confocal microscope.

[0023] like Figure 3 As shown in Figure A, after co-staining PANC-1 cells with O-SiNDs and the commercial dye MTG, the orange fluorescence emitted by O-SiNDs exhibited a clear linear structure and highly overlapped with the signal of MTG. The calculated Pearson correlation coefficient between the two was as high as 0.95, indicating that O-SiNDs can efficiently target mitochondria in live cells. Furthermore, to verify its specificity, the experiment also used the nuclear dye Hoechst for staining, and the results showed that its correlation coefficient with O-SiNDs was as low as 0.12 (…). Figure 3 B), thus confirming that O-SiNDs have no targeting effect on the cell nucleus and possess excellent mitochondrial labeling specificity.

[0024] In summary, this invention successfully prepared a novel silicon nanodot (O-SiNDs) that emits orange fluorescence and exhibits excellent photostability. Live-cell imaging results show that these O-SiNDs can specifically target mitochondria. Based on their unique optical properties, good biocompatibility, and targeting specificity, O-SiNDs show broad application prospects in fields such as bioimaging and sensing.

[0025] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for preparing orange fluorescent silicon nanodots, characterized in that, Includes the following steps: S1. Dissolve tetramethylrhodamine methyl ester in ultrapure water, then add N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, stir until homogeneous, and carry out hydrothermal reaction of the resulting mixture. S2. After the reaction is complete, the product is cooled and purified by chromatography. The product is eluted with eluent, collected, and dried to obtain orange fluorescent silicon nanodots.

2. The method for preparing orange fluorescent silicon nanodots according to claim 1, characterized in that, Step S1 is as follows: Tetramethylrhodamine methyl ester was dissolved in ultrapure water, and then N-(2-aminoethyl)-3-aminopropyltrimethoxysilane was added and stirred until homogeneous. The resulting mixture was then transferred to a reaction vessel and reacted at 150-200℃ for 5-20 hours.

3. The method for preparing orange fluorescent silicon nanodots according to claim 2, characterized in that, The hydrothermal reaction temperature in step S1 is 180℃ and the reaction time is 10 hours.

4. The method for preparing orange fluorescent silicon nanodots according to claim 1, characterized in that, Step S2 is as follows: After the reaction is completed, the product is cooled to room temperature, centrifuged to discard the precipitate, and then purified by silica gel chromatography column. The product is eluted with a mixture of dichloromethane and methanol. The elution solution is collected, freeze-dried, and orange fluorescent silicon nanodots are obtained.

5. The method for preparing orange fluorescent silicon nanodots according to claim 4, characterized in that, In the eluent of step S2, the volume ratio of dichloromethane to methanol is 5~20:

1.

6. The method for preparing orange fluorescent silicon nanodots according to claim 5, characterized in that, In the eluent of step S2, the volume ratio of dichloromethane to methanol is 10:

1.

7. The method for preparing orange fluorescent silicon nanodots according to claim 1, characterized in that, Includes the following steps: S1. Dissolve 0.2g of tetramethylrhodamine methyl ester in 20mL of ultrapure water, then add 1.0mL of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, stir until homogeneous, transfer the resulting mixture to a reaction vessel, and react at 180℃ for 10 hours. S2. After the reaction is complete, cool to room temperature, centrifuge the product to discard the precipitate, and then purify it using a silica gel chromatography column. The eluent consists of dichloromethane and methanol in a volume ratio of 10:

1. Collect the eluent, freeze-dry it, and obtain orange fluorescent silicon nanodots.

8. An orange fluorescent silicon nanodot, characterized in that, It is prepared by the method described in any one of claims 1-7.

9. The application of the orange fluorescent silicon nanodots as described in claim 8 in mitochondrial imaging within living cells.

10. The application of the orange fluorescent silicon nanodots as described in claim 8 in the preparation of fluorescent dyes targeting mitochondria.

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