Fluorescent carbon quantum dot, preparation method thereof and application of fluorescent carbon quantum dot in organelle and organoid imaging and mitochondrial membrane potential monitoring

Abstract

The invention discloses a fluorescent carbon quantum dot, a preparation method thereof and application of the fluorescent carbon quantum dot in organelle and organoid imaging and mitochondrial membrane potential monitoring, and the method comprises the following steps: S1, mixing 1, 5-diamino-anthraquinone, vanillic acid and ethidene diamine in a solvent, carrying out a hydrothermal reaction under heating, and cooling to room temperature after the reaction is completed; s2, centrifuging the product obtained in the step S1, taking the centrifugate, adding silica gel, performing rotary evaporation, eluting with an eluent, collecting the eluent, and performing freeze drying to obtain the fluorescent carbon quantum dots; wherein the solvent is deionized water or ethanol, and the eluent is a mixture of dichloromethane and methanol. The G-CDs and the R-CDs provided by the invention both show excellent optical characteristics and specific mitochondrial and lysosome targeting characteristics, so that the G-CDs and the R-CDs can be used for imaging and dynamic tracking of living organelles. Meanwhile, the carbon dots are verified to have the capability of realizing efficient biological imaging in an organoid system.

Description

Technical Field

[0001] The present invention relates to the field of biology and nanomaterials, and in particular to a fluorescent carbon quantum dot, a preparation method thereof, and an application thereof in organelle and organoid imaging and mitochondrial membrane potential monitoring. Background Art

[0002] Mitochondria play an important role in generating energy and regulating cell metabolism in cells. They produce adenosine triphosphate (ATP) through the process of oxidative phosphorylation to provide cells with necessary energy, regulate intracellular calcium balance and participate in cell apoptosis. When mitochondrial function is abnormal, it will have a serious impact on cells and the human body, leading to intracellular energy supply, oxidative stress, apoptosis and metabolic disorders, and have a serious impact on cell structure and function. Lysosomes are mainly involved in intracellular degradation and digestion, including the degradation of endocytosed substances and extracellular substances. Lysosomal abnormalities affect intracellular material metabolism and immune response, leading to lysosome-related diseases, such as lysosomal storage diseases. These diseases are usually characterized by the accumulation of specific substances in lysosomes, which may lead to impaired multi-organ function and pose a serious risk to human health. Therefore, long-term and accurate monitoring of mitochondria and lysosomes is particularly important, which not only helps researchers to deeply understand the normal physiological functions of these organelles and their relationship with the occurrence and development of diseases, but also provides an important basis for early diagnosis, treatment and prognosis evaluation of diseases.

[0003] Organoids are simple cell-based in vitro models of tissue engineering, which are miniaturized and simplified organ model systems in vitro. Studying cell and organ functions and their changes in disease states is also a common use of organoids. In order to further enhance the depth and breadth of organoid research, it is necessary to expand the library of probe tools suitable for organoid observation. Among them, fluorescent probes, as an important imaging tool, play an irreplaceable role in cell and tissue imaging. The present invention focuses on innovative research and development based on carbon dot fluorescent probes, aiming to develop new fluorescent probes with excellent optical properties, good biocompatibility and high targeting. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a fluorescent carbon quantum dot, a preparation method thereof and its application in organelle and organoid imaging and mitochondrial membrane potential monitoring in view of the deficiencies in the above-mentioned prior art.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: In a first aspect of the present invention, a method for preparing fluorescent carbon quantum dots is provided, comprising the following steps:

[0006] S1, mixing 1,5-diaminoanthraquinone, vanillic acid and ethylenediamine in a solvent, performing a hydrothermal reaction under heating, and cooling to room temperature after the reaction is completed;

[0007] S2, centrifuging the product obtained in step S1, taking the centrifuge liquid, adding silica gel powder, rotary evaporation, eluting with an eluent, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, then eluting with an eluent, collecting the eluate, and freeze-drying to obtain fluorescent carbon quantum dots;

[0008] The solvent is deionized water or ethanol, and the eluent is a mixture of dichloromethane and methanol.

[0009] Preferably, the method for preparing the fluorescent carbon quantum dots comprises the following steps:

[0010] S1, 0.05g 1,5-diaminoanthraquinone, 0.05g vanillic acid, and 1mL ethylenediamine were mixed in 30mL deionized water, and the mixture was hydrothermally reacted at 200°C for 8 hours, and then cooled to room temperature after the reaction was completed;

[0011] S2, centrifuging the product obtained in step S1, taking the centrifugal solution, adding 1.5 g of 300-400 mesh silica gel powder, rotary evaporation, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, and then eluting with an eluent, collecting the eluate that emits bright green fluorescence, freeze-drying, and obtaining green fluorescent carbon dots G-CDs;

[0012] The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane:methanol is 10:1.

[0013] Preferably, the method for preparing the fluorescent carbon quantum dots comprises the following steps:

[0014] S1, 0.05g 1,5-diaminoanthraquinone, 0.05g vanillic acid, and 1mL ethylenediamine were mixed in 30mL ethanol, and the mixture was hydrothermally reacted at 200°C for 8 hours, and then cooled to room temperature after the reaction was completed;

[0015] S2, centrifuging the product obtained in step S1, taking the centrifugal solution, adding 1.5 g of 300-400 mesh silica gel powder, rotary evaporation, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, and then eluting with an eluent, collecting the eluate that emits bright red fluorescence, freeze-drying, and obtaining red fluorescent carbon dots R-CDs;

[0016] The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 20:1.

[0017] The second aspect of the present invention provides green fluorescent carbon dots G-CDs, which are prepared by the method as described above.

[0018] The third aspect of the present invention provides red fluorescent carbon dots R-CDs, which are prepared by the method described above.

[0019] In a fourth aspect, the present invention provides a use of the green fluorescent carbon dots G-CDs as described above in organelle imaging, wherein the green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of mitochondria in living cells.

[0020] In a fifth aspect, the present invention provides an application of the green fluorescent carbon dots G-CDs as described above in organoid imaging, wherein the green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of mitochondria in organoids.

[0021] In a sixth aspect, the present invention provides a use of the green fluorescent carbon dots G-CDs as described above as a probe in monitoring mitochondrial membrane potential.

[0022] In a seventh aspect, the present invention provides an application of the red fluorescent carbon dots R-CDs as described above in organelle imaging, wherein the green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of lysosomes in living cells.

[0023] In an eighth aspect of the present invention, there is provided an application of the red fluorescent carbon dots R-CDs as described above in organelle imaging, wherein the green fluorescent carbon dots are used as fluorescent dyes for targeted imaging of lysosomes in organoids.

[0024] The beneficial effects of the present invention are:

[0025] The present invention uses the same precursor to synthesize two types of carbon dots through a "bottom-up approach", namely G-CDs emitting at 515nm and R-CDs emitting at 611nm, two types of carbon dots with different fluorescence properties; both G-CDs and R-CDs exhibit excellent optical properties, as well as specific mitochondrial and lysosomal targeting properties, which enable them to be used for imaging and dynamic tracking of living organelles. The G-CDs and R-CDs probes developed by the present invention show significant application potential in key cellular events such as dynamic monitoring of mitochondria and lysosomes, and also verify that these carbon dots also have the ability to achieve efficient biological imaging in organoid systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The morphology and particle size distribution results of G-CDs and R-CDs;

[0027] Figure 2 FTIR spectra of G-CDs and R-CDs;

[0028] Figure 3 XPS spectra of G-CDs and R-CDs;

[0029] Figure 4 are the excitation and emission spectra of G-CDs and R-CDs;

[0030] Figure 5 Cell images of HeLa cells stained for G-CDs and R-CDs with commercial dyes for mitochondria and lysosomes;

[0031] Figure 6 Comparison of the results of using G-CDs and TMRE to monitor changes in mitochondrial membrane potential;

[0032] Figure 7 The results of using G-CDs as a probe to monitor the process of mitophagy;

[0033] Figure 8 The results of R-CDs monitoring lysosome movement during lysosomal autophagy;

[0034] Fig. 9 Experimental results of fine-scale imaging of organoids using G-CDs and R-CDs. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0036] It should be understood that terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0037] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0038] Example 1

[0039] This embodiment provides a green fluorescent carbon dots G-CDs, and the preparation method thereof comprises the following steps:

[0040] S1, 0.05g 1,5-diaminoanthraquinone, 0.05g vanillic acid, and 1mL ethylenediamine were mixed in 30mL deionized water, and the mixture was hydrothermally reacted at 200°C for 8 hours, and then cooled to room temperature after the reaction was completed;

[0041] S2, centrifuging the product obtained in step S1, discarding the unreacted raw materials and by-products precipitated at the bottom, taking the centrifuge liquid, adding 1.5 g of 300-400 mesh silica gel powder, and fully drying by rotary evaporation to make the reaction product adhere to the silica gel powder, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, and then eluting with an eluent, collecting the eluate that emits bright green fluorescence, and freeze-drying to obtain green fluorescent carbon dots G-CDs;

[0042] The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:1.

[0043] This embodiment provides a red fluorescent carbon dot R-CDs, and the preparation method thereof is similar to that of the green fluorescent carbon dot G-CDs, except that the solvent and the eluent are different, and specifically comprises the following steps:

[0044] S1, 0.05g 1,5-diaminoanthraquinone, 0.05g vanillic acid, and 1mL ethylenediamine were mixed in 30mL ethanol, and the mixture was hydrothermally reacted at 200°C for 8 hours, and then cooled to room temperature after the reaction was completed;

[0045] S2, centrifuging the product obtained in step S1, taking the centrifugal solution, adding 1.5 g of 300-400 mesh silica gel powder, and fully drying by rotary evaporation to allow the reaction product to adhere to the silica gel powder, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, and then eluting with an eluent, collecting the eluate that emits bright red fluorescence, and obtaining red fluorescent carbon dots R-CDs;

[0046] The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane:methanol is 20:1.

[0047] In order to facilitate storage and subsequent experimental use, the prepared G-CDs and R-CDs were dissolved in water and ethanol solutions with a concentration of 10 mg / mL, respectively.

[0048] Performance Characterization and Testing

[0049] Reference Figure 1 , are the morphology and particle size distribution of G-CDs and R-CDs, Figure 1 In the figure, G-CDs ( Figure 1 a) and R-CDs( Figure 1 c) TEM image. Statistical analysis of the sizes of 50 particles showed that the average diameters of G-CDs and R-CDs were approximately 2.48 nm and 3.90 nm, respectively. In order to further explore the structural characteristics of the particles, high-resolution transmission electron microscopy was also used. Through the above characterization, it was found that the interplanar spacings of the two carbon dots were 0.210 nm and 0.209 nm, respectively, which is consistent with the (100) crystal plane of graphite.

[0050] Reference Figure 2 , are the FTIR spectra of G-CDs and R-CDs. In the FTIR spectrum of G-CDs, the stretching vibration of CH is present at 2928 and 2863 cm -1 The stretching vibration range of C=O is 1620.9cm -1 Up to 1512.2cm -1The absorption band caused by the stretching vibration of CN is located at 1307.0 cm -1 , and 817.5cm -1 The peak at 3312.6 cm-1 corresponds to the bending vibration of CH. In the spectrum of R-CD, the stretching vibrations of NH and CH are at 3312.6, 2972 ​​and 2880 cm-1, respectively. -1 The peak values ​​are at 1087.6 and 1045.7 cm -1 The peak at 879 cm -1 The sharp peak at corresponds to the bending vibration of CH. The surface groups of G-CDs and R-CDs were determined by infrared spectroscopy.

[0051] Reference Figure 3 , are the XPS spectra of G-CDs and R-CDs. In the XPS full spectrum analysis, G-CDs ( Figure 3 a) and R-CDs( Figure 3 The samples in b) all exhibit three significant peaks at C1s (284.8 eV and 285.1 eV, respectively), N 1s (399.1 eV), and O1s (532.0 eV and 532.1 eV, respectively), which reveal the presence of three main elements, C, O, and N, on the surface of these carbon dots.

[0052] Reference Figure 4 , are the excitation and emission spectra of G-CDs and R-CDs. It can be seen from the figure that the green carbon dots ( Figure 4 a) exhibits a strong emission peak at 515 nm, and its maximum excitation wavelength is located at 460 nm. In addition, red carbon dots ( Figure 4 b) exhibits an optimal emission wavelength at 611 nm and an optimal excitation wavelength at 560 nm.

[0053] G-CDs and R-CDs application performance test:

[0054] 1. G-CDs tracking mitochondrial dynamics study

[0055] In order to use G-CDs to monitor the changes in mitochondrial membrane potential during apoptosis and the dynamics of mitochondria during mitophagy, HeLa cells were first cultured in glass-bottomed culture dishes. Cells were pretreated with CCCP (10 mM) for 2 hours to induce apoptosis, which would cause changes in normal mitochondrial membrane potential. Then, G-CDs (100 μg / mL) were stained at 37°C for 60 minutes, and the fluorescence images of the cells were recorded.

[0056] Cells were initially incubated with G-CDs at 37°C for 60 min, and then treated with 5 μg / mL rapamycin to induce mitophagy. Fluorescence images were captured at different time points, including without rapamycin and 5, 10, and 20 min after the addition of rapamycin.

[0057] 2. Study on the dynamics of lysosomes tracked by R-CDs

[0058] In order to accurately record and analyze the dynamic changes of lysosomes during apoptosis and cell necrosis, and to better understand the role of lysosomes in these complex biological processes. A series of detailed experimental steps were designed in the experiment. HeLa cells were co-cultured with R-CDs at a concentration of 100μg / mL for 60 minutes. Next, in order to simulate the cell stress environment, 5μM chloroquine was used to stimulate the treated cells. Then, at specific time points after chloroquine stimulation, namely 0, 4, 8, 12 and 16 minutes, a series of cell image sequences were captured using a confocal microscope.

[0059] 3. Organoid Imaging of G-CDs and R-CDs

[0060] After the fresh tumor tissue was finely chopped, it was washed at least three times with ice-cold PBS mixed with 100U / mL penicillin / streptomycin and 0.25μg / mL amphotericin B (from Solarbio), 10μg / mL gentamicin (from Solarbio), and then digested in DMEM containing 10% FBS, 1.5mg / mL collagenase II (from Solarbio), 500U / mL collagenase IV (from Solarbio), 20μg / mL hyaluronidase (from Sigma) and 10μM Y27632 (from Biogems), and incubated at 37°C for 30 minutes. After digestion, the dissociated tissue was filtered through a cell strainer. Then, the cell suspension was centrifuged for 5 minutes. The cell pellet was resuspended with a solution containing BME (reduced growth factor provided by Biomaterial Engineering Company) and distributed to a 48-well cell culture plate, with 30μL BME added to each well. After culturing at 37°C for 20 min, pre-prepared culture medium was added to each well. After growing in the culture medium for 5 days, 100 μg / mL of carbon dot solution was added to the organoid samples for fluorescence imaging.

[0061] Reference Figure 5, cell images of HeLa cells stained with G-CDs and R-CDs and commercial dyes for mitochondria and lysosomes. The distribution of G-CDs in cells showed a high degree of consistency with the mitochondria stained with Mito Tracker Red, with a Pearson correlation coefficient of up to 0.82. The merged image of R-CDs and lysosomes stained with Lyso Tracker Green showed even more significant consistency, with a Pearson correlation coefficient of 0.87. The above experimental results not only demonstrate the potential of G-CDs and R-CDs to localize in specific organelles in the cytoplasm, but also demonstrate their effectiveness as cell imaging and organelle labeling tools through quantitative analysis.

[0062] Reference Figure 6 , is the comparison result of G-CDs and TMRE for monitoring the changes of mitochondrial membrane potential. After CCCP treatment, the green fluorescence intensity of G-CDs in the cell decreased, and the distribution range was no longer limited to the cytoplasm. At the same time, after CCCP treatment, the red fluorescence area stained by TMRE in the cell also showed a significant decrease in fluorescence intensity. Therefore, G-CDs has a similar fluorescence intensity attenuation trend compared with commercial probes for monitoring mitochondrial membrane potential.

[0063] Reference Figure 7 , which is the result of using G-CDs as a probe to monitor the process of mitochondrial autophagy. After 10 minutes of treatment, a decrease in fluorescence intensity can be observed, accompanied by the fragmentation of the mitochondrial filamentous structure. This observation indicates the activation of the process of mitochondrial autophagy. When the treatment time reaches 20 minutes, the obvious change in mitochondrial structure caused by rapamycin becomes particularly significant: the originally clear mitochondrial structure becomes small dots scattered inside the cell. Compared with the early observation points, this structural change is accompanied by a further decrease in fluorescence intensity. This phenomenon is likely the result of a series of changes triggered by rapamycin in the cellular microenvironment.

[0064] Reference Figure 8 , which is the result of R-CDs monitoring lysosome movement during lysosomal autophagy. Figure 8 ae are microscope images acquired at 0, 4, 8, 12, and 16 minutes, respectively. In order to more clearly show the movement trend of lysosomes, the images at different time points were combined, and the movement direction of lysosomes was marked with arrows in the small figure. The above experimental results prove that R-CDs can effectively monitor and reveal the dynamic changes of lysosomes in cells as a probe.

[0065] Reference Fig. 9, the results of a detailed imaging experiment using G-CDs and R-CDs on simulated miniature organs - organoids. By co-staining G-CDs with Mito Tracker Red, the mitochondria of intestinal organoids were labeled and observed. The imaging results are shown in the figure. At the same time, co-staining with R-CDs and Lyso Tracker Green was used to accurately locate and image the lysosomes in colon cancer organoids. The results of confocal microscopy are shown in Fig. 9 fj. Fig. 9 ac are images of intestinal organoids stained with Dapi, G-CDs and Mito-Tracker Red, respectively, and the merged images are: Fig. 9 e It can be observed that G-CDs have good overlap with the commercial mitochondrial staining probe; Fig. 9 fh are intestinal organoids stained with Dapi, R-CDs and Lyso-Tracker Green, respectively, and the merged images are: Fig. 9 j It can be observed that R-CDs can mark lysosomes in organoid samples;. Furthermore, G-CDs, R-CDs and Dapi were incubated with intestinal organoids, and detailed spatial structural images were obtained by three-dimensional fluorescence imaging technology, which are Fig. 9 k,l,m, Fig. 9 n is the merged image.

[0066] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing fluorescent carbon quantum dots, characterized in that: The following steps are involved: S1, mixing 1,5-diaminoanthraquinone, vanillic acid and ethylenediamine in a solvent, performing a hydrothermal reaction under heating, and cooling to room temperature after the reaction is completed; S2, centrifuging the product obtained in step S1, taking the centrifuge liquid, adding silica gel powder, rotary evaporation, eluting with an eluent, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, then eluting with an eluent, collecting the eluate, and freeze-drying to obtain fluorescent carbon quantum dots; The solvent is deionized water or ethanol, and the eluent is a mixture of dichloromethane and methanol.

2. The method for preparing fluorescent carbon quantum dots according to claim 1, characterized in that: The following steps are involved: S1, 0.05g 1,5-diaminoanthraquinone, 0.05g vanillic acid, and 1mL ethylenediamine were mixed in 30mL deionized water, and the mixture was hydrothermally reacted at 200°C for 8 hours, and then cooled to room temperature after the reaction was completed; S2, centrifuging the product obtained in step S1, taking the centrifugal solution, adding 1.5 g of 300-400 mesh silica gel powder, rotary evaporation, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, and then eluting with an eluent, collecting the eluate that emits bright green fluorescence, freeze-drying, and obtaining green fluorescent carbon dots G-CDs; The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane:methanol is 10:

1.

3. The method for preparing fluorescent carbon quantum dots according to claim 1, characterized in that: The following steps are involved: S1, 0.05g 1,5-diaminoanthraquinone, 0.05g vanillic acid, and 1mL ethylenediamine were mixed in 30mL ethanol, and the mixture was hydrothermally reacted at 200°C for 8 hours, and then cooled to room temperature after the reaction was completed; S2, centrifuging the product obtained in step S1, taking the centrifugal solution, adding 1.5 g of 300-400 mesh silica gel powder, rotary evaporation, adding the silica gel powder to the upper end of the stationary phase of the chromatography column, and then eluting with an eluent, collecting the eluate that emits bright red fluorescence, freeze-drying, and obtaining red fluorescent carbon dots R-CDs; The eluent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 20:

1.

4. A green fluorescent carbon dot G-CDs, characterized in that: It is prepared by the method according to claim 2.

5. A red fluorescent carbon dot R-CDs, characterized in that: It is prepared by the method according to claim 3.

6. A use of the green fluorescent carbon dots G-CDs as claimed in claim 4 in organelle imaging, characterized in that: The green fluorescent carbon dots were used as fluorescent dyes for targeted imaging of mitochondria in living cells.

7. A use of the green fluorescent carbon dots G-CDs as claimed in claim 4 in organoid imaging, characterized in that: The green fluorescent carbon dots were used as fluorescent dyes for targeted imaging of mitochondria in organoids.

8. Use of the green fluorescent carbon dots G-CDs as claimed in claim 4 as a probe in monitoring mitochondrial membrane potential.

9. A use of the red fluorescent carbon dots R-CDs as claimed in claim 5 in organelle imaging, characterized in that: The green fluorescent carbon dots were used as fluorescent dyes for targeted imaging of lysosomes in living cells.

10. A use of the red fluorescent carbon dots R-CDs as claimed in claim 5 in organelle imaging, characterized in that: The green fluorescent carbon dots were used as fluorescent dyes for targeted imaging of lysosomes in organoids.

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