A dual-color dual-targeting carbon quantum dot and a preparation method and application thereof

Abstract

This invention belongs to the field of fluorescent carbon quantum dot technology, specifically relating to a dual-color, dual-targeting carbon quantum dot, its preparation method, and its applications. The carbon quantum dots used in this invention have abundant surface functional groups, making them easily modifiable. Ion modification can alter their surface functional groups, transforming them from a single-targeting method targeting mitochondria to a dual-color, dual-targeting method targeting both mitochondria and lysosomes, resulting in excellent cell imaging and enabling the observation of interactions between different organelles. The dual-color, dual-targeting carbon quantum dots provided by this invention eliminate the need for simultaneous staining with two fluorescent probes with good restaining properties, simplifying multi-probe staining operations and reducing factors affecting cells. The dual-color, dual-targeting method provided by this invention exhibits accurate localization, strong photostability, and maintains good stability before and after ion modification. The dual-color, dual-targeting carbon quantum dots provided by this invention have low toxicity to live cells, making them suitable for long-term live-cell imaging.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent carbon quantum dot technology, specifically relating to a dual-color dual-targeting carbon quantum dot, its preparation method, and its application. Background Technology

[0002] Organelles are highly differentiated functional structures within cells, working together to maintain overall cellular function. Lysosomes, acidic cellular compartments responsible for intracellular digestion (endocytosis) and the renewal of cellular components (autophagy), ensure the stable renewal of the cellular environment. Mitochondria, double-membrane organelles, act as the cell's powerhouse, converting chemical energy into adenosine triphosphate (ATP) through cellular respiration. Mitochondria are also the cell's metabolic center, regulating cellular metabolism and signal transduction, and mediating apoptosis. Despite their different functions, organelles form a dynamic network through direct contact, regulation of autophagy, and coordinated movement, collectively determining cell fate. Therefore, real-time monitoring of organelle location and interactions is crucial for understanding complex intracellular physiological processes.

[0003] Existing cell imaging methods targeting mitochondria and lysosomes include three types: single-probe targeting mitochondria, single-probe targeting lysosomes, and single-probe simultaneous targeting of mitochondria and lysosomes. Single-probe targeting mitochondria or lysosomes requires two fluorescent probes with good restaining properties for simultaneous staining. While single-probe simultaneous targeting of mitochondria and lysosomes can achieve cell imaging, this single probe is unstable; when external stimuli are added to observe and study organelle responses, the cell imaging effect deteriorates or even disappears. Summary of the Invention

[0004] The purpose of this invention is to provide a dual-color dual-targeting carbon quantum dot, its preparation method, and its application. The dual-color dual-targeting carbon quantum dot provided by this invention has excellent stability and can be transformed into a dual-color dual-targeting mitochondrial and lysosomal dot under external stimuli, resulting in good cell imaging effects. At the same time, it does not require simultaneous staining with two fluorescent probes with good counterstaining properties.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing dual-color, dual-targeting carbon quantum dots, comprising the following steps:

[0007] 1) Carbon quantum dots were obtained by hydrothermal reaction of 3-diethylaminophenol, 4-sulfophthalic acid and water;

[0008] 2) The carbon quantum dots and HS - Ion mixing was used for ion modification to obtain the dual-color dual-target carbon quantum dots.

[0009] Preferably, the molar ratio of 3-diethylaminophenol to 4-sulfophthalic acid is 1:1.

[0010] Preferably, the mass ratio of 3-diethylaminophenol to water is 9-12:2000.

[0011] Preferably, the hydrothermal reaction temperature is 180°C and the holding time is 24 hours.

[0012] Preferably, the carbon quantum dots and HS - The mass ratio of ions is 1-3:300-1500.

[0013] Preferably, the ion modification temperature is 21–37°C, and the holding time is 1–5 minutes.

[0014] Preferably, the hydrothermal reaction further includes post-processing of the obtained product, the post-processing including the following steps: cooling the product of the hydrothermal reaction and then sequentially removing impurities and freeze-drying it.

[0015] Preferably, the freeze-drying temperature is -50℃ to 60℃, and the holding time is 24 to 48 hours.

[0016] The present invention also provides dual-color dual-targeting carbon quantum dots obtained by the preparation method described above.

[0017] This invention also provides the application of the dual-color dual-targeting carbon quantum dots described above as fluorescent probes.

[0018] This invention provides a method for preparing dual-color, dual-targeting carbon quantum dots (CDs1). The method employs a one-step hydrothermal process to prepare carbon quantum dots (CDs), which is simple, convenient, and reproducible, facilitating large-scale production and application. The carbon quantum dots prepared by this invention possess a positively charged surface, enabling them to target mitochondria within cells. Furthermore, their abundant surface functional groups make them easily modifiable, allowing for the specific recognition of ions (e.g., H+, S+, OH-). - By adding external factors (ion modification, such as HS), - Modification can alter the surface functional groups of carbon quantum dots, thereby affecting the targeting results of organelles in different channels, transforming it from single-targeting to dual-targeting of mitochondria and lysosomes. Specifically, this invention obtains dual-targeting carbon quantum dots through ion modification. The surface of these dual-targeting carbon quantum dots possesses thiol and amino groups. The thiol groups can form strong hydrogen bonds with the thiol groups of cysteine ​​residues on mitochondrial membrane proteins, thus achieving a more robust anchoring to mitochondria. The weakly basic amino groups are easily protonated in acidic lysosomes, leading to their aggregation within the lysosome and achieving lysosomal targeting. Ultimately, a single probe can meet multiple needs, resulting in excellent cell imaging and enabling the observation of interactions between different organelles.

[0019] This invention also provides dual-color, dual-targeting carbon quantum dots prepared by the method described above. The dual-color, dual-targeting carbon quantum dots prepared by this invention can simultaneously perform fluorescence imaging of mitochondria and lysosomes in living cells through different channels, eliminating the need for simultaneous staining with two fluorescent probes with good restaining properties, simplifying multi-probe staining operations and reducing influencing factors on cells. The dual-color, dual-targeting carbon quantum dots prepared by this invention have accurate positioning, strong photostability, and maintain good stability before and after ion modification. The dual-color, dual-targeting carbon quantum dots prepared by this invention have low toxicity to living cells and are suitable for long-term living cell imaging. The dual-color, dual-targeting carbon quantum dots provided by this invention can serve as an imaging tool for studying and observing organelle interactions, enabling long-term in-situ dynamic observation of organelles within living cells. This provides an efficient, convenient, and effective tool for studying the influence of external factors on organelles, multi-organelle interactions and their dynamic regulation, and related biological processes.

[0020] This invention also provides the application of the dual-color, dual-targeting carbon quantum dots described above as fluorescent probes. The dual-color, dual-targeting carbon quantum dots provided by this invention enable simultaneous fluorescence imaging of mitochondria and lysosomes using a single probe, with stable imaging results, and can be used as fluorescent probes for long-term imaging of mitochondria and lysosomes in living cells. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Transmission electron microscopy (TEM) images (a and b) and particle size distribution (c) of the carbon quantum dots prepared for this invention; wherein, the scale bar of a is 20 nm and the scale bar of b is 1 nm;

[0023] Figure 2 Fourier transform infrared (FTIR) spectrum of carbon quantum dots prepared in this invention;

[0024] Figure 3 The X-ray photoelectron spectroscopy (XPS) full spectrum (a), C1s high-resolution spectrum (b), N1s high-resolution spectrum (c), O1s high-resolution spectrum (d), and S2p high-resolution spectrum (e) of the carbon quantum dots prepared for this invention.

[0025] Figure 4The carbon quantum dots prepared in this invention are shown in the following figures under ultraviolet light irradiation: appearance (a-b), ultraviolet-visible absorption spectrum (a-b), fluorescence emission spectrum (c, d and g), fluorescence lifetime (e and h), and absolute quantum yield test results (f and i).

[0026] Figure 5 The graph shows the spectral stability test results of the carbon quantum dots prepared in this invention under different environmental conditions, including photostability under ultraviolet irradiation (a), stability in different pH environments (c), fluorescence stability in NaCl solution (b), and in the presence of common ions (d).

[0027] Figure 6 The results of cytotoxicity testing of the carbon quantum dots prepared in this invention (a) and cell imaging effects at different incubation times (b-g); scale bar is 20 μm;

[0028] Figure 7 The images shown are of the carbon quantum dots prepared in this invention under a confocal system; where a is the bright field imaging result, b is the green channel (488nm) imaging result, c is the red channel (561nm) imaging result, and d is the superimposed green and red channel imaging result; the scale bar is 10μm.

[0029] Figure 8 The images show the fluorescence stability of the carbon quantum dots prepared in this invention under continuous laser irradiation (561 nm) in HeLa cells; where a is the fluorescence stability after 0 laser irradiations, b is the fluorescence stability after 10 laser irradiations, c is the fluorescence stability after 20 laser irradiations, d is the fluorescence stability after 30 laser irradiations, e is the fluorescence stability after 40 laser irradiations, and f is the fluorescence stability after 50 laser irradiations; the scale bar is 40 μm.

[0030] Figure 9 The graphs show the test results of co-localization of carbon quantum dots prepared in this invention with lipid droplets, lysosomes, and mitochondrial commercial probes; wherein the scale bar of the test results of co-localization of carbon quantum dots with lipid droplets and lysosomes is 20 μm, and the scale bar of the test results of co-localization of carbon quantum dots with mitochondrial commercial probes is 10 μm.

[0031] Figure 10 This is a diagram verifying the mitochondrial targeting mechanism of the carbon quantum dots prepared in this invention; the scale bar is 20 μm.

[0032] Figure 11Imaging effect diagrams of carbon quantum dots provided by the present invention; wherein, a to f are imaging effect diagrams corresponding to 0, 2, 5, 10, 15 and 20 minutes, respectively, and g to j are imaging effect diagrams corresponding to bright field, green channel, red channel and superimposed green-red channel, respectively; the scale bar is 10 μm;

[0033] Figure 12 Transmission electron microscope images (a and b) and particle size distribution map (c) of dual-color dual-target carbon quantum dots provided for this invention; wherein, the scale bar of a is 50 nm and the scale bar of b is 5 nm;

[0034] Figure 13 Fourier transform infrared spectrum of dual-color dual-target carbon quantum dots provided for this invention;

[0035] Figure 14 The X-ray photoelectron energy spectrum of the dual-color dual-target carbon quantum dots provided for this invention includes (a), (b), (c), (d), and (e) high-resolution spectra of C1s, N1s, O1s, and S2p.

[0036] Figure 15 The UV-Vis absorption spectrum (a), fluorescence emission spectrum (b), fluorescence lifetime (c), and absolute quantum yield test results (d) of the dual-color dual-target carbon quantum dots prepared in Example 1 are shown.

[0037] Figure 16 The images shown are of the dual-color dual-target carbon quantum dots prepared in Example 1 under a confocal system; where a is the imaging effect of the green channel, b is the imaging effect of the red channel, and c is the imaging effect of the superimposed green and red channels; the scale bar is 10 μm.

[0038] Figure 17 The carbon quantum dots prepared in Example 1 were subjected to different concentrations of HS - Modified ζ-potential variation diagram;

[0039] Figure 18 This is a schematic diagram of the synthetic route for preparing carbon quantum dots according to the present invention. Detailed Implementation

[0040] This invention provides a method for preparing dual-color, dual-targeting carbon quantum dots, comprising the following steps:

[0041] (1) 3-Diethylaminophenol, 4-sulfophthalic acid and water were mixed and subjected to a hydrothermal reaction to obtain carbon quantum dots;

[0042] (2) The carbon quantum dots and HS - Ion mixing was used for ion modification to obtain the dual-color dual-target carbon quantum dots.

[0043] In this invention, 3-diethylaminophenol, 4-sulfophthalic acid, and water are mixed (denoted as the first mixture) to obtain a reaction solution. In this invention, the molar ratio of 3-diethylaminophenol to 4-sulfophthalic acid is 1:1.

[0044] In this invention, the water can be deionized water; the mass ratio of 3-diethylaminophenol to water can be 9 to 12:2000, specifically 11:2000.

[0045] In this invention, the first mixing can be carried out within a polytetrafluoroethylene liner.

[0046] In this invention, the first mixing method may include the following steps: premixing 3-diethylaminophenol and 4-sulfophthalic acid to obtain a premixed solution, and then mixing the premixed solution with water.

[0047] In this invention, the first mixing can be ultrasonic mixing; the power of the ultrasonic waves can be 100-300W, specifically 250W, and the duration of the ultrasonic waves can be 5 minutes.

[0048] After obtaining the reaction solution, the present invention performs a hydrothermal reaction on the reaction solution to obtain carbon quantum dots. In the present invention, the equipment for the hydrothermal reaction may include a high-pressure oven and a stainless steel autoclave placed inside the high-pressure oven; the stainless steel autoclave may be sealed.

[0049] In this invention, the temperature of the hydrothermal reaction can be 180°C, and the heat preservation time can be 24 hours.

[0050] In this invention, the hydrothermal reaction may further include post-processing of the resulting product; the post-processing may include the following steps: cooling the product of the hydrothermal reaction and then sequentially removing impurities and freeze-drying it.

[0051] In this invention, the cooling can be natural cooling; the final cooling temperature can be room temperature (25°C).

[0052] In this invention, the impurity removal process may include the following steps: sequentially centrifuging the cooled product, filtering the supernatant, and dialysis the filtrate; the centrifugation equipment may be a centrifuge. This invention removes large particulate impurities from the product through centrifugation.

[0053] In this invention, the pore size of the filter membrane used for supernatant filtration can be 0.22 μm.

[0054] In this invention, the molecular weight cutoff for the filtrate dialysis can be 500 Da; the dialysis can be performed in a dialysis bag filled with deionized water; and the dialysis time can be 48 hours. This invention, through dialysis, thoroughly removes precursor raw materials and small particulate impurities from the product.

[0055] In this invention, the freeze-drying temperature can be -50 to -60°C, specifically -60°C, and the holding time can be 24 to 48 hours, specifically 30 hours. This invention obtains solid powder of carbon quantum dots through freeze-drying.

[0056] After obtaining carbon quantum dots, the present invention combines the carbon quantum dots with HS - Ion mixing is used for ion modification to obtain the dual-color, dual-targeting carbon quantum dots. In this invention, the carbon quantum dots and HS... - The mass ratio of ions can be 1 to 3:300 to 1500, specifically 1:300.

[0057] In this invention, the temperature for ion modification can be 21–37°C, specifically 37°C, and the holding time can be 1–5 minutes, specifically 5 minutes. Through ion modification, this invention induces the deprotonation reaction of protonated amino groups (-NH3). + +HS - →-NH2+H2S), and the affinity substitution reaction of sulfonic acid groups (-SO3H+2HS). - →-SH+SO3 2- +2H2S).

[0058] In this invention, the ion modification can be performed in phosphate buffered saline (PBS); the HS - The ratio of the amount of ions to the volume of the phosphate buffer solution can be 5 mmol: 1 L.

[0059] The present invention also provides dual-color dual-targeting carbon quantum dots obtained by the preparation method described above.

[0060] The dual-color, dual-targeting carbon quantum dots provided by this invention can simultaneously perform fluorescence imaging of mitochondria and lysosomes in living cells through different channels. They can be used as an imaging tool to study and observe organelle interactions, enabling long-term in-situ dynamic observation of organelles within living cells. This provides an efficient, convenient, and effective tool for studying the influence of external factors on organelles, multi-organelle interactions and their dynamic regulation, and related biological processes.

[0061] This invention also provides the application of the dual-color dual-targeting carbon quantum dots described above as fluorescent probes.

[0062] The dual-color, dual-targeting carbon quantum dots provided by this invention enable simultaneous fluorescence imaging of mitochondria and lysosomes with a single probe, and the imaging effect is stable, enabling long-term imaging of mitochondria and lysosomes in living cells.

[0063] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] This embodiment prepares a dual-color, dual-targeting carbon quantum dot, and the specific steps are as follows:

[0066] (1) Weigh 0.165g of 3-diethylaminophenol and 0.246g of 4-sulfophthalic acid and add them to the polytetrafluoroethylene liner. Then add 30mL of deionized water and sonicate at 250W for 5 minutes to disperse it evenly. Place the polytetrafluoroethylene liner into a stainless steel autoclave.

[0067] (2) After sealing the stainless steel reactor, place it in a high-pressure oven and heat it to 180°C for 24 hours. After the reaction is completed, allow it to cool naturally to room temperature (25°C) to obtain the cooled product solution.

[0068] (3) Place the cooled product solution into a centrifuge and centrifuge the product solution at a speed of 10,000 rpm to remove large particulate impurities. Take the supernatant and filter the supernatant through a filter membrane with a pore size of 0.22 μm to obtain the filtrate.

[0069] (4) The obtained filtrate was placed into a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in deionized water for 48 hours to completely remove the precursor raw materials and small particulate impurities.

[0070] (5) The dialyzed product solution was freeze-dried at -60℃ and 10 Pa for 30 hours to obtain carbon quantum dots in the form of solid powder. The synthesis route of carbon quantum dots is as follows: Figure 18 As shown.

[0071] (6) The prepared carbon quantum dots were mixed with phosphate buffer solution to prepare a carbon quantum dot solution (concentration of 5 mg / mL). The carbon quantum dot solution and HS were then mixed. - Ion mixing was subjected to ion modification at room temperature for 5 minutes, involving carbon quantum dots and HS. - With an ion concentration ratio of 1:300, dual-color dual-target carbon quantum dots were obtained.

[0072] Example 2

[0073] The carbon quantum dots prepared in Example 1 were characterized physicochemically as follows: A 50 μg / mL carbon quantum dot solution was prepared using deionized water. Five drops of the solution were added dropwise to a 300-mesh copper mesh using a droplet method. Transmission electron microscopy (TEM) analysis was then performed. The results are shown below. Figure 1 As shown; the carbon quantum dots prepared in Example 1 were directly subjected to Fourier transform infrared spectroscopy (FTIR) testing, and the test results are as follows. Figure 2 As shown; the carbon quantum dots prepared in Example 1 were used to prepare a carbon quantum dot solution with a concentration of 5 mg / mL using deionized water, and X-ray photoelectron spectroscopy (XPS) was performed. The test results are shown below. Figure 3 As shown.

[0074] according to Figure 1 The transmission electron microscopy (TEM) images show that carbon quantum dots have a spherical morphology and good dispersion (e.g., Figure 1 (as shown in a); High-resolution TEM (such as...) Figure 1 As shown in b) it can be seen that carbon quantum dots have crystal planes and clear lattice fringes with a spacing of about 0.22 nm, corresponding to the (100) crystal plane of graphite; the particle size statistics show that the diameter of carbon quantum dots is about 2.2 ± 0.4 nm (as shown in b). Figure 1 (as shown in c).

[0075] according to Figure 2 The Fourier transform infrared (FTIR) spectra show that the surface of carbon quantum dots contains abundant functional groups: at 3480 cm⁻¹ -1 ~3280cm -1 The absorption peak at 3020 cm⁻¹ corresponds to the stretching vibrations of -OH and -NH₂; at 3020 cm⁻¹... -1 ~2872cm -1 The absorption peak at 1715 cm⁻¹ corresponds to the stretching vibration of CH; at 1715 cm⁻¹... -1 The sharp peak at 1606 cm⁻¹ corresponds to the C=O stretching vibration in the amide; -1 ~1480cm -1 The absorption peak at 1273 cm⁻¹ corresponds to the C=C vibration on the aromatic ring skeleton and the NH bending component; -1 ~1002cm -1 The absorption peak at that point corresponds to the stretching vibrations of CO and CN.

[0076] according to Figure 3 As can be seen from X-ray photoelectron spectroscopy (XPS), the full XPS spectrum (such as...) Figure 3As shown in (a) of the diagram, the presence of four peaks corresponds to C1s, N1s, O1s, and S2p, respectively, determining the elemental composition and content of the carbon quantum dot to be 63.63% C, 5.26% N, 27.29% O, and 3.82% S. The bonding modes of these four elements in the carbon quantum dot were then analyzed, and the high-resolution spectrum of the C1s orbital (as shown in Figure a) was obtained. Figure 3 As shown in b), there are three peaks, attributed to CC / C=C (284.8 eV), CN / CO (286.2 eV), and OC=O (289.0 eV), respectively; the high-resolution spectrum of N1s (as shown in b) Figure 3 As shown in c), two peaks exist, attributed to amino nitrogen (400.2 eV) and protonated amino nitrogen (401.8 eV), respectively. The main peak is observed to be protonated nitrogen, with only a small amount of amino nitrogen. This is likely because the 4-sulfophthalic acid in the raw material provides an acidic environment with a sulfonic acid group and two carboxyl groups, allowing most of the nitrogen in the carbon quantum dot to be protonated. The high-resolution O1s spectrum (as shown in c) shows... Figure 3 The spectrum (d in the image) contains two peaks, attributed to C=O (530.7 eV) and CO / OH (532.0 eV), respectively; the high-resolution spectrum of S2p (as shown in the image) shows... Figure 3 As shown in e), there are two peaks, which are attributed to -SO3H at S2p. 3 / 2 (167.9eV) and S2p 1 / 2 (169.1eV) Splitting peak of two spin orbitals.

[0077] Example 3

[0078] The color of the carbon quantum dot solution (prepared with deionized water, concentration 5 mg / mL) was observed under natural light and ultraviolet light excitation, and its absorption spectrum was recorded using a UV-Vis spectrophotometer. Its fluorescence emission spectrum was measured at different excitation wavelengths using a fluorescence spectrometer to study its emission characteristics. Then, the decay curves of its emission at 500 nm and 515 nm, as well as the absolute quantum yield at 500 nm and 515 nm emission, were determined using a fluorescence lifetime testing module. The test results are as follows: Figure 4 As shown.

[0079] according to Figure 4 It can be seen that carbon quantum dots exhibit obvious dual emission center characteristics in optical characterization; in this embodiment, two sets of corresponding excitation-emission curves were measured, showing that at an excitation wavelength of 350 nm, the emission spectrum of carbon quantum dots has an emission peak at 500 nm (e.g., Figure 4 As shown in a), and at an excitation wavelength of 515 nm, the emission spectrum of carbon quantum dots has an emission peak at 535 nm (as shown in a diagram). Figure 4(as shown in b); its aqueous solution is transparent pink under natural light, and under ultraviolet light excitation, carbon quantum dots exhibit green fluorescence (as shown in b); Figure 4 (as shown in the illustrations of a and b); UV-Vis absorption spectra (as shown in the illustrations of a and b); Figure 4 (As shown by the red curves in a and b) the absorption peak at 241 nm corresponds to π→π* for C=C, the absorption peak at 274 nm corresponds to n→π* for C=N, the absorption peak at 356 nm corresponds to n→π* for C=O / CN, and the peak at 507 nm is the exciton absorption band; the excitation-emission 3D fluorescence pattern of carbon quantum dots (as shown in the image) Figure 4 As shown in Figure c), carbon quantum dots can be excited by excitation light in the wavelength range of 300–560 nm. Furthermore, it can be clearly seen in the figure that carbon quantum dots have two distinct emission centers, with their respective maximum emission peaks located at 500 nm and 535 nm. Figure 4 The data in a and b are consistent; the emission peak position of carbon quantum dots fluctuates around 500 nm under different excitation wavelengths from 290 to 410 nm (e.g., Figure 4 (as shown in d); the fluorescence decay curve of carbon quantum dots at an emission wavelength of 500 nm was fitted, and its fluorescence lifetime was calculated to be 7.46 ns (as shown in d); Figure 4 (as shown in e); the quantum yield of carbon quantum dots at a 500 nm emission wavelength is 29.34% (as shown in e). Figure 4 (as shown in f); under different excitation wavelengths from 430 to 570 nm, the optimal emission wavelength of carbon quantum dots shifts with the excitation wavelength, exhibiting excitation-dependent luminescence (as shown in f). Figure 4 (as shown in g); the fluorescence decay curve of carbon quantum dots at an emission wavelength of 535 nm was fitted, and its fluorescence lifetime was calculated to be 4.49 ns (as shown in g). Figure 4 (as shown in h); the quantum yield of carbon quantum dots at an emission wavelength of 535 nm is 23.0% (as shown in h); Figure 4 (as shown in i in the diagram).

[0080] Example 4

[0081] The carbon quantum dots prepared in Example 1 were made into a carbon quantum dot solution (concentration of 50 μg / mL) and continuously irradiated under a 365 nm ultraviolet lamp for 1 h, and the changes in fluorescence intensity during the irradiation period were recorded. Simultaneously, the carbon quantum dots were added to NaCl solutions of different concentrations (0–200 mM) to simulate a physiological environment, and the changes in their fluorescence intensity were measured. The carbon quantum dots were also placed in an environment with pH values ​​ranging from 1 to 13, and the changes in their fluorescence intensity were measured. Furthermore, common ions (such as H₂S and H₂S) were added to the carbon quantum dot solution. - Cl - Fe 3+ Ca 2+ Zn 2+(etc.), record the fluorescence changes, and the test results are as follows: Figure 5 As shown.

[0082] according to Figure 5 As can be seen from 'a', after 1 hour of continuous irradiation at 365 nm, the fluorescence intensity of carbon quantum dots showed almost no decay, indicating that they have excellent anti-photobleaching properties. According to Figure 5 As shown in b, the fluorescence intensity of carbon quantum dots changes very little in NaCl solutions with concentrations ranging from 0 to 200 mM, indicating that carbon quantum dots have good stability in salt environments of different concentrations. According to... Figure 5 As can be seen from 'c', carbon quantum dots also exhibit good stability within the physiological pH range (5–9). Furthermore, according to… Figure 5 As can be seen from d, after adding different ions, only HS... - This significantly reduces the fluorescence intensity of carbon quantum dots, verifying the effect of carbon quantum dots on HS. - Specific selectivity.

[0083] Example 5

[0084] The cytotoxicity of carbon quantum dots to HeLa cells in Example 1 was evaluated using the CCK-8 assay. The specific steps were as follows: 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 carbon quantum dots (0, 50, 100, 150, 200, and 400 μg / mL) was then added to each well, and the cells were cultured for another 24 h. Subsequently, 100 μL of CCK-8 solution (CellCounting Kit-8) 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, 10, 15, and 20 min) to assess the optimal staining time. The test results are shown below. Figure 6 As shown.

[0085] according to Figure 6 As can be seen from 'a', carbon quantum dots are almost non-toxic to HeLa cells in the range of 0–400 μg / mL, and the cell viability remains above 90%, indicating that the cytotoxicity of carbon quantum dots is negligible, they have good biocompatibility, and are suitable for live-cell imaging in HeLa cells. According to Figure 6 As can be seen from b to g, carbon quantum dots can enter cells within 2 minutes, and the fluorescence basically stabilizes by 10 minutes. The imaging effect at 20 minutes is basically the same as that at 10 minutes, indicating that carbon quantum dots have the characteristic of rapidly staining cells and imaging them, making them suitable for real-time imaging of live cells.

[0086] Example 6

[0087] To study the imaging results of carbon quantum dots and their corresponding spectra, this embodiment observes the imaging effect of carbon quantum dots in the green and red channels under confocal microscopy, and continuously observes the fluorescence stability of carbon quantum dots under continuous laser irradiation (561 nm) in HeLa cells.

[0088] The specific steps are as follows: Replace the culture medium with DMEM solution (Dulbecco's Modified Eagle Medium) containing 50 μg / mL carbon quantum dots, and co-incubate with HeLa cells for 10 min. Then wash three times with PBS solution to remove residual carbon quantum dots and avoid background noise. Subsequently, place the culture dish on Nikon, CLSM (green channel: λ). ex =488nm, λ em =500~550nm; Red channel: λ ex =561nm, λ em Cell imaging was performed at a wavelength of 570–620 nm to determine the organelle targeting of carbon quantum dots. Based on the excitation-emission 3D fluorescence pattern of carbon quantum dots, it was found that carbon quantum dots have two distinct emission centers. Therefore, two different channels, the green and red channels, were used to observe cell imaging. The test results are as follows: Figure 7 As shown.

[0089] according to Figure 7 It can be seen that carbon quantum dots have obvious fluorescence signals in both the green and red channels. The fluorescence image obtained from the green channel basically overlaps with that of the red channel, and appears as an orange signal in the superimposed channel. The fluorescence signal of carbon quantum dots is distributed in the cytoplasm and has a filamentous shape, which is likely located in the mitochondria.

[0090] To ensure long-term observation and imaging during testing, this embodiment studies the stability of carbon quantum dots (CDs) in cells. After co-incubating CDs with HeLa cells, 50 consecutive images were taken using confocal microscopy, and the results were compared with the initial images. The results are as follows: Figure 8 As shown.

[0091] according to Figure 8 As can be seen, the fluorescence intensity and mitochondrial morphology of the image did not change significantly, indicating that carbon quantum dots are very stable in the cellular environment and can be used for live cell imaging.

[0092] Example 7

[0093] The targeting properties of the carbon quantum dots prepared in Example 1 were tested. The specific steps were as follows: HeLa cells were co-incubated with a carbon quantum dot solution (concentration 50 μg / mL) at 37°C for 10 min. Then, without removing the carbon quantum dots, 50 nm of commercial mitochondrial probes (Mito-Tracker Deep Red), 50 nm of commercial lysosomal probes (Lyso-Tracker Deep Red), and 100 nm of commercial lipid droplet probes (LDs-Tracker Deep Red) were added and co-incubated with the HeLa cells again for 15 min. After incubation, the cells were washed three times with PBS, and the cell imaging results were observed and recorded using a CLSM (excitation wavelength 641 nm). The commercial probes were used to collect deep red channel fluorescence under 640 nm excitation. Since the fluorescence images obtained in the green and red channels largely overlap, the red channel fluorescence of the carbon quantum dots was collected under 561 nm excitation in this example. Co-localization images were obtained by superimposing different channels, and the Pearson correlation coefficient (PCC) was calculated using ImageJ software to assess the degree of overlap between the carbon quantum dots and the dyes of each organelle. The test results are shown below. Figure 9 As shown.

[0094] according to Figure 9 It can be seen that after 10 min of incubation, when carbon quantum dots were co-stained with the commercial mitochondrial probe (Mito-Tracker Deep Red), the red and deep red signals highly overlapped, with a PCC of 0.93, indicating that the carbon quantum dots targeted mitochondria. Simultaneously, co-localization with other commercial probes was performed to confirm that no other organelles were involved in the fluorescence signal. When carbon quantum dots were co-stained with the lysosomal deep red fluorescent probe Lyso-Tracker Deep-Red (LYDR), the PCC was 0.34; when carbon quantum dots were co-stained with the lipid droplet deep red fluorescent probe Lipi-Deep Red (LDDR), the PCC was 0.17. Furthermore, the fluorescence intensity distribution of carbon quantum dots differed significantly from that of LYDR and LDDR in the linearly selected region, indicating that the carbon quantum dots did not target lysosomes and lipid droplets, but only mitochondria.

[0095] Example 8

[0096] The mechanism by which carbon quantum dots prepared in Example 1 target mitochondria was tested. After co-incubating carbon quantum dots with HeLa cells, the culture dish was treated with carbonyl cyanate-3-chlorophenylhydrazone (CCCP). CCCP is a mitochondrial depolarizer that can rapidly reduce mitochondrial membrane potential (MMP). MMP can be restored after CCCP is removed.

[0097] To verify whether MMP affects the targeting mechanism of carbon quantum dots, the specific steps were as follows: HeLa cells were co-incubated with 50 μg / mL carbon quantum dots for 10 min. Then, under a confocal microscope, 10 μM CCCP was used to replace the culture medium for 10 min to destroy MMP. Subsequently, the CCCP was removed, and fresh culture medium without CCCP was added for incubation for 5 min. This step was repeated to wash the CCCP twice. CLSM images were observed in situ during this process in real time. The results are as follows: Figure 10 As shown.

[0098] according to Figure 10 It can be seen that after CCCP treatment, mitochondria in the green and red channels detached from their target sites, and the fluorescence signal gradually shifted from mitochondria at 1 min to lysosomes and nucleoli at 3 min and 10 min. When carbon quantum dots enter the cell, due to the membrane potential difference of mitochondria, carbon quantum dots with a positive zeta potential preferentially bind to mitochondria and exhibit fluorescence. Upon addition of CCCP, mitochondrial MMPs dissipate rapidly, causing the MMP-dependent targeting mechanism to fail, thus preventing carbon quantum dots from binding to mitochondria. The carbon quantum dots then migrate to lysosomes and nucleoli, where they exhibit fluorescence, due to their less competitive binding. After CCCP removal, MMPs recover, and carbon quantum dots, due to their positive zeta potential, exhibit strong adsorption and re-aggregate at mitochondria, again showing fluorescence. This indicates that the mechanism by which carbon quantum dots target mitochondria is the electrostatic interaction between the positive zeta potential of carbon quantum dots and the negative membrane potential of the mitochondrial inner membrane.

[0099] Example 9

[0100] Carbon quantum dot pair prepared in Example 1 for HS - Exhibiting specific selectivity, this embodiment tests the imaging response to this selectivity. Carbon quantum dots were co-incubated with HeLa cells, and the culture dish was observed under a confocal microscope. The results are as follows. Figure 7 As shown, both red and green channels target mitochondria. Therefore, the specific steps in this embodiment are as follows: After co-incubating carbon quantum dots with HeLa cells, HS is used. - A mixture of (5mM) and PBS was used to replace the culture medium. The reaction process was observed at 2, 5, 10, 15, and 20 minutes, and the imaging results were recorded. The results are as follows: Figure 11 As shown.

[0101] according to Figure 11 As can be seen from a to f in the diagram, when HS is added... - Throughout the entire process, the fluorescence intensity of the carbon quantum dots did not change significantly, but rather increased with HS. - Over time, the targeting of carbon quantum dots gradually shifted from mitochondria to lysosomes. The organelle targeting status was recorded after 20 minutes, and the results are as follows: Figure 11As shown in g~j in the diagram. Figure 11 As can be seen from g~j, both red and green channels target lysosomes, and the fluorescence intensity of carbon quantum dots is similar to that of untreated HS. - The difference was not significant, and no fluorescence quenching phenomenon corresponding to the spectral data was observed.

[0102] Example 10

[0103] The physicochemical characterization of the dual-color, dual-targeting carbon quantum dots prepared in Example 1 was performed. The specific steps are as follows: The dual-color, dual-targeting carbon quantum dots prepared in Example 1 were prepared into a 50 μg / mL solution using deionized water. Five drops of the solution were added dropwise to a 300-mesh copper mesh using the droplet method. Transmission electron microscopy (TEM) analysis was then performed, and the test results are shown below. Figure 12 As shown; 30 mL of the dual-color dual-targeting carbon quantum dot solution prepared in Example 1 was freeze-dried (temperature: -60℃; pressure: 10 Pa) to obtain dual-color dual-targeting carbon quantum dot solid powder, and Fourier transform infrared spectroscopy (FTIR) was performed. The test results are shown below. Figure 13 As shown; the dual-color dual-targeting carbon quantum dots prepared in Example 1 were used to prepare a 5 mg / mL dual-color dual-targeting carbon quantum dot solution with deionized water, and X-ray photoelectron spectroscopy (XPS) was performed. The test results are shown below. Figure 14 As shown.

[0104] according to Figure 12 As can be seen from 'a', the morphology of the two-color, two-target carbon quantum dots is irregular and their sizes vary, which may be due to the addition of HS to the carbon quantum dots. - This causes multiple carbon quantum dots to randomly combine, resulting in irregular morphology and size of the two-color, dual-target carbon quantum dots. According to Figure 12 In the middle b (HR-TEM), the crystal planes and lattice of the dual-color, dual-target carbon quantum dots can be seen, and the image shows that its lattice spacing is 0.21 nm; according to Figure 12 According to the statistical analysis, the particle size distribution of the dual-color dual-target carbon quantum dots is between 4 and 13 nm, with an average particle size of 8.5 ± 4 nm.

[0105] according to Figure 13 Fourier transform infrared spectroscopy reveals that the surface of the dual-color dual-target carbon quantum dots contains abundant functional groups: at 3530 cm⁻¹ -1 ~3270cm -1 The absorption peak at this point corresponds to the stretching vibrations of -OH and -NH2, and is related to the carbon quantum dot at this point (see...). Figure 2 The significant increase and broadening compared to the previous description indicates an increase in surface amine groups; at 3000 cm⁻¹ -1 ~2840cm -1The absorption peak at 2600 cm⁻¹ corresponds to the stretching vibration of CH, and is slightly weaker compared to that at carbon quantum dots, indicating that the framework remains essentially unchanged; -1 ~2480cm -1 The occurrence of -SH stretching vibration at this point indicates that HS - Introducing the formation of -SH or covalent CS; at 1620 cm -1 ~1500cm -1 The absorption peak at 1270 cm⁻¹ corresponds to the C=C vibration and NH bending on the aromatic ring framework. The shape has changed compared to that of the carbon quantum dot at this point, indicating a change in the electronic environment and hydrogen bonding. -1 ~1010cm -1 The absorption peak at this point corresponds to the stretching vibrations of CO, CN, and CS, which are significantly enhanced compared to carbon quantum dots at this point, and a new absorption band appears.

[0106] according to Figure 14 The X-ray photoelectron spectroscopy (XPS) spectrum shows that the four peaks still correspond to C1s, N1s, O1s, and S2p, confirming the elemental composition and content of the dual-color dual-target carbon quantum dots as 50.01% C, 3.7% N, 35.9% O, and 10.39% S. Further analysis of the bonding modes of these four elements in the dual-color dual-target carbon quantum dots reveals three peaks in the high-resolution spectrum of the C1s orbital, attributed to CC / C=C (284.8 eV), CN / CO (286.4 eV), and OC=O (288.4 eV). The high-resolution spectrum of the N1s orbital shows two peaks, attributed to amino nitrogen (399.3 eV) and protonated amino nitrogen (401.3 eV), with the dominant peak being amino nitrogen and only a small amount of protonated nitrogen. The change compared to carbon quantum dots may be due to the addition of HS. - After that, HS - Hydrolysis in water makes the solution alkaline, deprotonating most of the protonated nitrogen in the carbon quantum dots, resulting in an increase in the content of amino nitrogen, leaving only a small portion of the ring nitrogen in a protonated state; the high-resolution spectrum of O1s has three peaks, attributed to C=O (531.5 eV), CO / OH (533.3 eV), and -SO3H (535.6 eV), respectively. The newly added high-binding-energy component is due to surface adsorption of water, making it more hydrophilic; the high-resolution spectrum of S2p has four peaks, the first two of which are attributed to -SO3H in S2p 3 / 2 (168.2 eV) and S2p 1 / 2 (169.4 eV) Splitting peaks of two spin orbitals, the latter two newly added peaks are attributed to -SH in S2p. 3 / 2 (162.0 eV) and S2p 1 / 2(163.3 eV) Splitting peak of the two spin orbitals, after adding HS - The subsequent appearance of a double peak in both low and high valence states indicates partial oxidation of sulfur, introducing thiol groups (-SH). These thiol groups can form strong hydrogen bonds with thiol groups on cysteine ​​residues of mitochondrial membrane proteins, thus achieving a more robust anchoring and replacing the adsorption between potentials. Meanwhile, a large number of weakly basic amine groups are easily protonated in acidic lysosomes, and subsequently accumulate within the lysosome, achieving lysosomal targeting.

[0107] Example 11

[0108] The dual-color, dual-target carbon quantum dots prepared in Example 1 were optically characterized. The absorption spectra of the dual-color, dual-target carbon quantum dots were recorded using a UV-Vis spectrophotometer. Their fluorescence emission spectra and excitation-emission 3D fluorescence spectra were measured using a fluorescence spectrometer to study their emission characteristics. Then, the decay curve at 500 nm emission and the absolute quantum yield at 500 nm emission were determined using a fluorescence lifetime testing module. The results are as follows: Figure 15 As shown.

[0109] according to Figure 15 As can be seen from 'a', the red curve in the absorption spectrum of the dual-color dual-target carbon quantum dots shows an absorption peak at 236 nm corresponding to the π→π* of C=C, and an absorption peak at 360 nm corresponding to the n→π* of the surface C=O / NC=O; the dual-color dual-target carbon quantum dots have only one emission center, with an optimal emission peak at 500 nm at an excitation wavelength of 400 nm. Figure 15 (The black and blue curves in section a). According to Figure 15 As can be seen from b, the excitation-emission 3D fluorescence pattern of the dual-color dual-target carbon quantum dots also shows that the dual-color dual-target carbon quantum dots have an emission center, and the optimal emission peak position of the dual-color dual-target carbon quantum dots fluctuates around 500nm under different excitation wavelengths from 310 to 570nm. Figure 15 As can be seen from 'c', the fluorescence decay curve of the dual-color, dual-targeted carbon quantum dots at an emission wavelength of 500 nm was fitted, and its fluorescence lifetime was calculated to be 8.15 ns. According to... Figure 15 As can be seen from d, the quantum yield of the dual-color dual-target carbon quantum dots at an emission wavelength of 500 nm is 25.99%.

[0110] Example 12

[0111] The imaging effect of the dual-color dual-targeting carbon quantum dots prepared in Example 1 under a confocal system was tested. Corresponding to its spectrum, this example also observed the imaging effect of the dual-color dual-targeting carbon quantum dots in the green and red channels under confocal microscopy. The specific steps are as follows: The dual-color dual-targeting carbon quantum dots (concentration of 50 μg / mL) were mixed with DMEM and added to a glass culture dish seeded with HeLa live cells, and co-incubated with the HeLa live cells for 10 min. After incubation, the mixture was washed three times with PBS solution to remove residual dual-color dual-targeting carbon quantum dots. Imaging was also performed under a confocal laser scanning microscope (CLSM), and the fluorescence signals of the green and red channels were acquired. The test results are as follows. Figure 16 As shown.

[0112] according to Figure 16 It can be seen that the dual-color, dual-target carbon quantum dots exhibit significant fluorescence signals in both the green and red channels, compared to those without HS. - The carbon quantum dots, whose green channels are still mitochondria in the form of filamentous lines (such as...) Figure 16 As shown in a), but the red channels have become densely dotted lysosomes (as shown in a diagram). Figure 16 (as shown in b). Dual-color, dual-targeting carbon quantum dots are produced via HS... - The surface modification changed from targeting mitochondria with both red and green channels to targeting mitochondria with green channels and lysosomes with red channels.

[0113] Example 13

[0114] This example applies to different concentrations of H₂S. - The changes in zeta potential induced by carbon quantum dot modification were tested, and the specific steps were as follows: Different concentrations of (carbon quantum dots and HS) were added to the carbon quantum dots. - HS (volume ratio adjusted from 3:1 to 1:3) - The ζ-potential was measured, and the test results are as follows: Figure 17 As shown.

[0115] according to Figure 17 It can be seen that with the interaction of carbon quantum dots and HS - When the ratio (concentration ratio) was adjusted from 3:1 to 1:3, the zeta potential gradually decreased, from an initial 9.88 mV for carbon quantum dots to a final -53 mV. This negative zeta potential indicates that the mitochondrial targeting mechanism is no longer the electrostatic interaction between the positive zeta potential of carbon quantum dots and the negative membrane potential of the mitochondrial inner membrane. However, mitochondrial targeting still exists in the green channels, only now through strong hydrogen bonds formed between the thiol groups on their surface and the thiol groups of cysteine ​​residues on mitochondrial membrane proteins, achieving a more robust anchoring and replacing the adsorption effect between potentials.

[0116] As can be seen from the above embodiments, the dual-color dual-targeting carbon quantum dots provided by the present invention have excellent stability and can be transformed into dual-color dual-targeting mitochondria and lysosomes under external stimuli, resulting in good cell imaging effects, while eliminating the need for simultaneous staining with two fluorescent probes with good counterstaining properties.

[0117] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing dual-color, dual-targeting carbon quantum dots, characterized in that, Includes the following steps: 1) Carbon quantum dots were obtained by hydrothermal reaction of 3-diethylaminophenol, 4-sulfophthalic acid and water; 2) The carbon quantum dots and HS - Ion mixing was used for ion modification to obtain the dual-color, dual-targeted carbon quantum dots; the carbon quantum dots and HS - The mass ratio of ions is 1-3:300-1500.

2. The preparation method according to claim 1, characterized in that, The molar ratio of 3-diethylaminophenol to 4-sulfophthalic acid is 1:

1.

3. The preparation method according to claim 1, characterized in that, The mass ratio of 3-diethylaminophenol to water is 9-12:2000.

4. The preparation method according to claim 1 or 2, characterized in that, The hydrothermal reaction was carried out at a temperature of 180°C for 24 hours.

5. The preparation method according to claim 1, characterized in that, The ion modification temperature is 21–37°C, and the holding time is 1–5 minutes.

6. The preparation method according to claim 1, characterized in that, The hydrothermal reaction is followed by post-processing of the resulting product, which includes the following steps: cooling the product of the hydrothermal reaction and then sequentially removing impurities and freeze-drying it.

7. The preparation method according to claim 6, characterized in that, The freeze-drying temperature is -50 to -60°C, and the holding time is 24 to 48 hours.

8. The dual-color, dual-targeting carbon quantum dots obtained by the preparation method according to any one of claims 1 to 7.

9. The application of the dual-color dual-targeting carbon quantum dots as a fluorescent probe according to claim 8.

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