Nitrogen-doped carbon dots, preparation method thereof and application of nitrogen-doped carbon dots in pH detection and cell imaging
Nitrogen-doped carbon dots were prepared by hydrothermal reaction of Bengal rose red and diethylenetriamine, solving the problems of energy level distribution and biotoxicity in carbon dot preparation, and enabling efficient pH detection and cell imaging applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING UNIV
- Filing Date
- 2026-01-20
- Publication Date
- 2026-05-01
AI Technical Summary
Existing methods for preparing carbon dots suffer from challenges such as wide product energy level distribution, difficulty in controllable doping and in-situ passivation, insufficient resolution of traditional imaging techniques, high biotoxicity or poor specificity, and lack of simple pH detection methods.
Nitrogen-doped carbon dots were prepared via hydrothermal reaction using Bengal rose red and diethylenetriamine as precursors. The reaction conditions were optimized by combining dialysis purification and freeze drying to obtain carbon dots with uniform energy levels and low biotoxicity.
Nitrogen-doped carbon dots with high quantum yield and low biotoxicity were prepared. They exhibit pH-responsive characteristics, enabling fluorescence-colorimetric dual-mode detection and cell imaging. They are suitable for accurate detection in the pH range of 2.0-7.0 and stable application in strongly alkaline environments.
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Figure CN121950302A_ABST
Abstract
Description
A nitrogen-doped carbon dot, its preparation method, and its application in pH detection and cell imaging. Technical Field
[0001] This application relates to the field of nanomaterial preparation and application, and in particular to a nitrogen-doped carbon dot, its preparation method, and its application in pH detection and cell imaging. Background Technology
[0002] As the most widely used method for biovisualization, fluorescence-based analysis techniques have achieved great success in bioimaging, diagnostics, and biosensing due to their advantages such as fast response, high sensitivity, and simple operation. Among various fluorescent materials, carbon dots, as ultra-small zero-dimensional photoluminescent nanomaterials, have attracted widespread attention due to their unique optical properties and good biocompatibility. Compared with traditional semiconductor quantum dots and organic fluorescent dyes, carbon dots not only possess tunable photoluminescence properties and excellent stability, but also have outstanding advantages such as good biocompatibility, and are regarded as a new generation of luminescent materials. However, the preparation of carbon dots still faces several key bottlenecks, such as the wide energy level distribution of the product, the difficulty of controllable doping and in-situ passivation, and the cumbersome and complex functionalization steps.
[0003] pH is a crucial environmental parameter that reflects not only physiological and biochemical processes but is also closely related to various pathological states. For example, many physiological functions in the human body depend on acid-base balance, and various substances are exchanged and transported through the blood. However, developing precise and convenient in-situ visualization methods for pH sensing in complex environments remains a significant challenge. Meanwhile, cell imaging technology has evolved from traditional optical microscopy to a precise visualization technique capable of dynamic tracking at the molecular level, with high resolution and low invasiveness being its core requirements. In scientific research, cell imaging can be used to observe morphological changes in organelles, track the spatiotemporal distribution and interactions of biomolecules, and monitor the transport pathways of drug molecules within cells. However, traditional imaging techniques often suffer from insufficient resolution, high biotoxicity, or poor specificity, necessitating the development of novel probe materials to overcome these technological bottlenecks.
[0004] Therefore, how to prepare high-performance fluorescent carbon dots with uniform energy levels, good biocompatibility, and simple, controllable pathways and efficient doping passivation is an urgent problem to be solved. Meanwhile, carbon dots can alter fluorescence or color signals through interactions with target molecules or the environment; this photoactive property holds promise for overcoming limitations in environmental pH detection and bioimaging. Summary of the Invention
[0005] To address the deficiencies in existing technologies, this invention proposes a method for preparing nitrogen-doped carbon dots, comprising the following steps: S1 Precursor solution preparation: Bengal rose red (RB) and diethylenetriamine (DETA) are added to water and ultrasonically dispersed uniformly to obtain a precursor solution; S2 Hydrothermal reaction: The precursor solution is transferred to a reaction vessel lined with polytetrafluoroethylene and placed in an oven for a one-step hydrothermal reaction to obtain a mixed solution containing nitrogen-doped carbon dots (NCDs); S3 Purification treatment: The mixed solution containing nitrogen-doped carbon dots (NCDs) is purified by dialysis using a dialysis bag to obtain a carbon dot solution; S4 Preparation of carbon dot stock solution: The carbon dot solution is further freeze-dried to obtain nitrogen-doped carbon dot (NCDs) powder, and a carbon dot stock solution of a certain concentration is prepared.
[0006] Furthermore, the precursor, Bengal rose red (RB), is photosensitive, biotoxic, and has a low quantum yield due to the heavy atom effect; the precursor, Bengal rose red (RB), has a large conjugated planar structure, which is used to retain the framework as nitrogen-doped carbon dots after hydrothermal reaction; the precursor, Bengal rose red (RB), has reactive halogen sites, which are used to initiate directional nucleophilic substitution reactions and provide active sites for crosslinking and carbonization.
[0007] Furthermore, the precursor diethylenetriamine is an electron-rich polyamine, and the precursor diethylenetriamine is used as a multifunctional reagent in hydrothermal reactions. The precursor diethylenetriamine is used to achieve nitrogen doping, surface passivation and amino functionalization.
[0008] Furthermore, in step S1, the mass ratio of Bengal rose red (RB) to diethylenetriamine (DETA) in the precursor solution is 2:1 to 1:5.
[0009] Furthermore, the hydrothermal reaction temperature gradient is 120-220℃, and the reaction time is 0-15h.
[0010] A nitrogen-doped carbon dot, which is prepared by the method described in any of the above-mentioned methods.
[0011] An application of nitrogen-doped carbon dots in pH detection, wherein the fluorescence emission peak intensity and ultraviolet absorption peak intensity of the nitrogen-doped carbon dots are linearly correlated with the ambient pH, and the pH detection is specifically an application that can realize fluorescence-colorimetric dual-mode detection in the pH range of 2.0-7.0.
[0012] A nitrogen-doped carbon dot that maintains a high quantum yield under strongly alkaline conditions (pH 14.0).
[0013] A nitrogen-doped carbon dot exhibits excellent biocompatibility, weak ability to generate singlet oxygen, and low dark toxicity and phototoxicity.
[0014] An application of nitrogen-doped carbon dots in cell imaging, wherein the nitrogen-doped carbon dots can be used for fluorescence imaging of 4T1 cells.
[0015] Compared with the prior art, the advantages of this invention are: (1) It fully considers the precursor structure and chemical properties, and prepares high-performance nitrogen-doped carbon dots with high quantum yield, low biotoxicity and independent excitation through a simple preparation method and efficient passivation modification.
[0016] (2) The retention of the large conjugated structure of Bengal rose promotes the generation of carbon dots with uniform energy levels, and the multifunctional diethylenetriamine promotes efficient elemental doping and passivation modification, which greatly improves the performance of carbon dots. The removal of halogens during hydrothermal process directly weakens the heavy atom effect, significantly reduces the singlet oxygen generation capacity and biotoxicity, and inhibits nonradiative transitions.
[0017] (3) Nitrogen doping and in-situ passivation make the radiation transition channel dominant, which promotes the improvement of the quantum yield of nitrogen-doped carbon dots; at the same time, the large amount of surface amino groups improves water dispersibility and biocompatibility, which promotes the subsequent application of nitrogen-doped carbon dots in sensing and imaging.
[0018] (4) Nitrogen-doped carbon dots not only have acid-responsive characteristics and can achieve fluorescence-colorimetric dual-mode detection in the pH range of 2.0-7.0, but also maintain high quantum yield in a strongly alkaline environment.
[0019] (5) Nitrogen-doped carbon dots have high quantum yield, excitation independence, good stability and biocompatibility, and can be used as fluorescent probes in the field of cell imaging, with good application prospects. Attached Figure Description
[0020] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings: Figure 1 is a characterization diagram of nitrogen-doped carbon dots prepared in the embodiment of the present invention, wherein (a) is a transmission electron microscope image, and the inset is a size distribution diagram; (b) is a high-resolution transmission electron microscope image; Figure 2 shows the Raman spectra of nitrogen-doped carbon dots and Bengal rose red prepared in the embodiment of the present invention; Figure 3 shows the excitation, fluorescence emission, and ultraviolet absorption spectra of nitrogen-doped carbon dots prepared in the embodiment of the present invention; Figure 4 shows the excitation, fluorescence emission, and ultraviolet absorption spectra of Bengal rose red prepared in the embodiment of the present invention; Figure 5 shows the nitrogen-doped carbon dots prepared in the embodiment of the present invention. The fluorescence emission spectra at different excitation wavelengths; Figure 6 shows the 3D fluorescence spectra of nitrogen-doped carbon dots prepared in the embodiment of the present invention; Figure 7 shows the stability test of nitrogen-doped carbon dots prepared in the embodiment of the present invention; Figure 8 shows the fluorescence emission spectra, ultraviolet absorption spectra, and intensity changes of nitrogen-doped carbon dots prepared in the embodiment of the present invention at different pH values; wherein (a) fluorescence emission spectrum changes with pH; (b) fluorescence emission peak intensity changes with pH; (c) ultraviolet absorption spectrum changes with pH; (d) ultraviolet absorption peak intensity changes with pH; Figure 9 shows the fluorescence intensity change of nitrogen-doped carbon dots prepared in the embodiment of the present invention in an alkaline environment of 1M NaOH (pH 14.0); Figure 10 shows the ability of nitrogen-doped carbon dots and Bengali rose red to generate singlet oxygen in the embodiment of the present invention; Figure 11 shows (a) the dark toxicity and phototoxicity of Bengali rose red RB; (b) the dark toxicity and phototoxicity of nitrogen-doped carbon dot NCDs prepared in the embodiment of the present invention; Figure 12 shows the imaging of nitrogen-doped carbon dots prepared in the embodiment of the present invention in 4T1 cells. Detailed Implementation
[0021] To illustrate the present invention in more detail, the technical solution of the present invention will be further described below with reference to preferred embodiments and accompanying drawings.
[0022] Referring to Figures 1-12, this application proposes a method for preparing nitrogen-doped carbon dots and their application in pH detection and cell imaging. First, nitrogen-doped carbon dots are prepared through the following steps: Bengal rose red (RB) and diethylenetriamine (DETA) are added to water and sonicated for 10 minutes to disperse them evenly, obtaining a precursor solution; the precursor solution is transferred to a reaction vessel lined with polytetrafluoroethylene (PTFE) and subjected to a one-step hydrothermal reaction in an oven to obtain a mixed solution containing nitrogen-doped carbon dots (NCDs); finally, the mixed solution containing nitrogen-doped carbon dots (NCDs) is purified by dialysis using a dialysis bag to obtain a carbon dot solution, which is further freeze-dried to obtain carbon dot powder, and a carbon dot stock solution of a certain concentration is prepared. To obtain strongly emitted carbon dots (CDs) and to gain a deeper understanding of the influencing factors in the NCDs formation process, quantum yield was used as the optimization standard parameter, and multi-parameter optimization experiments were conducted, including the precursor ratio, temperature, and time, as shown in Table 1. Table 1 shows the optimized reaction conditions for preparing nitrogen-doped carbon dots. First, the precursor ratio was optimized by adding 0.5-5 times the mass of RB to DETA. Tests revealed that the quantum yield of the product was highest at a 1:1 mass ratio. Temperature significantly affected the condensation, crosslinking, and carbonization degree of the precursor. By setting a temperature gradient of 120-220℃, we found that at temperatures below 140℃, the product exhibited almost no fluorescence, with a PLQY as low as 9%. Similarly, excessively high temperatures (>180℃) easily led to over-carbonization of the product, further reducing the PLQY. This is consistent with the top-down synthesis process of CDs in hydrothermal reactions. Finally, we optimized the reaction time at a fixed temperature of 160℃. Tests showed that 0 h yielded a quantum yield close to that of the precursor RB, only 1.14%. With continuously extended reaction times, the precursor underwent sufficient reaction, and nitrogen-doped carbon dots (NCDs) with high quantum yields could be prepared within 6-12 h.
[0023] Table 1 The optical properties of the obtained NCDs were further characterized. The transmission electron microscope (TEM) image in Figure 1 shows that the NCDs are monodisperse spherical nanoparticles with an average particle size of 3.18 ± 0.57 nm. Furthermore, HR-TEM revealed distinct lattice fringes, measured to be 0.21 nm, consistent with the (100) plane of graphitic carbon. This indicates the formation of a highly crystalline, uniformly sized carbon quantum dot structure. Further Raman spectroscopy in Figure 2 revealed that the NCDs exhibited optical properties within a range of ~1300 cm⁻¹. -1 and ~1600cm -1 There are spectral peaks at these locations, belonging to D(sp) respectively. 3 Hybridization) and G-band (sp 2 Hybridization indicates that NCDs have undergone graphitization—transforming from a small-molecule derivative state of RB to graphitic carbon. The Ig of NCDs was calculated. D / IG The value of 0.95 indicates that NCDs have fewer defects and a more ordered structure compared to most carbon dots, which is attributed to the retention of the RB large conjugated framework as the carbon dot core and the efficient passivation modification of DETA.
[0024] The optical properties of NCDs are further characterized below using fluorescence and UV absorption spectra, as shown in Figures 3 to 6. As shown in Figure 3, the optimal fluorescence excitation of NCDs is located at 510 nm, with a narrow fluorescence emission peak at 537 nm. The UV absorption peak is located at 508 nm, exhibiting a pale yellow hue under white light and a bright green hue under UV light. These optical characteristics differ significantly from those of the precursor RB (Figure 4), whose optimal excitation / emission is located at 551 nm and 568 nm, and whose UV absorption peak is located at 548 nm. The low quantum yield results in only a weak purplish-red fluorescence, which appears distinctly purplish-red under sunlight. However, the spectral shape of NCDs is also very similar to that of RB, indicating the retention of the large conjugated framework of RB. The significant blue shift indicates changes in electron distribution and surface state energy levels caused by DETA doping / passivation modification. Meanwhile, as shown in Figures 5 and 6, the fluorescence spectra of NCDs at different excitation wavelengths (Figure 5) and the 3D fluorescence spectra of NCDs (Figure 6) demonstrate that, compared to most excitation-dependent carbon dots, the NCDs prepared in this invention are not excitation-dependent and are independently excitation-dependent. This avoids the inconvenience caused by fluorescence overlap when used simultaneously with other fluorescent probes for imaging or sensing. This also reflects the uniform energy level distribution and uniform luminescence centers of the product, which are closely related to the preservation of the carbon skeleton of RB.
[0025] Secondly, the stability of NCDs under various extreme environments was evaluated, verifying their stability against photobleaching, high salt concentrations, high temperatures, and long-term storage, as shown in Figure 7. The NCDs solution was irradiated with a 365 nm UV lamp, and the fluorescence emission peak intensity was measured at 5-minute intervals. The experiment showed that after 60 minutes of continuous irradiation, the fluorescence intensity of the NCDs remained at 97.8%, indicating good photobleaching resistance. Subsequently, NaCl solutions of different concentrations were prepared, and NCDs of the same concentration were added and mixed thoroughly. As shown in the figure, the fluorescence intensity of the NCDs hardly decreased in high-concentration salt solutions, maintaining a high intensity. Simultaneously, considering the influence of ambient temperature during use, we also verified the fluorescence quenching effect of different temperatures on the NCDs. The figure shows that the NCDs maintained the highest fluorescence intensity at room temperature, and the fluorescence intensity did not decrease even near the freezing and boiling points of the aqueous solution. Finally, we placed the NCDs in an environment of 4 °C to study the stability of the probe's aqueous solution under long-term storage conditions. The results showed that the fluorescence intensity of NCDs could still reach 93.2% after 60 days. In summary, this demonstrates the stability of NCDs under various extreme environments, which provides a guarantee for their use in sensing applications.
[0026] In summary, the method for preparing nitrogen-doped carbon dots proposed in this invention can produce nitrogen-doped quantum dots with high quantum yield, independent excitation, and good stability.
[0027] NCDs prepared under optimal conditions were used for pH sensing, and the trends of their fluorescence emission peak and ultraviolet absorption peak were recorded. As shown in Figure 8, the fluorescence of NCDs gradually weakens under acidic conditions, while exhibiting good and slightly enhanced green fluorescence emission in the neutral and alkaline range (pH 7.0–12.0). In particular, under pH 2.0–7.0 conditions, the fluorescence intensity of NCDs shows a linear increasing trend with pH, with the linear equation being Y = 972.33X – 1464.28, R... 2 =0.998 (pH2.0-4.5), Y=559.32X+390.30, R 2 =0.999 (pH 4.5-7.0). Simultaneously, its UV absorption peak intensity also exhibits a linear trend similar to the fluorescence emission peak. Under acidic conditions, with increasing pH, the intensity of its UV absorption peak at 508 nm continuously increases and shows a slight red shift, with the linear equation being Y = 0.076X – 0.171, R... 2 =0.995 (pH 2.5-5.0), Y=0.288X–1.225, R 2=0.998 (pH 5.0-7.0). This suggests that NCDs have good pH response and can be used as a fluorescent-colorimetric dual-mode probe for detecting acidic environments.
[0028] Meanwhile, most fluorescent probes (small molecule organic compounds, nanoparticles, etc.) are not stable enough under strongly alkaline pH conditions, thus limiting their application scenarios. Therefore, to verify the stability of NCDs under extremely alkaline conditions, this study dispersed NCDs in 1M NaOH (pH 14.0) and recorded the changes in their fluorescence intensity. As shown in Figure 9, the fluorescence intensity of NCDs hardly decreased over a time interval of up to 180 minutes. Furthermore, the quantum yield of NCDs was 74.92% at this time, indicating that the stable conjugated structure and surface modification enable NCDs to maintain extremely strong photostability in strongly alkaline conditions, overcoming the difficulties of small molecule fluorescent compound probes.
[0029] In summary, this invention also proposes an application of nitrogen-doped carbon dots in pH detection. The fluorescence emission peak intensity and ultraviolet absorption peak intensity of the nitrogen-doped carbon dots are linearly correlated with the ambient pH, enabling fluorescence-colorimetric dual-mode detection in the pH range of 2.0-7.0, and maintaining a high quantum yield under strongly alkaline conditions (pH 14.0).
[0030] Since the ability to generate singlet oxygen is usually associated with biotoxicity, we first verified the ability of NCDs and RB to generate singlet oxygen. 1,3-Diphenylisobenzofuran (DPBF) is a fluorescent probe indicating singlet oxygen with high specificity. It irreversibly forms an internal peroxide upon contact, and the intensity of its UV absorption peak decreases rapidly. As shown in Figure 10, after mixing RB and DPBF solutions, the UV absorption peak of DPBF decreases significantly with increasing irradiation time, indicating the large-scale generation of singlet oxygen. In contrast, the UV absorption peak intensity of NCDs decreases slowly under the same conditions, indicating a significant reduction in their ability to generate singlet oxygen. The difference between the two can be visually seen in the slope comparison in the figure, indicating a reduction in the biotoxicity of NCDs.
[0031] Further, using 4T1 cells, cell viability was recorded using the CCK8 assay to verify the phototoxicity and dark toxicity of RB and NCDs. Figure 11 shows that under dark conditions, the cell viability of RB-cultured cells decreased significantly with increasing concentration, especially at high concentrations. NCDs, on the other hand, remained relatively stable, maintaining a cell viability of over 95% even at the same high concentration, indicating reduced dark toxicity. To further verify phototoxicity, the cells were irradiated with 20W white light during the last half hour of culture. Figure 11 shows that the cell viability of RB-cultured cells dropped sharply, consistent with its extremely high photosensitivity; while NCDs only showed a slight decrease at high concentrations, a result consistent with singlet oxygen detection, indicating that NCDs have good biocompatibility and are expected to be used as fluorescent probes for cell visualization imaging.
[0032] Considering the characteristics of NCDs—small size, high quantum yield, good stability, and good biocompatibility—we verified their ability for cell imaging. After co-culturing 200 μg / mL NCDs with 4T1 cells for 24 h, cell images were recorded using a fluorescence inverted microscope in bright field, dark field, and mixed field. As shown in Figure 12, the NCDs were fully internalized by the cells and did not leak after prolonged staining, presumably because the weakly basic amino groups in the NCDs have acid affinity and can specifically aggregate within lysosomes. Compared to the control group, the NCDs exhibited good cell imaging performance in the dark field, with almost all cells showing bright green fluorescence, and they were clearly visible even in the mixed field. This indicates that NCDs can serve as fluorescent probes for cell imaging and hold promise for long-term tracking and localization imaging.
[0033] In summary, this invention proposes an application of nitrogen-doped carbon dots in cell imaging. The nitrogen-doped carbon dots have excellent biocompatibility, and their ability to generate singlet oxygen is significantly weakened, resulting in a significant reduction in both dark toxicity and phototoxicity, thus enabling fluorescence imaging of 4T1 cells.
[0034] Finally, it should be noted that the above embodiments are merely preferred examples for clearly illustrating the present invention, but they are not intended to limit the implementation of the present invention. Those skilled in the art should understand that the technical features in the above solutions can be combined arbitrarily, and other modifications or equivalent substitutions can be made to some technical features based on the above specific implementation methods. It is impossible to exhaustively list all implementation methods here. Therefore, any modifications, improvements, equivalent substitutions, etc., derived from the technical solutions of the present invention within the spirit and principles of the present invention should be within the scope of protection claimed by the present invention.
Claims
1. A method for preparing nitrogen-doped carbon dots, characterized in that, Includes the following steps: Preparation of S1 precursor solution: Bengal rose red (RB) and diethylenetriamine (DETA) were added to water and ultrasonically dispersed to obtain a precursor solution; S2 Hydrothermal reaction: The precursor solution is transferred to a reaction vessel lined with polytetrafluoroethylene and placed in an oven for a one-step hydrothermal reaction to obtain a mixed solution containing nitrogen-doped carbon dots (NCDs); S3 Purification treatment: The mixed solution containing nitrogen-doped carbon dots (NCDs) is purified by dialysis using a dialysis bag to obtain a carbon dot solution; S4 Preparation of carbon dot stock solution: The carbon dot solution is further freeze-dried to obtain nitrogen-doped carbon dot (NCDs) powder, and a carbon dot stock solution of a certain concentration is prepared.
2. The method for preparing nitrogen-doped carbon dots according to claim 1, characterized in that, The precursor, Bengal rose red (RB), is photosensitive, biotoxic, and has a low quantum yield due to the heavy atom effect. The precursor, Bengal rose red (RB), has a large conjugated planar structure, which is used to retain the framework as nitrogen-doped carbon dots after hydrothermal reaction. The precursor, Bengal rose red (RB), has reactive halogen sites for initiating directional nucleophilic substitution reactions and for providing active sites for crosslinking and carbonization.
3. The method for preparing nitrogen-doped carbon dots according to claim 1, characterized in that, The precursor diethylenetriamine is an electron-rich polyamine. The precursor diethylenetriamine is used as a multifunctional reagent in hydrothermal reactions. The precursor diethylenetriamine is used to achieve nitrogen doping, surface passivation and amino functionalization.
4. The method for preparing nitrogen-doped carbon dots according to claim 1, characterized in that, In step S1, the mass ratio of Bengal rose red (RB) to diethylenetriamine (DETA) in the precursor solution is 2:1 to 1:
5.
5. The method for preparing nitrogen-doped carbon dots according to claim 1, characterized in that, The hydrothermal reaction temperature gradient is 120-220℃, and the reaction time is 0-15h.
6. A nitrogen-doped carbon dot, characterized in that, It is prepared by the method described in any one of claims 1-5.
7. The application of nitrogen-doped carbon dots in pH detection as described in claim 6, characterized in that, The fluorescence emission peak intensity and ultraviolet absorption peak intensity of the nitrogen-doped carbon dots are linearly correlated with the ambient pH. The pH detection specifically refers to the application of fluorescence-colorimetric dual-mode detection within the pH range of 2.0-7.
0.
8. A nitrogen-doped carbon dot as described in claim 6, characterized in that, The nitrogen-doped carbon dots maintain a high quantum yield under strong alkaline conditions (pH 14.0).
9. A nitrogen-doped carbon dot as described in claim 6, characterized in that, The nitrogen-doped carbon dots exhibit excellent biocompatibility, have a weak ability to generate singlet oxygen, and exhibit low dark toxicity and phototoxicity.
10. The application of nitrogen-doped carbon dots in cell imaging as described in claim 9, characterized in that, The nitrogen-doped carbon dots enable the application of 4T1 cell fluorescence imaging.