N-CDs fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly discloses an N-CDs fluorescent probe as well as a preparation method and application thereof, and the fluorescent probe is prepared from p-phenylenediamine and L-tartaric acid by adopting a one-step hydrothermal method. The invention relates to a carbon quantum dot probe which has a two-photon absorption characteristic and can specifically target a cell nucleus and a lipid droplet organelle in a living cell at the same time so as to realize double-color and double-target fluorescence imaging. The probe has important application value in the aspects of long-time dynamic observation of living cells, multi-organelle interaction research and deep tissue imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescent probe, in particular to a dual-targeting two-photon imaging carbon quantum dot fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Fluorescence imaging technology is a key means for studying the structure and function of living cells, and its development greatly depends on the progress of high-performance fluorescent probes. In cell biology research, it is crucial to simultaneously observe the dynamic changes of multiple organelles and their interactions to understand the mechanisms of cell life activities. However, existing commercial fluorescent probes face significant limitations in such applications.

[0003] Firstly, in terms of targeting ability, commercial probes such as Hoechst (nucleus) and BODIPY (lipid droplet) can only label a single organelle. To achieve simultaneous observation of the nucleus and lipid droplets, researchers must perform multi-probe re-staining. This process is not only cumbersome to operate, but also prone to problems such as asynchronous imaging results and large localization deviations due to differences in the uptake efficiency, metabolic rate, and light stability of different probes in cells, making it difficult to accurately reveal the real-time interaction between organelles.

[0004] Secondly, in terms of optical performance, existing small organic molecule dyes generally have poor light stability and are prone to photobleaching under the continuous irradiation of laser confocal microscopy, which cannot meet the needs of long-term dynamic observation. In addition, the excitation wavelengths of these dyes are mostly in the ultraviolet or visible short-wave region, with high photon energy, which can cause significant phototoxicity to living cells and limit their application in thick tissue samples due to shallow tissue penetration depth.

[0005] Currently, some studies have reported CDs with single organelle (such as nucleus or lipid droplet) targeting ability. However, developing CDs that can achieve dual-organelle specific targeting using a single probe, especially simultaneously targeting the nucleus and lipid droplets, remains a challenge. More importantly, CDs probes with such dual-targeting ability and two-photon absorption characteristics are extremely rare.

[0006] Therefore, there is an urgent need in the art to develop a new type of CDs fluorescent probe that can overcome the limitations of existing technology, such as single function, insufficient light stability, and potential high phototoxicity, to achieve efficient and accurate dual-targeting and two-photon imaging. SUMMARY

[0007] The purpose of this invention is to provide an N-CDs fluorescent probe, its preparation method, and its applications. This probe has abundant surface functional groups, enabling it to specifically target the cell nucleus and lipid droplets, and can achieve two-color, dual-target imaging and two-photon imaging under a confocal system. Using this probe, organelles can be observed in living cells for extended periods, in situ, and dynamically, thus providing an efficient and convenient new analytical tool for studying the influence of external factors on organelles, the interactions between multiple organelles, and their dynamic regulatory processes.

[0008] To achieve the above objectives, the present invention provides an N-CDs fluorescent probe, which is prepared by a one-step hydrothermal method using p-phenylenediamine and L-tartaric acid.

[0009] Preferably, the elemental composition and content of the N-CDs fluorescent probe are 64.26% C, 8.62% N and 27.11% O.

[0010] Preferably, the N-CDs fluorescent probe has a particle size of 1.75±0.04nm, a positively charged surface, a Zeta potential of +11.4mV, and two-photon absorption characteristics, enabling it to simultaneously and specifically target the cell nucleus and lipid droplets, achieving dual-color fluorescence imaging of the blue and red channels under single-photon excitation.

[0011] This invention also provides a method for preparing an N-CDs fluorescent probe, comprising the following steps: Step 1: Weigh 0.108g of p-phenylenediamine and 0.25g of L-tartaric acid and add them to the polytetrafluoroethylene liner. Then add 20ml of deionized water and sonicate the mixture to make it evenly dispersed. Place the liner in a stainless steel autoclave and put the whole thing into a high-pressure oven. Step 2: After sealing the reaction vessel, place it in an oven and heat it to 200°C for 20 hours. After the reaction is complete, allow it to cool naturally to room temperature. Step 3: Place the reaction mixture into a centrifuge and centrifuge the solution at high speed to remove large particulate impurities. Take the supernatant and then filter the supernatant through a 0.22μm filter membrane. 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 48 hours to remove impurities. Step 5: The dialyzed solution is freeze-dried to obtain N-CDs solid powder for later use.

[0012] This invention also provides an application of N-CDs fluorescent probes for dual-target two-photon imaging that simultaneously targets the cell nucleus and lipid droplets.

[0013] Preferably, the cell staining conditions in dual-target two-photon imaging are 50 µg / mL N-CDs staining for 30 min.

[0014] The advantages and beneficial effects of the present invention using the above-mentioned N-CDs fluorescent probe, its preparation method, and its application are as follows: 1. The carbon quantum dot (N-CDs) fluorescent probe prepared in this invention has a positive charge on its surface, which enables it to achieve specific targeting of the cell nucleus within the cell. At the same time, the N-CDs exhibit a blue shift in excitation and emission spectra and enhanced fluorescence intensity in organic solvents, indicating that they have significant lipophilicity, thereby enabling them to effectively target lipid droplets and achieve clear imaging in the blue channel.

[0015] 2. The N-CDs fluorescent probe developed in this invention possesses upconversion luminescence properties, enabling photoluminescence processes with long-wavelength excitation and short-wavelength emission, and exhibits two-photon absorption characteristics. After co-incubation with HeLa cells, this probe, under 808nm femtosecond laser excitation, can simultaneously image in the green and red channels of a confocal system, achieving multi-color dual-target two-photon imaging.

[0016] 3. The N-CDs fluorescent probe of the present invention has the characteristics of accurate positioning and excellent photostability, and can realize dual-color, dual-organelle synchronous targeted imaging of cell nuclei and lipid droplets, providing an effective tool for studying the interaction between organelles and related dynamic biological processes.

[0017] 4. This N-CD fluorescent probe has low toxicity to live cells and good biocompatibility, making it suitable for long-term live cell imaging; a single probe can label multiple organelles, simplifying the traditional multi-probe restaining process and reducing interference with cell state.

[0018] 5. This invention uses a one-step hydrothermal method to prepare N-CDs fluorescent probes. The process is simple, convenient, and highly reproducible, which is conducive to large-scale preparation and practical application.

[0019] 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

[0020] Figure 1 These are transmission electron microscope (TEM) images and particle size distribution statistics of the N-CDs fluorescent probe of the present invention, where A is a low-magnification TEM image, B is a high-resolution TEM image, and C is a particle size distribution statistics. Figure 2 This is the Fourier transform infrared (FTIR) spectrum of the N-CDs fluorescent probe of the present invention; Figure 3 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the N-CDs fluorescent probe of this invention, where A is the full spectrum, B is the C1s high-resolution spectrum, C is the N1s high-resolution spectrum, and D is the O1s high-resolution spectrum. Figure 4These are the zeta potential measurement results of the N-CDs fluorescent probe of this invention; Figure 5 A shows the three-dimensional fluorescence spectrum of the N-CDs fluorescent probe at a concentration of 10 ug / ml; B shows the appearance photograph under ultraviolet light; C shows the ultraviolet-visible absorption spectrum; D shows the fluorescence emission spectrum; E shows the fluorescence lifetime decay curve and the absolute quantum yield measurement results; F shows the three-dimensional fluorescence spectrum at a concentration of 100 ug / ml and compares the emission spectra and appearance photographs of 10 ug / ml and 100 ug / ml CDs solutions under 561 nm excitation. Figure 6 In the image, A represents the three-dimensional fluorescence spectrum of the N-CDs fluorescent probe in water, B represents the three-dimensional fluorescence spectrum of the N-CDs fluorescent probe in DMSO, C represents the three-dimensional fluorescence spectrum of the N-CDs fluorescent probe in DMF, and D represents a comparison between the emission spectrum and appearance of the N-CDs fluorescent probe under 405 nm excitation. Figure 7 In the figure, A represents the emission spectrum of the N-CDs fluorescent probe under 760–860 nm femtosecond laser excitation, B represents the fluorescence intensity of the N-CDs fluorescent probe under 808 nm excitation, and C represents the double logarithmic fitting curve of the laser power of the N-CDs fluorescent probe with a slope of n≈2.02. Figure 8 The results of fluorescence stability tests of the N-CDs fluorescent probe of this invention under different environmental conditions are shown. A and B are common ions, C is NaCl solution, D is continuous ultraviolet irradiation, and E is different pH environments. Figure 9 The results of the cytotoxicity test of the N-CDs fluorescent probe of this invention; Figure 10 Imaging comparison of N-CDs fluorescent probes in HeLa cells at different incubation times, where A is 0 min, B is 6 min, C is 12 min, D is 18 min, E is 24 min, and F is 30 min; Figure 11 Confocal imaging results after HeLa cells were incubated with N-CDs fluorescent probes at different concentrations. A represents 1 μg / mL, B 10 μg / mL, C 20 μg / mL, D 30 μg / mL, E 40 μg / mL, and F 50 μg / mL. Figure 12 These are confocal microscopy images of the N-CDs of this invention under excitation at 405 nm and 561 nm. Figure 13 The results of two-photon confocal microscopy imaging of the N-CDs fluorescent probe of this invention under 808 nm excitation light; Figure 14The results of the co-localization experiment of the N-CDs fluorescent probe of this invention with commercial cell nuclear and lipid droplet dyes; Figure 15 This is to verify the behavior of N-CDs fluorescent probes targeting the cell nucleus after HeLa cells were treated with DNA-cutting enzymes and RNA-cutting enzymes; Figure 16 The results show the fluorescence stability of the N-CDs fluorescent probe of this invention in HeLa cells under continuous irradiation with 405nm and 561nm lasers, where A is 0 min, B is 10 min, C is 20 min, D is 30 min, E is 40 min, and F is 50 min. Figure 17 The results of the study on the cellular uptake mechanism of the N-CDs fluorescent probe of this invention are shown in the figures. A is the autofluorescence control group, B is the 4℃ low temperature inhibition group, C is the 37℃ normal group, and D is the 2-deoxy-D-glucose metabolism inhibition treatment group. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] 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.

[0023] 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.

[0024] Example 1 A method for preparing an N-CDs fluorescent probe includes the following steps: Step 1: Weigh 0.108g of p-phenylenediamine and 0.25g of L-tartaric acid and add them to the polytetrafluoroethylene liner. Then add 20ml of deionized water and sonicate the mixture to make it evenly dispersed. Place the liner in a stainless steel autoclave and put the whole thing into a high-pressure oven.

[0025] Step 2: After sealing the reaction vessel, place it in an oven and heat it to 200°C for 20 hours. After the reaction is complete, allow it to cool naturally to room temperature.

[0026] Step 3: Place the reaction mixture in a centrifuge and centrifuge at high speed to remove large particulate impurities. Take the supernatant and filter it through a 0.22μm filter membrane.

[0027] 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 48 hours to remove raw materials and small particulate impurities.

[0028] Step 5: The dialyzed solution is freeze-dried to obtain N-CDs solid powder for later use.

[0029] The prepared N-CDs fluorescent probes were characterized physicochemically. Sample preparation methods were as follows: For transmission electron microscopy (TEM), the original N-CDs probe solution was diluted 50-fold with deionized water, and then 5 drops of the probe solution were added dropwise onto a 300-mesh copper grid using the droplet method before imaging. For Fourier transform infrared spectroscopy (FTIR), 30 mL of N-CDs probe solution was freeze-dried (temperature: -60℃; pressure: 10 Pa) to obtain N-CDs solid powder for testing. X-ray photoelectron spectroscopy (XPS) was performed directly using the original N-CDs probe solution. Zeta potential was measured using a 50 mg / mL N-CDs solution.

[0030] The characterization results are as follows: Figure 1 The transmission electron microscopy (TEM) images show that N-CDs have a spherical morphology and good dispersion. Figure 1 (A). High-resolution TEM ( Figure 1 (B) As can be seen, N-CDs have crystal planes and clear lattice fringes with a spacing of approximately 0.21 nm, corresponding to the (100) crystal plane of graphite. Particle size statistics show that its diameter is approximately 1.75 ± 0.04 nm (…). Figure 1 (C)

[0031] Fourier transform infrared (FTIR) spectrum ( Figure 2 ), indicating that its surface contains abundant functional groups: at 3250cm -1 -3650cm -1 The broad absorption peak at 1723 cm⁻¹ corresponds to the stretching vibrations of OH and NH; at 1723 cm⁻¹... -1 The sharp absorption peak at 1610 cm⁻¹ corresponds to the C=O stretching vibration in the amide bond; -1 The absorption peak at 1513 cm⁻¹ corresponds to the NH bending vibration in the amide bond; at 1513 cm⁻¹ -1 The absorption peak at 1422 cm⁻¹ corresponds to the C=C vibration on the aromatic ring skeleton; -1 The absorption peak at 1000 cm⁻¹ corresponds to the bending vibration of OH; at 1000 cm⁻¹... -1 -1300cm -1 The absorption peak at that point corresponds to the stretching vibrations of CO and CN.

[0032] X-ray photoelectron spectroscopy (XPS) analysis: XPS full spectrum ( Figure 3(A) shows the presence of three peaks, corresponding to C1s, N1s, and O1s respectively. Figure 3 The elemental composition of N-CDs was determined by using the BD (Digital Boolean) method, with contents of 64.26% C, 8.62% N, and 27.11% O. The bonding modes of these three elements in N-CDs were then analyzed. The high-resolution spectrum of the C1s orbital showed three peaks, attributed to CC / C=C (284.8 eV), CO / CN (286.14 eV), and C=O (288.10 eV), respectively. The high-resolution spectrum of the N1s orbital showed two peaks, attributed to amino nitrogen (399.33 eV) and graphitic carbon / protonated amino nitrogen (401.15 eV), respectively. The high-resolution spectrum of the O1s orbital showed two peaks, attributed to C=O (532.13 eV) and CO (533.47 eV), respectively.

[0033] The zeta potential of N-CDs was obtained by electrophoretic light scattering measurement. Figure 4 The zeta potential of N-CDs is +11.4 mV, indicating that their surface is positively charged. This is mainly attributed to the formation of -NH3 groups on the N-CDs surface by nitrogen atoms capturing protons. + Group.

[0034] Example 2 The color of a 10 μg / mL N-CDs aqueous solution was observed under natural light and ultraviolet light excitation, and its absorption spectrum was recorded using a UV-Vis spectrophotometer. Subsequently, its fluorescence emission spectrum was scanned at different excitation wavelengths using a fluorescence spectrometer to obtain a three-dimensional fluorescence spectrum for studying its emission characteristics. The fluorescence decay curve of N-CDs at an emission wavelength of 545 nm was determined using a fluorescence lifetime testing module, and its fluorescence lifetime was calculated; simultaneously, the absolute quantum yield at this wavelength was measured.

[0035] To further investigate the effect of concentration on the optical properties of N-CDs, the three-dimensional fluorescence spectrum of N-CDs at a concentration of 100 μg / mL was measured and compared with the emission spectrum of N-CDs at 10 μg / mL under 561 nm excitation. Finally, the effect of solvent polarity on the optical behavior of N-CDs was studied. N-CDs were dispersed in water, DMSO, and DMF, respectively, and the three-dimensional fluorescence spectra in each solvent were measured. The emission behavior under 405 nm excitation and the appearance color of the solutions were compared.

[0036] Optical characterization results show that N-CDs possess stable luminescent centers. For example... Figure 5 As shown in Figure A, the emission center position remains constant in the three-dimensional fluorescence spectrum of 10 μg / mL N-CDs. The solution is transparent and pale yellow under natural light, and emits yellow fluorescence under ultraviolet light excitation. Figure 5 Illustration B). Ultraviolet-Visible Absorption Spectrum (Figure 5 The (b, magenta curve) shows that at 272 nm, a π→π curve belonging to C=C appears. The transition absorption peak at 410 nm corresponds to the n→π transition of C=O. Transition absorption peak. The optimal excitation and emission wavelengths for N-CDs are 390 nm and 545 nm, respectively. Figure 5 (Medium B, dark gray and yellow curves). At an emission wavelength of 545 nm, its fluorescence decay curve, calculated through fitting, yields a fluorescence lifetime of 2.66 ns (…). Figure 5 (C), absolute quantum yield is 18.84% ( Figure 5 (D).

[0037] As the concentration increases, the excitation and emission wavelengths of N-CDs exhibit a red shift. The three-dimensional fluorescence spectrum of 100 μg / mL N-CDs ( Figure 5 (E) shows that emission light near 650 nm can be observed within the 450–650 nm excitation range, with both excitation and emission wavelengths showing a significant redshift compared to the 10 μg / mL sample. Under 561 nm excitation, 100 μg / mL N-CDs exhibit a distinct emission center ( Figure 5 (Middle F).

[0038] Furthermore, N-CDs exhibit differentiated excitation and emission behaviors in different solvents. Their three-dimensional fluorescence spectra in water, DMSO, and DMF (…) Figure 6 (AC) showed that both the excitation and emission wavelengths of N-CDs exhibited a blue shift in low-polarity solvents. Under 405 nm excitation, N-CDs emitted yellow light in aqueous solution, with the emission center located at 545 nm; while in DMSO and DMF, the solution appeared pale blue, with the emission center blue-shifted to 440 nm. Figure 6 (D).

[0039] The N-CDs prepared in this invention exhibit excellent two-photon fluorescence properties. Within the 760-860 nm femtosecond laser excitation range, the N-CDs produce a significant emission peak in the 500-650 nm wavelength range. Figure 7 (A), where bright orange light emission can be observed in aqueous solution excited at 808 nm. To further investigate its luminescence mechanism, the dependence of fluorescence intensity on excitation power was systematically studied (…). Figure 7 (B) Linear fitting of fluorescence intensity (If) and excitation power (P) on a double logarithmic coordinate system yields a slope n≈2.02 ( Figure 7The value of C (indicated by 2) clearly confirms that its luminescence process conforms to the two-photon absorption mechanism (If ∝ P^n). We hypothesize that nitrogen doping effectively modulates the electronic structure of N-CDs, introducing intermediate energy levels into the band gap, promoting the synergistic absorption of low-energy photons by N-CDs, thus achieving efficient two-photon luminescence. This characteristic allows N-CDs to effectively avoid damage to biological samples from ultraviolet light when using near-infrared excitation sources, significantly expanding their application prospects in the field of in vivo imaging.

[0040] Example 3 To evaluate the applicability of N-CDs fluorescent probes in complex environments, this study systematically investigated their fluorescence stability under different chemical and light conditions. First, N-CDs solutions were reacted with various common ions (including NO2). - Cl - Fe 3+ Ag + Ca 2+ Zn 2+ The N-CDs solutions were mixed separately, and their fluorescence intensity changes were monitored. Then, the N-CDs solutions were placed in NaCl solutions of different concentrations (0-210 mM) to simulate the physiological saline environment, and the fluorescence intensity was measured as a function of saline concentration. The N-CDs solutions were then continuously irradiated under a 365 nm UV lamp for 30 minutes, and the fluorescence intensity was recorded as a function of time. Finally, the response characteristics of the fluorescence intensity to pH changes were systematically studied by placing the N-CDs solutions in different buffer systems with pH values ​​of 2-11.

[0041] Stability test results showed that the fluorescence intensity of N-CDs remained stable in the presence of most ions, with significant quenching only occurring in a few specific ion environments, indicating that they possess good resistance to ion interference. Figure 8 China A Figure 8 (B) Within the physiologically relevant salt concentration range (0-210 mM NaCl), the fluorescence intensity of N-CDs showed minimal variation, demonstrating their excellent stability under physiological conditions. Figure 8 (C). After 30 minutes of continuous UV irradiation, no significant attenuation of the fluorescence intensity of N-CDs was observed, demonstrating excellent anti-photobleaching properties. Figure 8 (D). Furthermore, N-CDs maintained stable fluorescence emission over a wide pH range of 1-11, indicating their broad pH adaptability. Figure 8 These results collectively confirm that N-CDs fluorescent probes possess reliable fluorescence stability under complex operating conditions, laying a solid foundation for their practical application in the field of bioimaging.

[0042] Example 4 To systematically evaluate the biocompatibility of N-CDs fluorescent probes, their cytotoxicity to HeLa cells was detected using the CCK-8 assay. HeLa cells were seeded at an appropriate density in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours to allow for full cell adhesion. The culture medium was then replaced with fresh medium containing different concentrations of CDs (0, 25, 50, 100, 200, and 400 μg / mL), and cultured for another 24 hours. Subsequently, 100 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated by comparing the absorbance of the experimental and control groups. The results of the cytotoxicity assay are shown below. Figure 9 As shown, within the concentration range of 0-400 μg / mL, the survival rate of HeLa cells treated with N-CDs remained above 80%, indicating that the probe had no significant cytotoxicity within this concentration range, possessed good biocompatibility, and met the requirements of live cell imaging experiments.

[0043] To determine the optimal conditions for using N-CDs fluorescent probes in live-cell imaging, cell fluorescence images were further observed using confocal microscopy at different incubation times (0, 6, 12, 18, 24, and 30 minutes) and different N-CDs concentrations (1, 10, 20, 30, 40, and 50 μg / mL). The time-gradient experiments showed that N-CDs could rapidly enter cells, and their fluorescence signal gradually increased with increasing incubation time, reaching a steady state at 30 minutes. Figure 10 (AF). Concentration gradient experiments showed that as the N-CDs concentration increased from 1 μg / mL to 50 μg / mL, the signal-to-noise ratio and clarity of cell imaging gradually improved, with the optimal imaging effect obtained at 50 μg / mL. Figure 11 (AF). Based on the combined results of cytotoxicity and imaging quality analysis, staining with 50 μg / Mln-CDs for 30 minutes was ultimately determined as the standard condition for all subsequent cell imaging experiments.

[0044] Example 5 To systematically evaluate the performance of N-CDs fluorescent probes in cell imaging, a two-color imaging study under single-photon excitation mode was first conducted. HeLa cells were co-incubated with DMEM complete medium containing 50 μg / mL N-CDs for 30 minutes. After thorough washing with PBS buffer to remove uninternalized probes, observations were performed under a confocal microscope. Imaging parameters were set as follows: the blue channel was excited by a 405 nm laser, collecting emission signals in the wavelength range of 425-475 nm; the red channel was excited by a 561 nm laser, collecting emission signals in the wavelength range of 570-620 nm. Based on previous characterization results, N-CDs have a positively charged surface and can be attracted by the negative potential of the nuclear membrane through electrostatic interactions, thus specifically accumulating in the cell nucleus and exhibiting fluorescence in the red channel under 561 nm excitation. Simultaneously, due to the significant lipophilicity of N-CDs, they can effectively target lipid droplet structures, and their excitation and emission spectra undergo a blue shift in the low-polarity environment inside the lipid droplets, thus displaying fluorescence in the blue channel under 405 nm excitation. Imaging results (…) Figure 12 The results show that N-CDs exhibit clearly distributed and sufficiently intense fluorescence signals in both the blue and red channels, with virtually no spatial overlap between the two channels: the blue channel fluorescence is uniformly distributed in a dotted pattern in the cytoplasm, highly consistent with the morphological characteristics of lipid droplets; while the red channel fluorescence is concentrated in the nucleus, displaying a typical intranuclear distribution pattern. This result fully demonstrates that N-CDs can achieve simultaneous dual-color, dual-target imaging of both the nucleus and lipid droplets.

[0045] To further investigate the imaging performance of N-CDs under long-wavelength excitation, a two-photon imaging experiment was subsequently conducted. HeLa cells incubated with 50 μg / mL N-CDs for 30 minutes were observed under 808 nm femtosecond laser excitation. The results showed significant fluorescence signals in both the green and red channels. Figure 13 As shown, the green channel allows for simultaneous imaging of lipid droplets and cell nuclei, while the red channel signal primarily originates from the cell nucleus region. After fusing the dual-channel images, a clear two-color imaging effect is achieved, with lipid droplets appearing green and cell nuclei displaying orange. This demonstrates that N-CDs, under two-photon excitation mode, not only maintain excellent dual-targeting capabilities but also achieve clear differentiation and simultaneous display of cell nuclei and lipid droplets, validating their application potential in multiphoton imaging systems.

[0046] Example 6 To verify the specific targeting ability of N-CD fluorescent probes to cell nuclei and lipid droplets, this experiment used commercial organelle dyes for co-localization analysis. First, HeLa cells were co-incubated with 50 μg / mL N-CDs at 37°C for 30 minutes. Then, without removing the CDs, 50 nM of the nuclear dye Hoechst 33342 or 100 nM of the lipid droplet dye LDs-Tracker Deep Red were added, and incubation continued for 15 minutes. After incubation, the cells were washed three times with PBS buffer to remove unbound dye, and then observed using confocal microscopy. In this study, the nuclear dye Hoechst 33342 was used to acquire signals in the blue channel (425-475nm) under 405nm laser excitation, while the red channel (570-620nm) fluorescence of N-CDs was acquired using 561nm laser excitation in the same field of view. The lipid droplet dye LDs-TrackerDeepRed was used to acquire signals in the far-red channel under 640nm laser excitation, while the blue channel fluorescence of N-CDs under 405nm excitation was also acquired. Finally, co-localization results were obtained by overlaying the fluorescence images of each channel, and the Pearson correlation coefficient (PCC) was calculated using ImageJ software to quantitatively assess the spatial overlap between the CD fluorescence signals and the organelle dyes.

[0047] Colocalization experiment results are as follows Figure 14 As shown, when N-CDs were co-stained with the nuclear dye Hoechst 33342, their red fluorescence signal and Hoechst's blue signal exhibited high spatial overlap, with a calculated Pearson correlation coefficient of 0.92, indicating that N-CDs can specifically target the cell nucleus. In the co-staining experiment with the lipid droplet dye LDs-TrackerDeepRed, the fluorescence signal of N-CDs in the blue channel also showed good overlap with the far-red signal of the lipid droplet dye, with a Pearson correlation coefficient of 0.87, indicating that N-CDs can effectively target intracellular lipid droplet structures. The above quantitative co-localization analysis results jointly confirm that the N-CD fluorescent probe possesses the dual localization ability to simultaneously and specifically target both the cell nucleus and lipid droplets, providing experimental evidence for its application in dual-color dual-target cell imaging.

[0048] Example 7 To elucidate the specific targeting mechanism of N-CDs fluorescent probes in the cell nucleus, selective digestion of nuclear nucleic acid components was performed using an enzymatic digestion method. HeLa cells were first fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by treatment with 0.1% Triton X-100 permeabilization buffer at room temperature for 20 minutes to enhance membrane permeability. The experimental groups were treated with 100 μg / mL DNase I and 100 μg / mL RNase A at 37°C for 1 hour to ensure sufficient degradation of nuclear DNA or RNA; the control group was treated with an equal volume of PBS to maintain nucleic acid integrity. After enzyme treatment, all cells were co-incubated with 50 μg / mL N-CDs fluorescent probes for 30 minutes, followed by labeling with the nuclear DNA-specific dye Hoechst 33342. Finally, confocal microscopy was used to simultaneously acquire fluorescence signals of N-CDs in the red channel (λex=561nm, λem=570-620nm) and Hoechst in the blue channel (λex=405nm, λem=425-475nm), and the targeting behavior of N-CDs in the cell nucleus under different enzyme treatment conditions was systematically analyzed.

[0049] Experimental results are as follows Figure 15 As shown, after DNase I treatment, nuclear DNA was effectively degraded, as evidenced by a significant decrease in the Hoechst blue fluorescence signal compared to the control group. Meanwhile, the red fluorescence of N-CDs was still partially retained in the nucleolar region, indicating that it could still target nuclear RNA components even in the absence of DNA. Conversely, after RNase A treatment, the RNA component was specifically removed, while the Hoechst blue fluorescence remained intact. Furthermore, the red fluorescence of N-CDs and the Hoechst signal highly overlapped in the co-localization image, demonstrating that N-CDs could still specifically bind to DNA even in the absence of RNA. These results indicate that the N-CDs fluorescent probe possesses the dual recognition capability of simultaneously targeting both nuclear DNA and RNA, and can achieve effective nuclear localization under different nucleic acid component conditions.

[0050] Example 8 To systematically evaluate the photostability of N-CDs fluorescent probes during live-cell imaging, HeLa cells were co-incubated with complete culture medium containing 50 μg / mL N-CDs for 30 minutes, followed by continuous irradiation under a confocal microscope using 405 nm and 561 nm lasers, respectively. The attenuation of fluorescence signals in the blue channel (425-475 nm) and red channel (570-620 nm) was monitored to quantitatively analyze the anti-photobleaching properties of N-CDs in the cellular environment. This experiment simulated the conditions of prolonged laser irradiation during actual imaging, effectively evaluating the optical stability of the probes under physiological conditions.

[0051] Continuous irradiation experiments showed that N-CDs exhibited excellent photostability at both excitation wavelengths. For example... Figure 16 As shown in the image, during 50 minutes of continuous laser irradiation, the fluorescence signals of N-CDs remained stable in both the blue and red channels, and the fluorescence images targeting the cell nucleus and lipid droplets showed clear outlines with a high signal-to-noise ratio. Particularly after 50 minutes of continuous irradiation with a 561 nm laser, the fluorescence imaging of N-CDs in the cell nucleus region remained clear; similarly, under prolonged irradiation with a 405 nm laser, the fluorescence signal of N-CDs in the lipid droplet region also remained stable. These results indicate that N-CDs possess excellent resistance to photobleaching while maintaining their specific targeting performance to the cell nucleus and lipid droplets, providing crucial assurance for their application in long-term dynamic observation of live cells.

[0052] Example 9 To elucidate the molecular mechanism of N-CDs entry into living cells, an energy-dependent uptake experiment was designed. First, HeLa cells were pretreated at 4°C for 30 minutes to inhibit cellular energy metabolism-related activities, followed by co-incubation at 37°C for 30 minutes in DMEM complete medium containing 50 μg / mL N-CDs. In another experiment, HeLa cells were first co-incubated at 37°C for 30 minutes in PBS solution containing 100 μg / mL 2-deoxy-D-glucose, then washed with PBS to remove residual inhibitors, and subsequently incubated for another 30 minutes in medium containing 50 μg / mL N-CDs under the same conditions. Fluorescence images of both groups of cells were acquired using confocal microscopy in the blue channel (405 nm excitation) and red channel (561 nm excitation), respectively. The cellular uptake mechanism of N-CDs was comprehensively analyzed by systematically comparing the results with those of the normal staining group and the autofluorescence control group.

[0053] Experimental results show that ( Figure 17 Compared with the normal staining group, the fluorescence intensity of N-CDs in both the 4℃ low-temperature treatment group and the 2-deoxy-D-glucose treatment group was significantly reduced, resulting in a significant decrease in the clarity and signal-to-noise ratio of cell imaging. Low-temperature treatment effectively blocked the active transport function of cells by inhibiting cell membrane fluidity and energy metabolism processes; while 2-deoxy-D-glucose, as a glucose analog, interfered with the cell's energy supply by competitively inhibiting glycolysis. Significant inhibition of N-CD uptake was observed in both groups, indicating that the process of N-CDs entering the cell depends on the cell's energy state, consistent with an energy-dependent passive transport mechanism. This finding provides an important theoretical basis for understanding the behavioral characteristics of N-CDs in living cells.

[0054] Therefore, the present invention employs the above-mentioned N-CDs fluorescent probe, its preparation method and application, which has abundant surface functional groups, can specifically target cell nuclei and lipid droplets, and can realize dual-color, dual-target imaging and two-photon imaging under a confocal system.

[0055] 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. An N-CDs fluorescent probe, characterized in that: The fluorescent probe was prepared by a one-step hydrothermal method using p-phenylenediamine and L-tartaric acid.

2. The N-CDs fluorescent probe according to claim 1, characterized in that: The elemental composition and content of the N-CDs fluorescent probes are 64.26% C, 8.62% N, and 27.11% O.

3. The N-CDs fluorescent probe according to claim 1, characterized in that: The N-CDs fluorescent probe has a particle size of 1.75±0.04nm, a positively charged surface, a Zeta potential of +11.4mV, and two-photon absorption characteristics. It can simultaneously and specifically target the cell nucleus and lipid droplets, realizing dual-color fluorescence imaging of the blue and red channels under single-photon excitation.

4. A method for preparing an N-CDs fluorescent probe according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Weigh 0.108g of p-phenylenediamine and 0.25g of L-tartaric acid and add them to the polytetrafluoroethylene liner. Then add 20ml of deionized water and sonicate the mixture to make it evenly dispersed. Place the liner in a stainless steel autoclave and put the whole thing into a high-pressure oven. Step 2: After sealing the reaction vessel, place it in an oven and heat it to 200°C for 20 hours. After the reaction is complete, allow it to cool naturally to room temperature. Step 3: Place the reaction mixture into a centrifuge and centrifuge the solution at high speed to remove large particulate impurities. Take the supernatant and then filter the supernatant through a 0.22μm filter membrane. 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 48 hours to remove impurities. Step 5: The dialyzed solution is freeze-dried to obtain N-CDs solid powder for later use.

5. The application of an N-CDs fluorescent probe according to any one of claims 1-3, characterized in that: It is applied to dual-target two-photon imaging that simultaneously targets the cell nucleus and lipid droplets.

6. The application of the N-CDs fluorescent probe according to claim 5, characterized in that: The cell staining conditions in dual-target two-photon imaging were 50 µg / mL N-CDs staining for 30 min.

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