Single carbon dot-based triple-targeting multicolor fluorescent probe and preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescent probes, and specifically discloses a triple-targeting multicolor fluorescent probe based on single carbon dots, a preparation method and application thereof, and the preparation method comprises the following steps: weighing tetramethyl julolidine, dissolving the tetramethyl julolidine in anhydrous ethanol, and placing the tetramethyl julolidine in a high-pressure reaction kettle; after the reaction kettle is sealed, the reaction kettle is placed in a constant-temperature oven, heated and kept for 6 hours; the reaction mixture is subjected to high-speed centrifugation to remove precipitates and filtration; the filtrate is loaded into a dialysis bag, and dialysis is performed in deionized water for 24 hours to remove unreacted precursors and small molecular impurities, so that the fluorescent probe is obtained. The triple-targeting multicolor fluorescent probe based on single carbon dots and the preparation method and application thereof are used, a carbon quantum dot fluorescent probe is prepared by using a one-step solvothermal method, the fluorescent probe can simultaneously and specifically target and label lipid droplets, lysosomes and mitochondria in living cells, and reliable differentiation of the three cell organelles can be realized under multiband confocal fluorescence imaging.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and in particular to a single-carbon-dot triple-targeting multicolor fluorescent probe, its preparation method, and its application. Background Technology

[0002] Organelles are highly differentiated functional structures within cells, working together to maintain overall cellular function. Among them, lipid droplets are dynamic lipid reservoirs regulating cellular metabolism; lysosomes are acidic cellular compartments responsible for the degradation and recycling of biomolecules; and mitochondria serve as the cell's energy factories and mediate apoptosis. Despite their different functions, these organelles form dynamic networks through direct contact, fusion-division events, and coordinated movement, which are crucial for maintaining cellular homeostasis. Therefore, real-time monitoring of the spatial distribution and interactions of organelles is essential for understanding complex intracellular physiological processes.

[0003] Existing multi-organelle imaging methods mainly rely on multiple dye labeling, but these methods suffer from problems such as complex operation, overlapping fluorescence spectra, and poor fluorescence stability. To overcome these limitations, various multicolor fluorescent probes have been developed in recent years, which can generate multiple emission signals in a single probe, enabling simultaneous imaging of multiple organelles. For example, some small molecule probes, through polarity-sensitive or pH-responsive structural design, can distinguish lipid droplets, lysosomes, and mitochondria and achieve tricolor imaging. However, these probes are usually complex to synthesize, have insufficient photostability, and exhibit certain cytotoxicity, limiting their application in long-term live-cell imaging.

[0004] Therefore, developing a single carbon dot three-target fluorescent probe that can be prepared in one step and has multicolor emission, low toxicity and high photostability has important scientific value and application prospects for realizing real-time dynamic imaging of multiple organelles. Summary of the Invention

[0005] The purpose of this invention is to provide a single-carbon-dot triple-targeting multicolor fluorescent probe, its preparation method, and its applications. A carbon quantum dot fluorescent probe is prepared using a one-step solvothermal method. This probe can simultaneously and specifically target and label lipid droplets, lysosomes, and mitochondria within living cells, and reliably distinguish these three organelles under multi-band confocal fluorescence imaging. Using this probe, organelles can be dynamically observed in situ within living cells over a long period, thus providing an efficient and convenient new tool for studying multi-organelle interactions and their dynamic regulation.

[0006] To achieve the above objectives, this invention provides a method for preparing a triple-targeted multicolor fluorescent probe based on a single carbon dot, comprising the following steps:

[0007] Step 1: Weigh tetramethyljulonidine, dissolve it in anhydrous ethanol, mix thoroughly and place it in a 50mL stainless steel high-pressure reactor lined with polytetrafluoroethylene.

[0008] Step 2: After sealing the reaction vessel, place it in a constant temperature oven, heat and maintain for 6 hours, and allow it to cool naturally to room temperature after the reaction is complete.

[0009] Step 3: Centrifuge the reaction mixture at high speed to remove the precipitate, and then filter the supernatant through a 0.22μm filter membrane to remove large particulate impurities;

[0010] Step 4: The obtained filtrate is placed into a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in deionized water for 24 hours to remove unreacted precursors and small molecule impurities, thereby obtaining the fluorescent probe.

[0011] Preferably, in step 1, 0.10 g of tetramethyljulonidine is weighed and dissolved in 20 mL of anhydrous ethanol.

[0012] Preferably, in step 2, the temperature is raised to 180°C.

[0013] Preferably, in step 3, the centrifugation conditions are 10,000 rpm for 15 min.

[0014] The present invention also provides a triple-targeted multicolor fluorescent probe based on a single carbon dot, which is prepared by the above-described preparation method.

[0015] This invention also provides the application of a triple-targeting multicolor fluorescent probe based on a single carbon dot for imaging of multiple organelles in living cells.

[0016] The advantages and beneficial effects of the above-mentioned single-carbon-dot-based triple-targeting multicolor fluorescent probe, its preparation method, and its application are as follows:

[0017] 1. This invention prepares a multi-target fluorescent probe (TriTarget-CDs) capable of simultaneously targeting and labeling lipid droplets, lysosomes, and mitochondria. Its surface simultaneously possesses positive charge, lipophilicity, and weakly basic amine groups. These functional groups enable the probe to selectively locate the three organelles in different microenvironments within the cell: the positive charge promotes mitochondrial accumulation, the lipophilicity enhances binding with lipid droplets, and the weakly basic amine groups are easily protonated in acidic lysosomes, achieving lysosomal targeting.

[0018] 2. The TriTarget-CDs fluorescent probes prepared in this invention have accurate positioning, strong photostability, and multicolor emission in cells, enabling simultaneous visualization of three organelles, providing an effective tool for studying the interactions between organelles and related biological processes.

[0019] 3. The TriTarget-CDs fluorescent probe prepared in this invention generates blue, green and red fluorescence in environments with different polarities, pH and local concentrations, enabling a single probe to distinguish and image multiple organelles.

[0020] 4. The TriTarget-CDs fluorescent probes prepared in this invention have low toxicity to live cells, making them suitable for long-term live cell imaging. They can simplify multi-probe staining operations and reduce potential interference to cells.

[0021] 5. The process for preparing TriTarget-CDs fluorescent probes in this invention is simple, easy to operate, and reproducible, making it convenient for large-scale preparation and application.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 The images show transmission electron microscopy (TEM) images and particle size distributions of the TriTarget-CDs fluorescent probe of this invention, where (a) is a TEM image, (b) is a high-resolution TEM image, and (c) is a particle size distribution diagram.

[0024] Figure 2 The results of ζ-potential measurement of the TriTarget-CDs fluorescent probe of this invention;

[0025] Figure 3 The Fourier transform infrared (FTIR) spectrum of the TriTarget-CDs fluorescent probe of this invention is shown below.

[0026] Figure 4 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the TriTarget-CDs fluorescent probe of this invention, where A is the X-ray photoelectron spectroscopy (XPS) analysis, B is the elemental characterization result of C, C is the elemental characterization result of N, and D is the elemental characterization result of O.

[0027] Figure 5 The solution appearance of the TriTarget-CDs fluorescent probe of the present invention without light irradiation, wherein A is natural light, B is 405nm laser, C is 488nm laser, and D is 561nm laser.

[0028] Figure 6 The UV-Vis absorption spectrum and fluorescence emission spectrum of the TriTarget-CDs fluorescent probe of this invention are shown, along with the test results of fluorescence lifetime. A is the UV-Vis absorption spectrum, B is the emission under different excitations, C is the short-wavelength emission, D is the fluorescence decay of the short-wavelength emission, E is the optimal emission, and F is the fluorescence decay of the optimal emission.

[0029] Figure 7The results of the spectral characteristics test of the TriTarget-CDs fluorescent probe of the present invention under different environmental conditions include photostability under ultraviolet irradiation and fluorescence stability in the presence of NaCl solution, common ions and amino acids. Wherein A is the fluorescence intensity under ultraviolet irradiation, B is the fluorescence intensity in NaCl solution of different concentrations, C is the fluorescence intensity in different amino acids, and D is the fluorescence intensity in different ions.

[0030] Figure 8 The results of the cytotoxicity test of the TriTarget-CDs fluorescent probe of the present invention, and the cell uptake of the probe at different incubation times, are shown in A, where cytotoxicity is represented and cell uptake at different incubation times is represented in B.

[0031] Figure 9 The images are laser scanning confocal (CLSM) images of the TriTarget-CDs fluorescent probe of this invention in HeLa cells under three excitation conditions for 20 min of continuous scanning.

[0032] Figure 10 The fluorescence intensity changes of different channels in HeLa cells were continuously scanned for 20 minutes under three excitation conditions, as shown in this invention. A is the blue channel, B is the green channel, and C is the red channel.

[0033] Figure 11 The images show the CLSM of the TriTarget-CDs fluorescent probe of the present invention in HeLa cells under different temperature treatment conditions and intervention by various endocytosis inhibitors.

[0034] Figure 12 The diagram shows the fluorescence intensity changes of different channels in HeLa cells under different temperature treatment conditions and intervention by multiple endocytosis inhibitors, where A is the blue channel, B is the green channel, C is the red channel, and D is a schematic diagram of the cellular uptake pathway.

[0035] Figure 13 The images show the TriTarget-CDs fluorescent probes of this invention in cells and their co-localization with organelles. (a) and (b) are cell images without commercial dyes, (c) shows the co-localization with lipid droplets, (d) shows the co-localization with lysosomes, and (e) and (f) show the co-localization with mitochondria.

[0036] Figure 14 For based on Figure 13 The distribution curves of normalized fluorescence intensity as a function of distance obtained from the colocalization experiment were used for quantitative evaluation in conjunction with the Pearson correlation coefficient (PCC). The PCC values ​​for A were 0.81, B was 0.84, C was 0.93, and D was 0.82.

[0037] Figure 15 This diagram shows the spectral response of the TriTarget-CDs fluorescent probe of the present invention to different microenvironmental factors (solvent polarity, pH, and probe concentration). A and B correspond to the relationship between normalized fluorescence intensity and maximum emission wavelength as a function of solvent polarity, respectively. C and D are graphs showing the changes in normalized fluorescence intensity of the probe under different pH conditions. E and F are graphs showing the changes in normalized fluorescence intensity and fluorescence intensity ratio of the probe at different concentrations.

[0038] Figure 16 This is a schematic diagram of the preparation method of the single carbon dot triple-targeting multicolor fluorescent probe of the present invention. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0040] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0041] The following examples are not intended to limit the invention, but are only for illustration. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] Example 1

[0043] Preparation methods based on single-carbon-dot triple-targeting multicolor fluorescent probes, such as Figure 16 As shown, it includes the following steps:

[0044] Step 1: Weigh 0.10g of tetramethyljulonidine, dissolve it in 20mL of anhydrous ethanol, mix thoroughly and place it in a 50mL stainless steel high-pressure reactor lined with polytetrafluoroethylene.

[0045] Step 2: After sealing the reaction vessel, place it in a constant temperature oven and heat it to 180°C for 6 hours. After the reaction is completed, allow it to cool naturally to room temperature (approximately 25°C).

[0046] Step 3: Centrifuge the reaction mixture at high speed (10,000 rpm, 15 min) to remove the precipitate, and then filter the supernatant through a 0.22 μm filter membrane to remove large particulate impurities.

[0047] Step 4: The obtained filtrate is placed into a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in deionized water for 24 hours to remove unreacted precursors and small molecule impurities, thereby obtaining a single carbon dot triple-targeted multicolor fluorescent probe (TriTarget-CDs fluorescent probe).

[0048] Step 5: The dialyzed solution is dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution for subsequent live cell experiments.

[0049] Example 2

[0050] The TriTarget-CDs fluorescent probes prepared in Example 1 were subjected to physicochemical characterization.

[0051] The sample preparation methods are as follows: For transmission electron microscopy (TEM) samples, the original TriTarget-CDs probe solution was diluted 50 times with deionized water, and 5 drops of probe solution were added dropwise onto a 300-mesh copper grid using the droplet method before imaging; for zeta potential samples, the original TriTarget-CDs probe solution was used directly; for Fourier transform infrared (FTIR) samples, 5 mL of TriTarget-CDs probe solution was dried in a 60°C oven to obtain a powder sample for testing; for X-ray photoelectron spectroscopy (XPS), the original TriTarget-CDs probe solution was used directly for testing.

[0052] The characterization results are as follows: Figure 1 Transmission electron microscopy (TEM) images show that TriTarget-CDs are quasi-spherical particles, with uniform particle size and distribution. Figure 1 (a) indicates that the probe has good dispersibility. High-resolution TEM ( Figure 1 (b) shows that the particles have clear lattice fringes with a spacing of approximately 0.21 nm, corresponding to the (100) crystal plane of graphite. Particle size statistics show that their diameter is approximately 2.43 ± 0.54 nm. Figure 1 (c)

[0053] Figure 2 The image shows the zeta potential of the TriTarget-CDs fluorescent probe, which has a zeta potential of +10.68 mV.

[0054] Figure 3 The Fourier transform infrared (FTIR) spectrum of TriTarget-CDs shows that its surface contains abundant functional groups: 3438 cm⁻¹ -1 The peak of the O–H stretching vibration is located at 2954–2855 cm⁻¹. -1 It is an aliphatic C–H stretching vibration; 1428 cm⁻¹ -1 and 1381cm -1 Corresponding to –CH3 bending vibration, 1606 cm -1 and 1312cm -1 Corresponding to C=C / C=N and C–N bonds, 1264cm -1 It is a C–O stretching vibration.

[0055] X-ray photoelectron spectroscopy (XPS) analysis Figure 4 Figure A shows that TriTarget-CDs mainly contain C, O, and N elements, with contents of 83.5%, 11.6%, and 4.9%, respectively. The C element is present in the form of C=C / C=N (284.8 eV). Figure 4 It exists in the forms of B and C–O / C–N (285.8 eV); nitrogen exists in the forms of pyridine nitrogen and graphitic nitrogen (399.4, 400.5 eV). Figure 4 In the middle (C); the O element mainly exists as a C–O bond (532.6 eV). Figure 4 (D). The above characterization results show that TriTarget-CDs are nitrogen-doped ultrasmall graphitic carbon dots with various oxygen / nitrogen functional groups on their surface and positive charges, which endows them with good dispersibility, stability and interaction ability in biological environments.

[0056] Example 3

[0057] In natural light ( Figure 5 (A) and 405nm ( Figure 5 (Middle B), 488nm ( Figure 5 (C) and 561nm ( Figure 5 The color of the TriTarget-CDs solution was observed under D) excitation. Subsequently, its absorption spectrum was recorded using a UV-Vis spectrophotometer. Fluorescence emission spectra were measured at different excitation wavelengths on a fluorescence spectrometer to investigate excitation-dependent emission characteristics. The optimal excitation wavelengths and corresponding emission spectra for 500 nm and 585 nm emission were obtained using excitation-emission two-dimensional scans. Next, the decay curves of emission at 500 nm and 585 nm were determined using a fluorescence lifetime testing module.

[0058] TriTarget-CDs exhibit distinct multicolor emission characteristics in their optical performance. Their aqueous solution appears transparent pink under natural light. Figure 5 (A) Ultraviolet-Visible Absorption Spectroscopy ( Figure 6 Image A shows strong π–π* absorption peaks at approximately 270 nm and 297 nm, and a weaker n–π* shoulder peak at 360 nm attributed to the C=N group, with a low-intensity broad peak appearing at approximately 565 nm in the long-wavelength range, suggesting the presence of a structure with long-wavelength emission. Under excitation at 405 nm, TriTarget-CDs exhibit weak cyan-green fluorescence (…). Figure 5 (B). This short-wavelength emission exhibits a slight excitation dependence ( Figure 6 (B) The optimal excitation / emission wavelength is approximately 426 / 500 nm. Figure 6 The fluorescence decay is double exponential, with an average lifetime of approximately 2.6 ns. Figure 6(Middle D). Under excitation at 488nm or 561nm, TriTarget-CDs produce orange-yellow fluorescence ( Figure 5 (C, D) The long-wavelength emission does not change with the excitation wavelength. Figure 6 (B) indicates that the emission center is stable and uniform. The excitation-emission spectrum shows that the optimal excitation / emission is 567 / 585 nm. Figure 6 The decay of the medium E is a single exponential decay, with an average lifetime of approximately 4.3 ns. Figure 6 (F). TriTarget-CDs have two distinct emission channels (blue-green and yellow-orange) and stable photophysical properties, making them suitable for multicolor live-cell imaging.

[0059] Example 4

[0060] The prepared TriTarget-CDs solution (50 μg / mL) was continuously irradiated under a 365 nm UV lamp for 1 h, and the fluorescence intensity changes during irradiation were recorded. Simultaneously, TriTarget-CDs were added to NaCl solutions of different concentrations (0–200 mM), and their fluorescence intensity changes were measured. Further, common ions (such as Fe) were added to the probe solution. 3+ Ca 2+ Zn 2+ The fluorescence changes of amino acids (such as Gly, His, etc.) were recorded.

[0061] like Figure 7 As shown in Figure A, after continuous irradiation at 365 nm for 1 hour, the fluorescence intensity of TriTarget-CDs showed almost no decay, indicating its excellent anti-photobleaching properties. Furthermore, the fluorescence intensity changed very little in 0–200 mM NaCl solutions. Figure 7 (B) indicates that the probe has good stability in a salt environment. Additionally, the addition of different ions or amino acids ( Figure 7 C and Figure 7 The fluorescence intensity of TriTarget-CDs remained essentially unchanged, indicating that TriTarget-CDs have high tolerance to common biomolecular interference.

[0062] Example 5

[0063] The cytotoxicity of TriTarget-CDs on HeLa cells was evaluated using the CCK-8 assay. HeLa cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. Fresh culture medium containing different concentrations of TriTarget-CDs (0, 50, 100, 150, 200, and 250 μg / mL) was added, and the cells were cultured for another 24 h. Then, 100 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Fluorescence images were captured at different incubation times (2.5, 5, 10, and 20 min) to assess cell uptake efficiency. HeLa cells were co-incubated with 50 μg / mL TriTarget-CDs for 10 min, and then continuously scanned with a laser at 405 nm, 488 nm, and 561 nm excitation for 0–20 min. Fluorescence images in the blue, green, and red channels were recorded, and the changes in fluorescence intensity in the three channels were measured.

[0064] like Figure 8 As shown in Figure A, TriTarget-CDs exhibited almost no toxicity to HeLa cells within the range of 0–250 μg / mL, with cell viability remaining above 85%, indicating good biocompatibility. Figure 8 As shown in Figure B, TriTarget-CDs can enter cells within 2.5–5 minutes, and the fluorescence basically stabilizes by 10 minutes, indicating that the probe has rapid cell uptake characteristics and is suitable for real-time imaging of live cells. Figure 9 and Figure 10 Figures A, B, and C show that the fluorescence intensity under the three excitation conditions did not show significant attenuation within 20 minutes of continuous scanning, demonstrating that TriTarget-CDs have good anti-photobleaching ability in cells.

[0065] Example 6

[0066] like Figure 11 As shown, to investigate the uptake pathway of TriTarget-CDs in cells, cryotherapy and various endocytosis inhibitors were used as interventions. Figure 11As shown, HeLa cells were pre-cooled to 4°C for 30 min and then co-incubated with TriTarget-CDs (50 μg / mL) for 10 min to determine whether their uptake was energy-dependent. As a control group, cells were incubated at 37°C under the same conditions. Subsequently, HeLa cells were pretreated at 37°C for 30 min–1 h with different endocytosis inhibitors: 2-deoxyglucose (2-DG, 30 mM, ATP depletion), chlorpromazine (CPZ, 10 μM, inhibiting clathrin-mediated endocytosis), methyl-β-cyclodextrin (MβCD, 2 mM, disrupting lipid raft structure), amiloride (AMI, 100 μM, inhibiting macropinocytosis), and genistein (GEN, 200 μM, inhibiting pitot-mediated endocytosis). TriTarget-CDs (50 μg / mL) were then added and incubated for another 10 min in the presence of the inhibitors. Immediately after incubation, the fluorescence of the blue, green, and red channels in each treatment group was recorded under a confocal microscope; at the same time, the uptake of TriTarget-CDs in different treatment groups was quantitatively analyzed by flow cytometry.

[0067] At 4℃ or after treatment with 2-DG, the fluorescence in all three channels was significantly reduced. Figure 12 The diagrams (A, B, C) indicate that TriTarget-CD uptake is ATP-dependent, representing an energy-dependent process. The MβCD treatment group showed a significant decrease in the three-channel signal, indicating that lipid raft-mediated endocytosis is the primary cellular uptake pathway. CPZ treatment resulted in decreased fluorescence in the green and red channels, suggesting that clathrin-mediated endocytosis plays a supporting role in lysosomal and mitochondrial transport. AMI and GEN treatments had minimal impact on fluorescence signals, indicating that macropinocytosis and micropinocytosis-mediated endocytosis play a weaker role in TriTarget-CD endocytosis. In summary, as shown in the schematic diagram of the cellular uptake pathway (…),… Figure 12 As shown in D), TriTarget-CDs mainly enter cells via energy-dependent lipid raft-mediated endocytosis, with clathrin-mediated endocytosis playing a secondary role.

[0068] Example 7

[0069] HeLa cells were co-incubated with TriTarget-CDs (50 μg / mL) at 37°C for 10 min. Then, without removing the probes, 100 nM of lipid droplet dye Lipi-DeepRed, 50 nM of lysosomal dye LysoTrackerDeepRed, and 100 nM of mitochondrial dye MitoTrackerDeepRed were added, and incubation continued for 15 min. After incubation, cells were washed with PBS to remove unbound dyes, and then imaged under a confocal laser scanning microscope. Commercial dyes were used to collect deep red channel fluorescence at 640 nm excitation, while TriTarget-CDs were used to collect blue, green, and red channel fluorescence at 405, 488, and 561 nm excitations, respectively. Colocalization images were obtained by overlaying different channels, and the Pearson correlation coefficient (PCC) was calculated using ImageJ software to assess the overlap between TriTarget-CDs and the organelle dyes.

[0070] like Figure 13 As shown in (a), (b), (c), (d), (e), and (f), TriTarget-CDs can simultaneously label lipid droplets, lysosomes, and mitochondria after 10 min of incubation, displaying corresponding fluorescence signals in the blue, green, and red channels, respectively. The signals in each channel are clearly distinguishable, exhibiting good spatial resolution. In the blue channel, lipid droplets appear as discrete small spots, not overlapping with the green and red signals; the bright-field plot shows that these spots correspond to dark, high-refractive-index spherical structures, consistent with the typical characterization of lipid droplets (LDs). When co-stained with the lipid droplet dye Lipi-DeepRed, the blue signal highly overlaps with the deep red signal, as shown in... Figure 14 In the middle channel (A), the PCC is 0.81, indicating that the blue emission originates from lipid droplets. According to the probe targeting mechanism, TriTarget-CDs enhance lipid solubility through surface hydrophobic functional groups (such as –CH3), thus preferentially accumulating within the neutral core of lipid droplets, achieving high affinity labeling of lipid droplets. The green channel shows a unique speckled structure, with a PCC of 0.84 when co-stained with LysoTrackerDeepRed. Figure 14 (B) indicates that the green signal mainly originates from lysosomes. Its targeting mechanism involves the protonation of weakly basic amino groups on the surface in the acidic environment of the lysosome, thereby promoting probe retention within the lysosome. The red channel and part of the green channel signals exhibit a filamentous distribution and partially overlap with MitoTrackerDeepRed (red PCC = 0.93, green PCC = 0.82). Figure 14 Figures C and D show that TriTarget-CDs can be localized in mitochondria and produce significant green and red fluorescence. This localization mechanism stems from the electrostatic attraction between the probe's positive charge (ζ potential +10.68 mV) and the mitochondrial membrane potential (Δψm), thereby promoting its adsorption onto the mitochondrial membrane.

[0071] Example 8

[0072] To elucidate the mechanism by which TriTarget-CDs generate blue, green, and red fluorescence in different organelles, their fluorescence emission spectra were tested under different solvent polarities, pH values, and probe concentrations. Specifically, TriTarget-CDs (50 μg / mL) solutions were prepared in solvent systems with different polarities, and their emission spectra were recorded under 405 nm excitation. The probe solutions were adjusted to different pH values ​​(3–9), and the changes in fluorescence intensity at 496 nm and 585 nm were monitored. Simultaneously, the relative intensity of short-wavelength (496 nm) and long-wavelength (585 nm) emission was compared by adjusting the probe concentration (10–90 μg / mL).

[0073] like Figure 15 As shown in Figures A and B, the short-wavelength emission peak of TriTarget-CDs exhibits a significant blue shift in low-polarity solvents and a red shift in high-polarity systems, indicating that its emission wavelength is polarity-modulated. This phenomenon can explain the blue fluorescence observed in lipid droplets and the green fluorescence observed in lysosomes and mitochondria, which have higher polarity. The fluorescence gradually weakens as the solution pH increases. Figure 15 (C and D) indicates that an acidic environment enhances green emission, consistent with the bright green signal of lysosomes. Furthermore, long-wavelength (585nm) emission is significantly quenched under high probe concentration conditions, while short-wavelength signal shows little change. Figure 15 The values ​​in E and F indicate that higher local concentrations tend to suppress red emission. This is consistent with the fact that TriTarget-CDs show minimal changes in blue / green channel intensity even when locally enriched in lipid droplets and lysosomes, and that their dispersed distribution in mitochondria preserves red emission. In summary, the blue, green, and red luminescence of TriTarget-CDs is jointly regulated by the specific polarity, acidity, and local concentration of organelles, thus endowing this probe with the ability to selectively recognize three types of organelles in a single system.

[0074] Therefore, this invention utilizes the aforementioned method for preparing a single-carbon-dot triple-targeting multicolor fluorescent probe and its applications. A carbon quantum dot fluorescent probe is prepared using a one-step solvothermal method. This probe can simultaneously and specifically target and label lipid droplets, lysosomes, and mitochondria within living cells, and reliably distinguish these three organelles under multi-band confocal fluorescence imaging. Using this probe, organelles can be dynamically observed in situ within living cells over extended periods, providing an efficient and convenient new tool for studying multi-organelle interactions and their dynamic regulation.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a triple-targeting multicolor fluorescent probe based on a single carbon dot, characterized in that, Includes the following steps: Step 1: Weigh tetramethyljulonidine, dissolve it in anhydrous ethanol, mix thoroughly and place it in a 50mL stainless steel high-pressure reactor lined with polytetrafluoroethylene. Step 2: After sealing the reaction vessel, place it in a constant temperature oven, heat and maintain for 6 hours, and allow it to cool naturally to room temperature after the reaction is complete. Step 3: Centrifuge the reaction mixture at high speed to remove the precipitate, and then filter the supernatant through a 0.22μm filter membrane to remove large particulate impurities; Step 4: The obtained filtrate is placed into a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in deionized water for 24 hours to remove unreacted precursors and small molecule impurities, thereby obtaining the fluorescent probe.

2. The method for preparing a triple-targeted multicolor fluorescent probe based on a single carbon dot according to claim 1, characterized in that: In step 1, weigh 0.10 g of tetramethyljulonidine and dissolve it in 20 mL of anhydrous ethanol.

3. The method for preparing a triple-targeted multicolor fluorescent probe based on a single carbon dot according to claim 1, characterized in that: In step 2, heat to 180°C.

4. The method for preparing a triple-targeted multicolor fluorescent probe based on a single carbon dot according to claim 1, characterized in that: In step 3, the centrifugation conditions are 10,000 rpm for 15 min.

5. A triple-targeting multicolor fluorescent probe based on a single carbon dot, characterized in that: It is prepared by the preparation method described in any one of claims 1-4.

6. The application of the triple-targeting multicolor fluorescent probe based on a single carbon dot as described in claim 5, characterized in that: It is applied to the imaging of multiple organelles in living cells.

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