Traditional Chinese medicine derived carbon dot-based fluorescent probe and hydrogel dressing for treating wounds

Abstract

The invention discloses a traditional Chinese medicine derived carbon dot-based fluorescent probe and a hydrogel dressing for treating wounds, orange emission carbon dots RC-CDs are successfully synthesized through fine optimization by adopting a solvothermal method and utilizing coptis chinensis powder and p-phenylenediamine (p-PDA) as precursors, the RC-CDs emit an orange spectrum at 606 nm, and the fluorescence probe has the advantages that the fluorescence probe and the hydrogel dressing are used for treating the wounds; the RC-CDs prepared by excitation at the wavelength of 490 nm show a remarkable burst response to Cu, and the detection limit is 68.85 nM. The recovery rate of Cu detected in environmental water and food is stable (96.68-109.34%), and the relative standard deviation (RSD) is less than 4.71%, which shows that the method has good environmental tolerance and practical application potential. The hydrogel dressing remarkably improves the wound healing efficiency by promoting epidermal tissue regeneration and accelerating collagen deposition.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanosynthesis and detection technology, and specifically relates to a traditional Chinese medicine derived carbon dot-based fluorescent probe, and the fluorescent probe is used for recognizing Cu 2+ and its antibacterial performance is used for a hydrogel dressing for antibacterial promotion of wound healing. BACKGROUND

[0002] With the rapid development of industry and the increasing diversification of human activities, food contamination and water pollution problems are becoming increasingly prominent in modern society. The potential harm of iron ions and mercury ions to food safety has attracted widespread attention. Mercury ions are known for their high toxicity and are widely distributed in water, air, soil, and even the food chain. Excessive accumulation in the human body can seriously damage the digestive, reproductive, and nervous systems, and in extreme cases, even threaten life [Chakraborty A, Rajana V K, Saritha C, et al. A new Eosin Y-based ‘turn-on’ fluorescent sensor for ratiometric sensing of toxic mercury ion (Hg²⁺) offering unaided eye detection and its antibacterial activity [J]. Journal of Hazardous Materials, 2024, 470: 134207. Alizadeh S R., Biparva P, Ebrahimzadeh M A. A novel quinoxaline-based multifunctional probe: Sensitive and selective naked-eye detection of Fe³⁺ and fluorescent sensing of Cu²⁺ and its application [J]. Arabian Journal of Chemistry, 2024, 17: 105724.]. Divalent copper ions (Cu 2+ ) play an important role in catalysis and maintaining the stability of the nervous system. However, due to biological enrichment, excessive Cu 2+can cause human body damage and may indirectly cause some serious kidney or liver diseases [Zhang L C, Yang Y M, Liang L, et al. Lighting up of carbon dots for copper(II) detection using an aggregation-induced enhanced strategy[J]. Analyst, 2022, 147: 417-422.]. Therefore, it is very important to develop effective analysis methods to selectively detect Cu 2+ in the environment. Fluorescence method has the advantages of convenient detection, fast response speed, high sensitivity and in-situ monitoring in various environments, and has been widely studied and applied in food and biological sample detection [He H, Sun D W, Wu Z, et al. On-off-on fluorescent nanosensing: Materials, detection strategies and recent food applications[J]. Trends in Food Science & Technology, 2022, 119: 243-256.]. In view of this, developing a method that can rapidly and accurately detect Cu 2+ on-site has extremely important practical significance for ensuring food safety and water quality safety.

[0003] So far, people have been trying to create efficient heavy metal ions (including Cu 2+Ion) detection techniques such as polymerase chain reaction, atomic absorption spectrometry, high performance liquid chromatography and inductively coupled plasma spectrometry. However, most of the current detection methods require complex sample pretreatment procedures, professional equipment and personnel, are expensive and time-consuming, and cannot achieve timely and rapid detection. Therefore, it is essential to develop a portable, sensitive and rapid detection method. Fluorescent sensor method is known for its high selectivity, high sensitivity, rapid response and environmental friendliness, and is considered as a very promising method for heavy metal pollutant detection. At present, many nanomaterials with unique optical properties, such as carbon dots (CDs), metal organic frameworks (MOFs), metal nanoparticles (MNPs) and metal nanoclusters (MNCs) etc. Among them, the low production cost of CDs, the rich surface functional groups and the excellent optical properties make them can be used as fluorescent probes for the detection and monitoring of analytes, and the fluorescent probes based on CDs have the advantages of in-situ and real-time detection of target analytes. Wound healing is an important physiological process of human tissue repair, but chronic wounds (such as diabetic ulcers, burn infections and traumatic wounds) are often affected by bacterial infection, inflammatory response disorder and cell regeneration disorder, which often leads to delayed healing or even tissue necrosis, seriously threatening the quality of life of patients. According to statistics, the number of chronic wound patients worldwide has exceeded 40 million, and with the aging of the population and the rising incidence of metabolic diseases, the demand for treatment is increasingly urgent. Traditional treatment methods (such as antibiotics, growth factors and dressings) can partially alleviate symptoms, but still face limitations such as proliferation of drug-resistant bacteria, low bioavailability and significant side effects, and there is an urgent need to develop new therapies with high efficiency of antibacterial, anti-inflammatory and regeneration functions. However, how to optimize the surface functionalization of carbon dots to balance their biological safety and efficacy is still a core challenge for clinical translation.

[0004] To the best of our knowledge, there is no literature report on the synthesis of carbon dots using Coptis as a carbon source and p-phenylenediamine as a nitrogen source for the detection of Cu 2+ Therefore, in this experiment, RC-CDs were synthesized using p-phenylenediamine PPD and Coptis powder as precursors to achieve rapid and portable detection of Cu 2+ The orange fluorescent probe can stably maintain the fluorescence properties and be used for detecting Cu 2+ In addition, RC-CDs also have certain antibacterial and wound healing effects on Escherichia coli and Staphylococcus aureus, as well as cell imaging effects, and have better application prospects in the future. SUMMARY

[0005] The purpose of the present application is to solve the above problems, and provide a traditional Chinese medicine derived carbon dot-based fluorescent probe and its application in the detection of Cu 2+The RC-CDs have the application of the aspect, and since the RC-CDs are carbon dots taking the Coptis powder as a precursor, so the RC-CDs retain the effective active ingredients of the Coptis for resisting inflammation and resisting bacteria, have the antibacterial performance, and are made into the antibacterial hydrogel dressings, can realize the effects of promoting wound healing of the living mice and accelerating tissue growth.

[0006] The traditional Chinese medicine derived carbon dot based fluorescent probe RC-CDs are synthesized by a solvothermal method taking p-phenylenediamine and Coptis powder as precursors;

[0007] The solvothermal method comprises the following steps:

[0008] 1) 0.0800-0.1404 g of p-phenylenediamine and 0.3-0.6 g of Coptis powder are dispersed in 5-10 mL of ethanol, and then transferred into a high-pressure reaction kettle, and heated at a temperature of 200-240 DEG C for 2-10 h;

[0009] 2) cooled to room temperature, and then purified by dialysis to obtain RC-CDs;

[0010] In step 1), the p-phenylenediamine is 0.1080 g-0.1104 g, the Coptis powder is 0.45-0.55 g, and the ethanol is 8-10 mL;

[0011] In step 1), the p-phenylenediamine is 0.1080 g, the Coptis powder is 0.4 g, and the ethanol is 9 mL;

[0012] In step 1), the temperature is 220 DEG C, and the heating is 5-7 h;

[0013] In step 2), the dialysis purification is filtration through a 0.22 mu m microporous filter membrane and dialysis purification through a dialysis bag with a molecular weight cut-off of 1000 Da.

[0014] Another object of the present application is to provide a method for detecting Cu 2+ by using the traditional Chinese medicine derived carbon dot based fluorescent probe RC-CDs. 2+ The carbon dot RC-CDs with the luminescence characteristics can detect trace Cu 2+ in environmental water samples and food samples through a fluorescence mode.

[0015] The method for detecting Cu 2+ by using the traditional Chinese medicine derived carbon dot based fluorescent probe RC-CDs comprises the following steps:

[0016] 1) drawing of a standard curve: mixing RC-CDs solution with an equal volume of ultrapure water, measuring the fluorescence intensity value I0 of the blank group, and taking RC-CDs solution mixed with different known concentrations of Cu 2+After mixing the solution, the fluorescence intensity value I at 606 nm was measured, and the corresponding relative fluorescence intensity value I / I0 was calculated to establish the standard curve of the relative fluorescence intensity value I / I0 versus the Cu 2+ concentration.

[0017] 2) Fluorescence detection of Cu 2+ : After mixing the RC-CDs solution with the sample solution to be tested, the fluorescence intensity value I was measured, and the relative fluorescence intensity value I / I0 was calculated, and the concentration of Cu 2+ in the sample solution to be tested was obtained according to the above standard curve.

[0018] The concentration of Cu 2+ in the sample solution to be tested is 0.05-20 μM.

[0019] The mixing time of the RC-CDs solution with the sample solution to be tested is 5 min.

[0020] The reaction is carried out at room temperature.

[0021] The fluorescence detection conditions are: excitation wavelength 390 nm, excitation slit and emission slit width 3 nm, and the emission intensity of RC-CDs at 606 nm is recorded.

[0022] Broad-spectrum antibacterial performance of traditional Chinese medicine-derived carbon dot-based fluorescent probe RC-CDs:

[0023] The antibacterial performance experiment of RC-CDs selected Escherichia coli and Staphylococcus aureus as model strains, and cultured in Luria-Bertani broth (LB) liquid medium at 37 ℃ with constant temperature oscillation at a speed of 200 rpm for 3 hours. 100 μL of bacterial suspension was inoculated in 5 mL of fresh culture medium, and 100 μL of RC-CDs solution with concentration gradient of 0-300 μg / mL was added, and the oscillation culture was continued for 3 hours. Then, the culture was diluted to a final concentration of 10 6CFU / mL, 100 μL was uniformly coated on the surface of agar plates and incubated in a 37 ℃ constant temperature incubator for 16 hours. The relative bacterial survival rate was determined by plate colony counting method, and all experiments were set up in triplicate biological replicates. The experimental results showed that RC-CDs exhibited significant growth inhibition effect on both gram-negative bacteria and gram-positive bacteria, and had significant difference compared with the control group without adding RC-CDs. At the concentration of 250 μg / mL, the antibacterial effect of RC-CDs on E. coli and S. aureus was 89% and 97% respectively, while the inhibition rate of Coptis extract on E. coli at the same concentration was significantly lower, which was 34%, and the inhibition rate on S. aureus was 31%. Nano-sized RC-CDs showed stronger antibacterial performance and were excellent antibacterial agents.

[0024] It is another object of the present application to provide a hydrogel dressing of RC-CDs.

[0025] The hydrogel dressing of RC-CDs is prepared by the following method:

[0026] First, 1-5%wt of RC-CDs solution and 0.5-2.0%wt of sodium alginate solution are stirred by a magnetic stirrer for 3-6h, and then cross-linked with 1-5%wt of calcium ion solution to form SA@RC-CDs.

[0027] The use of the hydrogel dressing of RC-CDs in the prevention of wound infection and rapid healing.

[0028] This invention discloses a traditional Chinese medicine-derived carbon dot-based fluorescent probe and a hydrogel dressing for wound treatment. Using a solvothermal method with Coptis chinensis powder and p-phenylenediamine (p-PDA) as precursors, we successfully synthesized orange-emitting carbon dot RC-CDs through meticulous optimization. The RC-CDs emit an orange spectrum at 606 nm, and the prepared RC-CDs, excited at 490 nm, exhibit a significant burst response to Cu²⁺, with a detection limit of 68.85 nM. The recoveries of Cu²⁺ in environmental water and food were stable (96.68%-109.34%), with a relative standard deviation (RSD) of less than 4.71%, indicating good environmental tolerance and practical application potential. The hydrogel dressing significantly improves wound healing efficiency by promoting epidermal tissue regeneration and accelerating collagen deposition. Of particular interest is the successful development of a RC-CDs-based test strip, which is used as a portable sensor for rapid detection of Cu²⁺. The detection limit of 70.5 nM shown by this test strip represents a convenient and effective solution in the field of environmental monitoring. The minimum bactericidal concentration of RC-CDs against Escherichia coli is 250 μg / mL. Notably, the study confirmed that RC-CDs possess significant broad-spectrum antibacterial activity. Based on this property, researchers developed a novel RC-CDs hydrogel wound dressing. Experimental data show that this dressing significantly improves wound healing efficiency by promoting epidermal tissue regeneration and accelerating collagen deposition. This conclusion is reliably supported by data from both in vitro bacterial experiments and in vivo mouse model studies, fully validating the broad-spectrum antibacterial efficacy of RC-CDs. Given its excellent bioactivity, RC-CDs show broad application prospects in food safety testing, environmental monitoring, and biomedical treatment, opening up new directions for research in related fields.

[0029] The present invention has the following advantages:

[0030] 1. The carbon dots of the present invention have good photoluminescence properties.

[0031] 2. The carbon dot fluorescent probe constructed in this invention can directly detect Cu. 2+ Detection, Cu 2+ The fluorescence of carbon dots is quenched, resulting in intuitive, visual, and easily observable results.

[0032] 3. The fluorescent probe provided by this invention for detecting Cu 2+ It features high sensitivity and good selectivity, with a detection limit of 68.85 nM.

[0033] 4. This invention is simple to operate, and the detection results are intuitive and easy to read, enabling rapid detection of analytes.

[0034] 5、 The application uses traditional Chinese medicine Huanglian as a carbon source, has good antibacterial performance, can improve the antibacterial performance around a wound, and enables the wound to be quickly healed. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Optimization of synthesis of RC-CDs (a) RC-CDs synthesized at different reaction temperatures. (b) RC-CDs synthesized at different reaction times, (c) Fluorescence spectra of RC-CDs synthesized at different amounts of p-phenylenediamine. (d) Fluorescence spectra of RC-CDs synthesized at different volume ratios of ethanol and Huanglian powder.

[0036] Figure 2 (a) TEM image of RC-CDs, and the inset is the corresponding HRTEM image. (b) Ex, Em fluorescence spectra and UV-Vis absorption spectra, and the insets are the images under natural light (left) and UV light (right), respectively. (c) Fluorescence emission spectra at 450-550 nm excitation wavelength, and (d) Three-dimensional contour plot.

[0037] Figure 3 (a) XPS full spectrum of RC-CDs, and (b) High-resolution XPS energy spectrum of C 1s, (c) N 1s and (d) O 1s.

[0038] Figure 4 (a) Fluorescence spectra of RC-CDs mixed with different kinds of metal ions, and (b) corresponding images under UV light. (c) Fluorescence intensity ratio I / I0of RC-CDs under different light irradiation times.

[0039] Figure 5 (a) Fluorescence spectra of RC-CDs mixed with different concentrations of Cu 2+ (b) Linear relationship between the fluorescence intensity ratio of RC-CDs and the concentration of Cu 2+ (0.05-20 μM).

[0040] Figure 6 Schematic diagram of multi-element detection of RC-CDs fluorescence probe.

[0041] Figure 7 (a) RC-CDs +Cu 2+ test paper detection flowchart. (b) Sensing selectivity of RC-CDs-based test paper and smartphone-assisted sensing platform. (c) Detection of Cu 2+ concentration range (0.3-400 μM). (d) Linear regression curve fitting between B / R and Cu 2+ horizontal concentration, and the detection limit is 68.85 nM.

[0042] Figure 8 Photos of E. coli colonies treated with control and different concentrations of Coptis powder and RC-CDs.

[0043] Figure 9 Confocal microscope images of RC-CDs treated with MCF-7 cells.

[0044] Figure 10 ICR name is derived from the Institute of Cancer Research, which represents the therapeutic effect of mouse acute wound closure. (a) Schematic diagram of ICR mouse experimental scheme. (b) Representative photos of wound healing process of mice treated with control group, pure hydrogel, SA@RC-CDs (3wt%) gel at 0, 3, 6, 9 and 12 days. (c) Wound traces of each treatment within 12 days. (d) Statistical analysis of wound closure rate (n=5). (e) Hematoxylin-eosin staining (H&E) and Masson staining of acute wound tissue at 12 days. (f) Quantification of skin thickness (n=3). (g) Statistical analysis of collagen deposition (n=3). Data are expressed as mean ± standard error of the mean. Statistical significance was calculated by one-way ANOVA. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). DETAILED DESCRIPTION

[0045] Example 1 Preparation of RC-CDs with orange emission

[0046] RC-CDs were prepared by solvothermal method. First, the flaky Coptis from Guangxi was crushed into powder with a cell wall crusher and sieved with a 200-mesh sieve. 0.108 g of p-phenylenediamine was dispersed in 9 mL of ethanol with 0.4 g of Coptis powder, and then the mixture was transferred to a 50 mL high-pressure reaction kettle. After heating at 220 °C for 6 h and cooling to room temperature, a blood-red solution containing RC-CDs was obtained. After purification by 0.22 μm microfiltration membrane and dialysis bag with a molecular weight cutoff of 1000 Da, RC-CDs were obtained. The fluorescence intensity of carbon dots synthesized under different conditions was tested by fluorescence spectrum (Figure 1), and the optimal synthesis conditions were as follows: the mass of p-phenylenediamine was 0.108 g, the Coptis powder was 0.4 g, the ethanol was 9 mL, the solvothermal reaction temperature was 220 °C, and the reaction time was 6 h. The product obtained was RC-CDs prepared under the optimized conditions.

[0047] Example 2 Preparation of RC-CDs with orange emission

[0048] The same solvothermal method was used to prepare RC-CDs, and other conditions were kept constant. When the ratio of Coptidis Rhizoma powder to ethanol solvent was adjusted to 0.5:9.5, only weak fluorescence could be obtained from the solution. When the ratio of Coptidis Rhizoma powder to ethanol solvent was adjusted to 2:8, almost no sample with fluorescence emission could be obtained. As shown in FIG. 1d, only when 0.4 g of Coptidis Rhizoma powder was dispersed in 9 mL of ethanol, RC-CDs with strong fluorescence emission could be obtained, which could be further used as a fluorescence probe and antibacterial material. Figure 1

[0049] Example 3 Preparation of RC-CDs with orange emission

[0050] The same solvothermal method was used to prepare RC-CDs, and other conditions were kept constant. When the reaction temperature was set to 140 °C, the product obtained had almost no fluorescence. When the temperature was increased to 160 °C, only weak fluorescence was obtained. Until the temperature was increased to 180 °C, the fluorescence of the product was only weak, and there was no obvious enhancement. When the temperature was increased to 200 °C, the fluorescence intensity was greatly enhanced, and reached the highest intensity at 220 °C. When the temperature was further increased to 240 °C, the fluorescence intensity decreased again. Therefore, the RC-CDs synthesized at 220 °C were the best conditions.

[0051] Example 4 Preparation of RC-CDs with orange emission

[0052] The same solvothermal method was used to prepare RC-CDs, and other conditions were kept constant. Different amounts of p-phenylenediamine (0.3 mM-1.3 mM) were used to adjust the synthesis of RC-CDs, from Figure 1 As can be seen from FIG. 1c, when the amount of p-phenylenediamine was 0.3 mM, the fluorescence intensity of the obtained RC-CDs was very low. When the amount of p-phenylenediamine was gradually increased, the fluorescence intensity of the RC-CDs gradually increased. When the amount of p-phenylenediamine was 1.0 mM, the obtained RC-CDs were the strongest fluorescent product. When the amount of p-phenylenediamine was further increased to 1.3 mM, the fluorescence began to decrease. Therefore, the product was the RC-CDs prepared under the optimized conditions, and all the conditions were verified by experiments, not by prediction.

[0053] Example 5 Characterization of RC-CDs with orange emission

[0054] (1) Transmission electron microscopy (TEM)

[0055] In order to characterize the size and morphology of RC-CDs, TEM characterization was performed. As can be seen from FIG. 2a, the morphology of the synthesized RC-CDs was spherical, and the average diameter was about 2.9 ± 0.32 nm (FIG. 2b). Figure 2 a, the morphology of the synthesized RC-CDs was spherical, and the average diameter was about 2.9 ± 0.32 nm (FIG. 2b). Figure 2 ​a inset). HRTEM images of RC-CDs further show (Fig. 1 a inset) the presence of lattice fringes of 0.21 nm, consistent with the (100) plane of graphite. The above results demonstrate the successful synthesis of RC-CDs. Figure 2 a inset), the presence of lattice fringes of 0.21 nm, consistent with the (100) plane of graphite. The above results demonstrate the successful synthesis of RC-CDs.

[0056] (2) UV-Vis absorption spectrum

[0057] The UV-Vis absorption spectrum of RC-CDs (Fig. 2c red part), an intense absorption peak at 290 nm was observed, which was attributed to the π - π* transition of aromatic groups.

[0058] (3) Fluorescence spectrum

[0059] From the fluorescence excitation and emission spectra of RC-CDs, the strongest emission peak was observed at 606 nm under the optimal excitation wavelength of 490 nm, and the position of the fluorescence emission peak did not change with the change of excitation wavelength, indicating that RC-CDs had no excitation dependence, which indicated that the size distribution and surface state distribution of carbon dots were uniform (Fig. 2c). In addition, the three-dimensional (3D) mapping spectrum showed that RC-CDs showed a robust red photoluminescence center (Fig. 2d). The measurement results of XPS revealed the electronic state of RC-CDs. The three main peaks were located at 284.80 eV, 399.16 eV, 532.42 eV (Fig. 2e) Figure 3 a) were divided into C1s, N 1s, O 1s, respectively, in which C (77.1 %), N (16.2 %), O (6.7 %). High-resolution C 1s (Fig. 2e a) Figure 3 b) had four peaks at 284.80 eV, 285.76 eV, 287.42 eV and 289.36 eV, corresponding to C-C / C=C, C-O, C=O and -COOH groups, respectively, indicating the distribution of abundant carbon-based groups. The N 1s spectrum of RC-CDs (Fig. 2e c) Figure 3 c) showed two types of nitrogen, pyrrolic nitrogen at 399.16 eV and graphitic nitrogen at 400.42 eV, and the O 1s spectrum (Fig. 2e d) Figure 3 d) three peaks at 531.25 eV, 532.42 eV and 533.42 eV, were attributed to C=O, C-O-C and C-OH bonds, respectively. Therefore, the above data verified the formation of a rich surface functional group modification, including C=O, -NH2 and -OH, -COOH, which provided effective coordination. The surface of these RC-CDs had abundant hydroxyl, carboxyl, amino and amide hydrophilic functional groups, which made them have good water solubility.

[0060] The above optical properties and morphology characterization can prove the successful preparation of RC-CDs, and have excellent optical properties.

[0061] Example 6: Direct detection of Cu using RC-CDs as fluorescent probes 2+

[0062] Using RC-CDs to detect Cu 2+ The synthesized RC-CDs were diluted 50 times and used as a fluorescent detection probe. Then, an equal volume of the sample solution to be tested was added, and the fluorescence change was observed under a UV lamp, as shown in Figure 4b. When Cu... 2+ In the presence of Cu, the fluorescence of the RC-CDs probe solution is significantly quenched. This is observed by measuring the fluorescence spectrum of the sample and noting the decrease in RC-CDs fluorescence intensity (Figure 4a). 2+ At that time, the fluorescence spectral intensity of the solution was related to Cu. 2+ The ion concentration (0.05–20 μM) showed a reliable linear dependence, allowing for the quantitative determination of Cu. 2+ Concentration, and when Cu in the solution 2+ At higher concentrations (≥100 μM), RC-CDs fluorescent probes exhibit complete quenching, achieving control over Cu. 2+ The detection (Figure 4b).

[0063] (1) Detection of Cu by fluorescence method 2+ Selectivity and anti-interference assessment

[0064] After analyzing the effect of ethanol on fluorescence, RC-CDs diluted 30 times with ethanol were used as probes for subsequent heavy metal detection studies. To discuss the effect of RC-CDs on Cu... 2+ The specificity of Cu ions, for equal amounts of ions 2+ Hg 2+ Ag + Fe 2+ Fe 3+ Co 2+ Ca 2+ Cr 3+ Cd 2+ Ni 2+ Mn 2+ Mg 2+ Ba 2+ Zn 2+ Pb 2+ The fluorescence of RC-CDs was measured and analyzed at 100 μM. 2+ Significant quenching ( Figure 4 a) While the fluorescence changes of other fluorescence sources under the same conditions are negligible, this indicates that RC-CDs have a significant effect on Cu. 2+ The detection exhibits good specificity. The corresponding image under ultraviolet light ( Figure 4b) Shows orange fluorescence by Cu 2+ Significant quenching further demonstrates its good selectivity. To clearly assess the selectivity, we analyzed and compared the I / I0 ratio for all samples (I0 and I represent the fluorescence intensity of missing and present ions in RC-CDs, respectively). Figure 3 As shown in c, with 100 μM Cu 2+ The I / I0 ratio of the mixed RC-CDs decreased significantly, consistent with the results of the fluorescence histogram (Figure 4c). The relative ratio (I / I0) and anti-interference data indicated that the signal response of RC-CDs to anions was negligible. These results confirm that RC-CDs are effective against Cu... 2+ High selectivity and anti-interference performance.

[0065] (2) Detection of Cu by fluorescence method 2+ Sensitivity assessment

[0066] RC-CDs were used as fluorescent probes to detect Cu. 2+ At that time, Cu 2+ The concentrations of the solution samples ranged from 0.05 to 20 µM, and the fluorescence spectra of each sample were measured to verify the probe's sensitivity. The fluorescence spectra show that as Cu... 2+ As the concentration increases, the fluorescence intensity gradually decreases (Figure 5a). The fluorescence intensity is related to Cu. 2+ The fluorescence intensity of the mixed solution is inversely proportional to the concentration, as can be observed in the corresponding experimental phenomena images; the fluorescence intensity increases with Cu concentration. 2+ The fluorescence intensity ratio (I / I0) of RC-CDs decreases with increasing concentration and is related to Cu. 2+ There was a good linear relationship between the concentrations (0.05~20 μM) (Figure 5b), and the detection limit was calculated to be 68.85 nM.

[0067] (3) Cu in actual water samples 2+ Recovery rate analysis

[0068] To further evaluate the detection of Cu in real samples 2+ To ensure accuracy and reliability, recovery rate tests were conducted on actual water samples (tap water, Changbai Mountain water, and Yulong Snow Mountain snowmelt) and food samples (fish and shrimp). Typically, due to various complex interferences in real-world environments, matrix effects should be considered in actual sample testing, as matrix compounds can interfere with the accurate quantification and identification of target species. Removing or minimizing the presence of matrix compounds is crucial to ensure reliable and accurate analysis. In this study, since nitric acid was used as the solvent in the extraction process, the nitric acid effect in the detection can be ignored, and the recovery rate was determined in relation to Cu. 2+ The solutions were identical. As shown in Table 1, the RC-CDs fluorescent probes detected Cu in real water samples.2+ The recoveries of Cu2+ were in the range of 96.68%~109.34% with RSD values less than 4.71% (Table 1). RC-CDs fluorescent probe in food Cu2+ 2+ The recoveries of Cu2+ were in the range of 98.06%~102.85% with RSD values less than 3.55% (Table 1). The detection results showed that the fluorescent analysis method had good detection performance, and could be widely applied to the rapid analysis of Cu2+ 2+ content in actual samples, and had high accuracy and reliability, further proving that RC-CDs probe had good precision and environmental tolerance. 2+

[0069] .

[0070] Example 7 Broad-spectrum antibacterial performance of RC-CDs

[0071] Based on the unique pharmacological activity of Coptis precursor, the antibacterial performance of RC-CDs was systematically evaluated, and a carbon dot-based antibacterial gel was developed for wound treatment. Escherichia coli and Staphylococcus aureus were selected as model strains, and were incubated in Luria-Bertani broth (LB) liquid medium at 37 ℃ with constant shaking at 200 rpm for 3 hours. 100 μL of bacterial suspension was inoculated into 5 mL of fresh culture medium, and 100 μL of RC-CDs solution with a concentration gradient of 0~300 μg / mL was added, and the shaking culture was continued for 3 hours. Then, the culture was serially diluted to a final concentration of 10 6 CFU / mL, 100 μL was uniformly coated on the surface of the agar plate, and incubated in a 37 ℃ constant temperature incubator for 16 hours. The relative bacterial survival rate was determined by plate colony counting method, and all experiments were set up in triplicate. The experimental results showed that RC-CDs exhibited significant growth inhibition effect on both gram-negative and gram-positive bacteria, and had significant difference compared with the control group without adding RC-CDs (P<0.001), at a concentration of 250 μg / mL, the antibacterial effect of RC-CDs on Escherichia coli and Staphylococcus aureus was 89% and 97% respectively, in comparison, the inhibition rate of Coptis extract on Escherichia coli was significantly lower at the same concentration, the inhibition rate was 34%, and the inhibition rate on Staphylococcus aureus was 31%, in general, RC-CDs was an excellent antibacterial agent. Figure 8

[0072] Example 8 Cell imaging of RC-CDs ​​

[0073] MCF-7 cells, a breast cancer cell line, were used to discern the difference in perception of RC-CDs between normal and cancer cells. To examine the cellular uptake of RC-CDs, cells were incubated with cell culture medium containing RC-CDs for 2 h. After that, cells were washed with DPBS for 3 times and fresh cell culture medium was added for observation. The results showed that all naked cells showed minimal fluorescence, as shown in Figure 9. Subsequently, an attempt was made to enhance cellular uptake by modifying the cells with RC-CDs. Compared with the fluorescence exhibited by naked RC-CDs, RC-CDs-treated cells showed a significant enhancement in the green channel under UV (465-495 nm) illumination (Figure 9). This fluorescence can be detected by differential interference contrast (DIC) fluorescence microscopy.

[0074] Example 9 Preparation of hydrogel dressing

[0075] A 3% wt solution of RC-CDs was first stirred with a 1.0% wt solution of sodium alginate using a magnetic stirrer for 6 h. After mixing well, it was crosslinked with a 3% wt solution of calcium ions to form SA@RC-CDs. When the two solutions were mixed, a gel was quickly formed, which can be used as a dressing for treatment on wounds.

[0076] Example 10 Study on skin wound healing performance of hydrogel dressing after using in mouse antibacterial model in vivo

[0077] Initially, 5-6 weeks old female ICR mice (24-26 g) were anesthetized with isoflurane (0.7 mL / 100 g body weight) by intraperitoneal injection of the following drugs. The back hair of the mice was carefully shaved. An acute wound model of 0.8 cm x 0.8 cm was formed on the back of each rat by surgery. All wounds were inoculated with 100 uL of 10 8 CFU mL -1Staphylococcus aureus cell suspension 24 hours. Then the mice were randomly divided into five groups. The experimental setup included a control group without intervention and two treatment groups, each with 5 mice. Each treatment group used a different hydrogel formulation: control, pure hydrogel, SA@RC-CDs (3% wt) hydrogel. These hydrogels were sprayed on the surface of the mouse wounds and a solution of calcium ions (3% wt) was sprayed, which due to their cross-linking effect, they were applied directly to the wound in situ forming a gel dressing at the mouse wound. Subsequently, these mice were kept in separate cages and received daily care. The wound healing process was monitored and recorded in detail on days 0, 3, 6, 9, and 12. After treatment, the mice were anesthetized and euthanized. The wound tissue was fixed with 4% paraformaldehyde for histopathological examination. After 90% wound healing, the entire skin layer was taken for H&E and Masson staining. To conduct a comprehensive experiment, each group was excised for analysis.

[0078] Figure 10 In (b, c), the wound closure rate of SA@RC-CDs was accelerated by 20.46% compared with the control group. In particular, the mice in the SA@RC-CDs treatment group had the highest closure rate of 96% on day 12, while the control group only reached a closure rate of 75% on day 12 (Figure 10d). In addition, we performed H&E and Masson staining maps (Figure 10e) to evaluate the SA@RC-CDs acute wound healing gel. However, the thickness of the skin in the SA@RC-CDs gel group was lower than that in the other gel treatment groups, indicating that there was a large amount of proliferative granulation tissue in the gel treatment groups, which gradually transformed into mature collagen deposition in the SA@RC-CDs gel group. Therefore, the SA@RC-CDs hydrogel showed excellent performance in wound healing applications, and the SA@RC-CDs gel did not significantly enhance the thickness of the collagen deposition in the mouse skin, without dressing (control group), pure hydrogel (blank group), and SA@RC-CDs treatment group compared with the control group (Figure 10f). After 12 days of treatment with no dressing (control group), pure hydrogel (blank group), SA@RC-CDs gel, inflammatory cell infiltration was reduced, and fibroblasts in the wound area increased, indicating that healing entered the proliferation stage. Notably, after SA@RC-CDs gel treatment, there was almost no inflammatory cell infiltration, but a large number of fibroblasts were observed. In addition, using Masson trichrome staining to quantitatively determine collagen deposition, wounds treated with SA@RC-CDs gel also showed significantly higher collagen deposition than other treatments (Figure 10g). Figure 10 g). The above results show that the SA@RC-CDs gel significantly reduces tissue inflammation, promotes epithelial cell growth, promotes collagen deposition, and accelerates acute wound healing, thus demonstrating the good antibacterial performance of carbon dot-based gels and good applicable prospects.

Claims

1. A traditional Chinese medicine derived carbon dot-based fluorescent probe RC-CDs, characterized in that: is prepared by a solvothermal method using p-phenylenediamine and Coptis powder as precursors. 2.The traditional Chinese medicine derived carbon dots-based fluorescent probe RC-CDs of claim 1, characterized in that: The solvent method comprises: 1) dispersing 0.0800-0.1404 g of p-phenylenediamine and 0.3-0.6 g of Coptis powder in 5-10 mL of ethanol, transferring into a high-pressure reaction kettle, heating at 140-240 ℃ for 2-10 h; 2) cooling to room temperature, and obtaining RC-CDs after purification by dialysis. 3.The traditional Chinese medicine derived carbon dots-based fluorescent probe RC-CDs of claim 2, characterized in that: In step 1), the p-phenylenediamine is 0.1080-0.1104 g, the Coptis powder is 0.45-0.55 g, and the ethanol is 8-10 mL. 4.The traditional Chinese medicine derived carbon dots-based fluorescent probe RC-CDs of claim 3, characterized in that: In step 1), the p-phenylenediamine is 0.1080 g, the Coptis powder is 0.4 g, and the ethanol is 9 mL.

5. The traditional Chinese medicine derived carbon dot-based fluorescent probe RC-CDs according to claim 1, 2, 3 or 4, characterized in that: In step 1), the temperature is 200-240 ℃, and the heating is 5-7 h. 6.The traditional Chinese medicine derived carbon dots-based fluorescent probe RC-CDs of claim 5, characterized in that: In step 1), the purification by dialysis is filtration through a 0.22 μm microporous filter and purification by dialysis through a dialysis bag with a molecular weight cutoff of 1000 Da.

7. Detection of Cu by carbon-dot-based fluorescent probes derived from traditional Chinese medicine (RC-CDs) 2+ The method includes: 1) Standard curve drawing: take RC-CDs solution and mix with equal volume of ultrapure water, determine the fluorescence intensity value I0 of the blank group, take RC-CDs solution and mix with different known concentrations of Cu 2+ After mixing, the fluorescence intensity value I at 606 nm was measured, and the corresponding relative fluorescence intensity value I / I0 was calculated, and the standard curve of relative fluorescence intensity value I / I0 and Cu 2+ concentration was established; 2) Fluorescence detection of Cu 2+ : After mixing the RC-CDs solution with the sample solution to be tested, the fluorescence intensity value I is measured, and the relative fluorescence intensity value I / I0 is calculated. According to the above standard curve, the concentration of Cu 2+ in the sample solution to be tested is obtained. The RC-CDs are the RC-CDs of claim 1.

8. The method of claim 7, wherein the traditional Chinese medicine derived carbon dot-based fluorescent probe RC-CDs is used for detecting Cu 2+ characterized in that: Cu in the sample solution to be tested 2+ The concentration is 0.05~20μM; the mixing time between the RC-CDs solution and the sample solution is 5min; all reactions are carried out at room temperature; the fluorescence detection conditions are: excitation wavelength 390nm, excitation slit and emission slit width 3nm, and recording the emission intensity of RC-CDs at 606nm.

9. A hydrogel dressing of RC-CDs, which is prepared by the following method: First, 1-5% wt of RC-CDs solution and 0.5-2.0% wt of sodium alginate solution are stirred with a magnetic stirrer for 3-6 h, and then crosslinked with 1-5% wt of calcium ion solution to form SA@RC-CDs.

10. Use of the hydrogel dressing of RC-CDs of claim 9 in resisting wound infection and rapid healing.

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