All chemicals and reagents employed in this study were of analytical grade and compliant for experimental use. Yongchuan Xiuya tea was procured from a local supermarket in Chongqing, China. Deionized water was used throughout the experiments to ensure consistency and purity. Horseradish peroxidase (HRP) was obtained from Sinopharm Chemical Reagent Co., Ltd. Phosphate buffer solution (PBS) was sourced from Merck (Darmstadt, Germany). Hydroxypropyl acrylate (HPA) and 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT) were supplied by Aladdin Co., Ltd. All materials were used as received without further purification.

The CDs were synthesized via a one-step hydrothermal method. Briefly, 0.20 g of Yongchuan Xiuya tea was dispersed in 15 mL of deionized water under stirring to ensure homogeneity. The mixture was then transferred into a 25 mL Teflon-lined autoclave and subjected to hydrothermal treatment at 200 °C for 10 h. After the reaction, the autoclave was allowed to cool to room temperature naturally. The resulting suspension was centrifuged at 8,000 rpm for 10 min to remove large particles. The supernatant was further filtered through a 0.22 μm microporous membrane to obtain a clear solution. To purify the CDs, the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 500 Da for 12 h to remove any unreacted precursors or small impurities. Finally, the purified CDs solution was freeze-dried to yield a solid powder, which was stored at 4 °C for subsequent characterization and applications.

Transmission electron microscopy (TEM) images were acquired using a JEOL 2100F instrument (Tokyo, Japan) to analyze the morphology and size distribution of the synthesized CDs. Fourier-transform infrared (FT-IR) spectra were recorded on a Nicolet iS5 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) to identify the functional groups present on the surface of the CDs. For detailed elemental composition analysis, X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB Xi + spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The minimum inhibitory concentration (MIC) assay was conducted using a microplate reader from Awareness Technology (Florida, USA), with absorbance measurements taken at 600 nm. Additionally, the zeta potentials of both the CDs and bacterial cells were determined using a Nano Brook 90Plus Zeta Potential Analyzer (Malvern Instruments, UK) to assess surface charge characteristics. A relative quantum yield (QY) of CDs was determined with quinine sulfate (dispersed in 0.1 M H₂SO₄, QY = 54%) as a reference.

E. coli and S. aureus were cultured in Luria-Bertani (LB) broth medium under standard conditions. Each bacterial strain was inoculated into 5 mL of LB medium and incubated at 37 °C for 12 h with continuous shaking at 180 rpm to ensure optimal growth. The bacterial concentration was monitored by measuring the optical density at 600 nm (OD₆₀₀), and cultures were maintained within an OD600 range of 0.6–0.8 to ensure consistent growth phases. The bacterial suspensions were then diluted to a final concentration of 1.5 × 10⁷ colony-forming units per milliliter (CFU/mL). Following activation, the bacterial suspensions were stored at 4 °C in a refrigerator for subsequent experimental use.

The minimum inhibitory concentration (MIC) of CDs against E. coli and S. aureus was determined using the broth microdilution method in 96-well plates. Briefly, 100 μL of bacterial suspension (1.5 × 10⁷ CFU/mL) was mixed with 100 μL of varying concentrations of CDs in the wells. The plates were then incubated at 37 °C for 12 h. After incubation, bacterial growth was quantified by measuring the optical density at 600 nm (OD₆₀₀). To further assess cell viability, 10 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well, and the plates were incubated for an additional 20 min. All experiments were performed in triplicate to ensure reproducibility.

To evaluate the potential development of bacterial resistance, the initial MIC values of CDs against E. coli and S. aureus were determined. Bacteria from the 1/2 MIC concentration were then diluted to 1 × 10⁷ CFU/mL and subjected to a 24-h incubation at 37 °C. This process was repeated daily for 14 consecutive passages, with the MIC values of the CDs-bacteria mixtures recorded at each passage. This longitudinal study aimed to monitor any changes in bacterial susceptibility to CDs over time, providing insights into the potential for resistance development.

Zeta potential measurements were performed to evaluate the surface charge characteristics of the CDs and their interactions with bacterial cells. A diluted CDs solution and a mixture of the diluted CDs solution with activated bacterial suspension were analyzed using a zeta potential analyzer. This analysis provided insights into the electrostatic interactions between the CDs and bacterial surfaces, which are critical for understanding their antibacterial mechanisms.

Antioxidant activity was evaluated by DPPH free radical scavenging capacity, and ABTS free radical scavenging capacity comparing with the equivalent antioxidant capacity (TEAC) of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and the ferric reducing antioxidant power (FRAP).

Evaluation of Synthetic CDs’ Protective Effects Against H₂O₂-Induced Cell Damage. The cytoprotective effects of synthetic CDs against H₂O₂-induced oxidative stress were analyzed using the CCK-8 assay. First, the cytotoxicity of synthetic peptides on HepG2 cells was tested. Cells were seeded in a 96-well plate (1 × 10⁴ cells/well) and cultured for 24 h at 37 °C. After replacing the medium with fresh medium containing varying peptide concentrations, the cells were incubated for an additional 12 h. The medium was then replaced with 100 μL of fresh medium containing 10 μL of CCK-8 reagent, followed by a 2 h incubation. Absorbance was measured at 450 nm. For oxidative stress protection assessment, HepG2 cells were pretreated with peptides for 12 h before exposure to 175 μmol/L H₂O₂ for 2 h. All experiments were conducted in triplicate, and cell viability was calculated as a percentage relative to the control group.

HepG2 cells from the control, model and experimental groups were collected, lysed and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatants were collected and analyzed using commercial assay kits following the manufacturer’s instructions. ROS levels were quantified using a fluorescence-based assay.

The morphology and structural characteristics of the synthesized CDs were analyzed using transmission electron microscopy (TEM). As shown in Figure 2a, the CDs exhibited a particle size distribution ranging from 0.21 to 7.38 nm, with an average diameter of approximately 4.17 nm. High-resolution TEM (HRTEM) revealed a well-defined lattice spacing of 0.22 nm, indicating the crystalline nature of the CDs.

To further investigate the surface composition and functional groups of the CDs, Fourier-transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were employed. The FT-IR spectrum (Figure 2b) displayed characteristic peaks at 3,390 cm^-1 and 3,160 cm^-1, corresponding to the stretching vibrations of N–H and O–H groups, respectively. A peak at 1,620 cm^-1 confirmed the presence of C=O/C=C bonds, while the peak at 1,400 cm^-1 was attributed to C–N/O–H vibrations. Additionally, a peak at 775 cm^-1 was associated with C–H stretching.

XPS analysis (Figure 2c) revealed the elemental composition of the CDs, with C 1s (45.56%), O 1s (38.38%), and N 1s (16.12%) being the primary components. The high-resolution C 1s spectrum (Figure 2d) showed peaks at 283.8 eV and 286 eV, corresponding to C=O, C–N, and C=C bonds. The N 1s spectrum (Figure 2e) exhibited peaks at 397 eV, 399.8 eV, and 400 eV, assigned to R–NH₂, C–N–C, and N–H groups, respectively. The O 1s spectrum (Figure 2f) displayed peaks at 531.2 eV, 532.5 eV, and 534.7 eV, which were attributed to C=O, C–OH, and C–O bonds, respectively. Through elemental analysis, the elemental composition of this carbon dot was determined as follows: C: 44.73%, H: 6.12%, N: 15.35%.

Taking quinoline sulfate as a reference (Jiang et al., 2015), the quantum yield was 18.3%. These results collectively provide a comprehensive understanding of the structural and compositional properties of the synthesized CDs, highlighting their potential for various applications.

The minimum inhibitory concentration (MIC) of the CDs was determined using a 96-well cell culture plate and the microdilution method. Bacterial suspensions of E. coli and S. aureus were treated with varying concentrations of CDs and incubated for 12 h under consistent conditions. Bacterial viability was assessed by measuring the optical density at 600 nm (OD₆₀₀) and comparing the values across different treatment groups.

As illustrated in Figures 3a,b, the viability of both E. coli and S. aureus decreased progressively with increasing concentrations of CDs, demonstrating a dose-dependent antibacterial effect. The MIC values, defined as the lowest concentration of CDs that completely inhibited bacterial growth, were determined to be 0.62 mg/mL for E. coli and 0.85 mg/mL for S. aureus. These results highlight the potent antibacterial activity of the CDs against both Gram-negative and Gram-positive bacteria, underscoring their potential as effective antimicrobial agents. However, the extract of Yongchuan Xiuya did not show excellent antibacterial effects. This indicates that after converting Yongchuan Xiuya into carbon dots, the antibacterial activity was enhanced.

As summarized in Table 1, numerous studies have successfully synthesized fluorescent probes capable of inhibiting both Gram-positive and Gram-negative pathogens at low concentrations. Notably, carbon dots exhibit significantly enhanced antibacterial activity compared to other tested compounds.

The time-dependent antibacterial activity of the CDs was evaluated by monitoring the optical density at 600 nm (OD600) over time, as shown in Figures 4a,b. The results demonstrated that the antibacterial efficacy of the CDs was strongly concentration-dependent, with higher concentrations of CDs leading to a more significant reduction in bacterial growth. Notably, when treated with CDs at a concentration equivalent to 1× the minimum inhibitory concentration (MIC), the growth of both E. coli and S. aureus was completely inhibited within 24 h. This finding underscores the potent and rapid antibacterial action of the CDs, highlighting their potential as effective agents for controlling bacterial infections.

To further investigate the antibacterial mechanism of the CDs, the zeta potential of the CDs was examined. As shown in Figure 5, the zeta potentials of both the CDs and the bacterial cells were measured. The zeta potentials of E. coli and S. aureus were determined to be −19.63 mV and −26.76 mV, respectively, reflecting the negatively charged nature of their cell walls. In contrast, the CDs exhibited a positive zeta potential because of the presence of cationic groups such as amino groups, facilitating their electrostatic adsorption onto the negatively charged bacterial surfaces. This interaction is a critical step in the antibacterial activity of the CDs, as it enables close contact between the CDs and the bacterial cells.

The antioxidant activity of the CDs was evaluated using three different in vitro methods, DPPH (2,2-diphenyl-1picrylhydrazyl) radical scavenging and ferric reducing antioxidant power (FRAP) assay.

The objective of this experiment was to evaluate the free radical scavenging activity of the CDs using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The results demonstrated that the CDs possess significant antioxidant potential, with a DPPH radical scavenging activity of 91.32% ± 0.31%. This high scavenging efficiency highlights the strong ability of the CDs to neutralize free radicals, underscoring their potential as effective antioxidant agents. These findings suggest that the CDs could be valuable in applications requiring oxidative stress mitigation, such as in biomedical or environmental contexts.

In the FRAP assay, an antioxidant reduces Fe³⁺ to Fe²⁺ by donating electrons. The reducing power of the synthesized compounds measured using the FRAP test revealed that CDs has the best-reducing capacity. The results demonstrated that the CDs possess significant antioxidant potential, with FRAP radical scavenging activity of 1,320% ± 32%.

HepG2 cells, derived from human hepatocellular carcinoma, are widely used for antioxidant assay of peptides in vitro. Therefore, this study employed the HepG2 cell model to evaluate the antioxidant activity of CDs. Initially, we assessed the cytotoxicity of the CDs at concentrations of 125, 250, 500, and 1,000 μg/mL. As shown in Figure 6a, the cell viability remained above 90% at all tested concentrations, indicating that the CDs exhibited no significant cytotoxicity towards HepG2 cells at these concentrations. Therefore, the concentrations used in this study are considered safe and reasonable. Subsequently, HepG2 cells were pre-incubated with the CDs and H₂O₂ was used to simulate oxidative stress (Figure 6b). The results showed that exposure to H₂O₂ significantly decreased cell viability (p < 0.05) compared to the control group, indicating that oxidative stress induced cellular damage. In contrast, pre-incubation with CDs significantly improved cell viability in a concentration-dependent manner, demonstrating their protective effect against H₂O₂-induced oxidative damage.

The antioxidant effect of carbon dots can last for more than 3 days and can be explained by the hydrogen donor groups present on their surface (such as hydroxyl (-OH) and carboxyl (-COOH)). The conjugated sp² carbon structure inside the carbon dots enables the delocalization of electrons, which is conducive to the formation of free radical intermediates. The interaction between the free radicals and the functional groups on the carbon dots leads to the formation of stable free radical adducts, effectively neutralizing reactive oxygen species (ROS). Due to the stability of the sp² carbon core and the diversity of the surface functional groups, carbon dots can maintain their antioxidant activity for a long time.