The E. gracilis powder was purchased from Yunnan Baoshan Zeyuan Algal Health Technology Co., Ltd. Deionized (DI) water was prepared in the laboratory. A poly (tetrafluoroethylene) Teflon-lined autoclave was purchased from Beijing Kemet Technology Co., Ltd.

To synthesize E-CQDs, 4.8 g of dry E. gracilis powder was thoroughly mixed with 60 mL of DI water and stirred for 10 min. The resulting was then transferred to 100 mL Teflon-lined autoclave and heated it at 220 °C for 2.5 h in an electrothermal constant temperature drying oven. After cooling the autoclave to room temperature, the solution was filtered through a sulfone filter membrane with a 0.45 µm pore size to remove any insoluble residues. The filtrate was subsequently freeze-dried to yield a brown powder. The CQDs obtained from this process were designated as E-CQDs.

To optimize the synthesis of E-CQDs, we systematically varied the ratios of E. gracilis powder (2%, 4%, 6%, 8%, 10%), reaction temperatures (140 °C, 160 °C, 180 °C, 200 °C, 220 °C), and reaction times (0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h). The resulting solutions were filtered, and the filtrates were subsequently diluted to a suitable concentration. The absorbance and fluorescence of the diluted E-CQDs were measured at an excitation wavelength of 380 nm, following the standardized procedure outlined by Dong et al. (2021). Specifically, the absorbance at 380 nm was meticulously adjusted to fall within the range of 0.06–0.10 to ensure precise and reliable measurements. Thereafter, the absorbance and relative fluorescence intensity were comprehensively analyzed using a Spark Multifunctional Enzyme Marker (Tecan A-5082, Made in Austria) to thoroughly evaluate the optical properties of the synthesized E-CQDs. Relative Fluorescence Units (RFU) was used to qualitatively asses the efficiency of the synthesis method.

Fluorescence emission spectroscopy was performed using an F-4700 spectrophotometer (Hitachi, Tokyo). The crystalline phase was characterized by X-ray diffraction (XRD) using an Ultima IV instrument (Rigaku Corp.). Ultraviolet-visible (UV-vis) absorption spectra were acquired using a LAMBDA 1050+ UV/Vis Spectrophotometer (PerkinElmer). Transmission electron microscopy (TEM) was performed at 200 kV using a JEM-F200 microscope (JEOL). TEM image was analyzed using ImageJ^® software version 1.54 m. Fourier transform infrared (FTIR) spectra were recorded within the range of 4,000–500 cm^-1 using a Nicolet iS10 FTIR spectrometer (Thermo Fisher Scientific), and X-ray photoelectron spectroscopy (XPS) was performed using an AXIS Supra + spectrometer (Shimadzu).

To assess the bioimaging potential of E-CQDs, Chlorella, a widely recognized single-celled algal model organism, was selected for fluorescence imaging assays. Previous work by Nam et al. (2019) has demonstrated the utility of Chlorella as an organism model for fluorescence imaging. Based on this foundation, the present study utilized E-CQDs to conduct fluorescence imaging of Chlorella, thereby assessing the viability of CQDs for bioimaging applications.

Two 1 mL tubes of Chlorella suspensions were prepared and labeled as tube A (control) and tube B (E-CQDs treated). A solution of 20 mg/L E-CQDs was added to tube B, while DI water was added to tube A as a negative control. Both tubes were incubated at 25 °C with continuous shaking at 180 rpm. After 24 h of incubation, the culture solution was removed by centrifugation at 10,000 rpm then resuspended in DI water. The fluorescence effect was observed under a fluorescence microscope (Leica Microsystems CMS GmbH) after 30 min of Uv irradiation.

All experimental data were processed using Microsoft Excel 2021 (Microsoft Corp., United States) for preliminary calculations and normalization. Statistical significance was evaluated by one-way analysis of variance (ANOVA) performed with IBM SPSS Statistics 27.0 (IBM Corp., United States) at a 95% confidence level (p < 0.05 considered significant). Graphical representations were generated using OriginPro 2021 (OriginLab Corp., United States) with error bars denoting standard deviations (n ≥ 3).

E-CQDs were synthesized using a direct hydrothermal method with E. gracilis powder as the raw material. The schematic illustration of the synthesis process was summarized in Figure 1a. The synthesis parameters of E-CQD including additional amount, reaction temperature, and reaction time were optimized.

The influence of E. gracilis powder addition ratio ranging from 2% to 10% on the fluorescence intensity of E-CQDs was studied. As shown in Figure 1b, with reacting at 200 °C for 2 h, the fluorescence intensity first increased and then decreased with increasing E. gracilis powder addition ratio. The maximum value of RFU is obtained at 8% E. gracilis powder addition, which was adopted for further experiments. The influences of incubation temperature ranging from 140 °C to 220 °C were also examined, and the results are shown in Figure 1c. As the reaction temperature increased, a concomitant increase in the RFU of the E-CQDs was observed. When the reaction temperature was set at 220 °C, the RFU of the E-CQDs reached their maximum value. When the reaction was conducted at 220 °C, the influence of different reaction time (0.5 h–2.5 h) on RFU was also investigated. As depicted in Figure 1d, with the increase of incubation time, RFU of E-CQDs also significantly increased. As the reaction time was 2.5 h, maximum RFU could be obtained. To achieve the highest RFU and ensure a stable signal, the optimized synthesis conditions were determined to be the addition of 8% E. gracilis powder, followed by a reaction at 220 °C for 2.5 h. All E-CQDs used for subsequent in-depth studies were synthesized under these optimized conditions. It’s reported that the aggregation and interactions of CQD species in solution could inhibit full potential of light emitters, which named aggregation-caused quenching (ACQ) effect (Adsetts et al., 2020). The complex of CQDs and phthalimide crystals (CQDs/PC) could prevent CQDs from touching directly by embedding the CQDs in phthalimide crystal matrix in situ, which effectively reduced the ACQ effect (Zheng et al., 2020). In this study, concentration-dependent sedimentation was observed in the E-CQDs stock solution while diluted E-CQDs solutions maintained colloidal stability with no observed aggregation. In terms of reducing ACQ effect, the E-CQDs embedded in situ within PC may be also suggested.

The CQDs prepared by the hydrothermal method were observed with TEM. Figure 2a shows that the E-CQDs exhibited a globular morphology, with a relatively uniform distribution of particles and absence of distinct lattice fringes. A histogram of the size distribution was derived by counting 100 particles, revealing that the diameter distribution of the E-CQDs ranged from 6.5 to 10.5 nm, with an average diameter of 9.2 nm. These results indicated that the materials can be classified as CQDs. The crystalline phase purity of E-CQDs was elucidated via XRD analysis. Figure 2b depicts the XRD crystalline profile of E-CQDs. There is a broad peak at 23.89 °, which is attributed to the partial ordering in the carbon dots. This ordering may arise from the covalent crosslinking of the skeletal structure during the polymerization process. (Huang et al., 2024; Miao et al., 2025). The FTIR spectrum was recorded to explore the surface functional groups of the E-CQDs. As illustrated in Figure 2c, the FTIR spectrum of the synthesized E-CQDs exhibits peaked at 3,238.9 cm⁻¹, which is linked to the stretching vibrations of O-H or N-H bonds. Meanwhile, the peak of E-CQDs at 2,964.87 cm⁻¹ may show the presence of the stretching vibrations of C-H (Bao et al., 2015; Qandeel et al., 2024). The 1,667.13 cm⁻¹ peak is attributed to the absorption bands of C=O. It may originate from the amid bond (-CONH -) or the -COOH group (Hua et al., 2017; Zhu et al., 2013). The peak at 1,454.94 cm⁻¹ corresponds to C-H stretching vibration and the peak at 1,118.71 cm⁻¹ corresponds to C-N stretching vibration (Li et al., 2024; Mmelesi et al., 2024).

XPS was employed to analyze the microstructures and elemental composition of the synthesized CQDs. As depicted in Figure 2d, the four main peaks in this spectrum include C 1s, N 1s, O 1s, and Si 2p, which can be seen at 284, 399, 531, and 101 eV binding energies, respectively. The elemental composition of E-CQDs was determined to be C (68.58%), O (18.03%), N (11.94%), and Si (1.44%). Figure 2e shows the C 1s spectrum of the E-CQDs. The C 1s XPS spectrum reveals multiple peaks at 284.8 eV, 285.9 eV, 286.5 eV, and 288 eV, corresponding to C-C/C=C, C-N, C-O, and C=O bonds (Fatima et al., 2024; Gawal and Golder, 2024), respectively. The two peaks of N 1s at 400 eV and 401.6 eV, as shown in Figure 2f, are attributed to the C-N and N-H bonds, respectively (Tao et al., 2018). XPS analysis revealed that the surface composition of E-CQDs aligns well with the findings from FTIR spectroscopy. It suggests that E-CQDs possess a high density of oxygen- and nitrogen-containing functional groups, including hydroxyl, carboxyl, and amine groups. These functional groups endow E-CQDs with excellent water solubility.

Figure 3 shows the optical properties of the E-CQD. As shown in Figure 3a, the original synthesized aqueous solution of E-CQDs (prepared from a mixture of 4.8 g of dry E. gracilis powder and 60 mL of DI water) was filtered, and initially appeared brownish-black. However, upon dilution by a factor of 100, the solution turned light yellow. Figure 3b shows an image of the E-CQDs samples under UV light irradiation. Upon exposure to 365 nm ultraviolet (UV) light, the sample exhibited bright blue fluorescence. Consistent with the literature (Zapata-Hernandez et al., 2024), the blue luminescence of E-CQDs is likely attributed to the presence of oxygen-containing functional groups. These groups create distinct sp² energy levels between the π and π* bands, as evidenced by the FTIR spectrum of the CQDs. Figure 3c depicts the E-CQD’s photoluminescence with excitation wavelength variations ranging from 300 to 400 nm. The most significant emission peak was observed at 416.8 nm when an excitation wavelength of 330 nm was used. The photoluminescence in E-CQDs may stem from the separation of sp² domains by sp³ domains. The formation of sp³ domains is supported by the presence of oxygen-containing functional groups. Additionally, the excitation-wavelength-dependent emission behavior could be attributed to oxygen functionalities that induce surface defects, acting as surface energy traps (Zapata-Hernandez et al., 2024). The E-CQD’s UV-Vis absorption spectrum (Figure 3d) evidenced a peak in the UV region at approximately 252 nm, with a tail extending into the visible range. The absorbance band at 252 nm is related to the π–π* transition of the aromatic C=C bonds (Hua et al., 2017).

Disk diffusion assays, MIC, MBC, together with image analysis, were employed to evaluate the antibacterial activity of E-CQDs against E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria) (Figure 4). Figures 4a,b illustrate the observed inhibition zone diameters for different concentrations (0, 5, 10, 20, 50, 100, 200 mg/mL) of E-CQDs against E. coli and S. aureus, respectively. When the concentration is below 50 mg/mL, E-CQDs display no antibacterial activity against E. coli. However, at concentrations of 50, 100, and 200 mg/mL, distinct inhibition zones are observed (Figure 4a). At a concentration of 200 mg/mL, E-CQDs showed an inhibition zone value of 7.5 mm. Differently, There is no significant antibacterial activity against S. aureus when the concentration is below 100 mg/mL (Figure 4b). The 200 mg/mL E-CQDs inhibited the S. aureus growth with inhibition zone of 9 mm. The results were consistent with those reported by Amoon et al. (2024) and Ma et al. (2020), both of whom demonstrated that bacterial growth could be inhibited by treatment with suitable concentrations of CQDs. Figures 4c,e show MIC of the E-CQDs against E. coli while Figures 4d,f depict MBC of the E-CQDs against S. aureus, using broth microdilution method. As the concentration of E-CQDs increased, the antibacterial effect became increasingly pronounced. MIC of the E-CQDs against E. coli and S. aureus were determined to be 100 mg/mL. At a concentration of 200 mg/mL, only a minimal number of colonies were observable on LB agar plates, indicating bactericidal activities exceeding 95% against E. coli and 90% against S. aureus. In terms of MBC, Both E. coli and S. aureusdid can not grow at a concentration of 250 mg/mL and above, indicating the value of MBC was 250. mg/mL.

Nitrogen-doped carbon quantum dots (N-CQDs) with excellent antifungal performance were synthesized using chitosan quaternary ammonium salt (HACC) as the raw material, with a minimum inhibitory concentration (MIC) of 1.8 mg/mL (Wang Y. et al., 2024). Phosphorus-doped CQDs were prepared by a simple hydrothermal method using valine as a carbon source, triethylamine as a nitrogen source, and phosphoric acid as a phosphorus source. Their MIC values decreased from 0.71 to 0.51 to 0.18 mg/mL on E. coli and S. aureus with increasing phosphorus content (Chai et al., 2022). N-CQDs derived from Hedyotis diffusa Willd. Exhibited significant antimicrobial activity against both S. aureus and E. coli, with MIC values of 0.055 mg/mL and 0.038 mg/mL, respectively (Pei et al., 2025). Additionally, novel CMC/CuO NPs/CQDs were effective against S. aureus and E. coli, with MIC values of 25 mg/mL and >50 mg/mL, respectively (Amoon et al., 2024). Compared to these CQDs, the concentration of E-CQDs (100 mg/L) required to inhibit bacterial growth is higher, likely due to the absence of amino acids (e.g., Arg/Lys), metal doping (e.g., zinc (Zn), and copper (Cu)), or nanocomposite formation in E-CQDs. Certainly, the antibacterial activity of E-CQDs can not be denied. MIC of E-CQDs is relatively high compared to other natural extracts. For instance, the MIC of E-CQDs is significantly lower than that of arum ethanolic extracts (MIC: 500 mg/mL) (Al-Daghistani et al., 2021) and Scorzonera mackmeliana extracts (MIC: 341.85 mg/mL) (Sweidan et al., 2020), indicating the potential of E-CQDs as an effective antibacterial agent. Future research may explore the doping of E-CQDs with compounds such as valine, triethylamine, phosphoric acid, Zn, Fe, and Cu to enhance their antibacterial capabilities.

It is reported that the bactericidal mechanism of CQDs can be characterized by the disruption of the bacterial membrane, leading to the leakage of cytoplasmic contents and ultimately culminating in cell apoptosis (Pant et al., 2023). SEM was used to image the bacteria in the presence of the E-CQDs (Figure 5). The morphology of E. coli and S. aureus changed significantly prior to and post the treatment with E-CQDs. In the absence of CQDs, the bacteria exhibited their typical sizes and morphologies: E. coli appears as rod-shaped bacteria (Figure 5a), whereas S. aureus has a spherical appearance and formed grape-like clusters (Figure 5c). Both types of bacteria presented smooth, intact surfaces. The cell membrane of the bacterial group treated with E-CQDs exhibited severe damage, with the surface becoming rough, deformed and significantly shrunken (Figures 5b,d). These changes may strongly suggest cell membrane damage (Bacellar et al., 2014). There was a hypothesis that S. aureus was less susceptible after treatment of CQDs than E. coli (Nie et al., 2020). Consistent with this supposition, intact S. aureus was still observable in Figure 5d while E. coli was seriously damaged (Figure 5b). It could be explained by the fact that E-CQDs may have a reduced ability to penetrate into the interior of S. aureus. Thus, the observed morphological changes in the bacteria may indicate alterations in the permeability of their membranes, which would affect the regulation of transmembrane transport and result in cell death.

CQDs with multicolor fluorescence emissions may show great potential in applications such as molecular imaging and in vivo molecular tracking. For instance, in a study conducted by Manjubaashini et al. (2024), CQDs were utilized for in vivo bioimaging in zebrafish to evaluate their potential for bioimaging applications. Moreover, it has been reported that unicellular algae, as autotrophic biosystems, uniquely possess multiple functions, including oxygen generation, dynamic motility, fluorescence imaging, and programmable biosynthesis (Wang et al., 2025). Chlorella, a widely utilized single-celled alga, is a model organism commonly employed in biological and biotechnological studies.

In this research, Chlorella pyrenoidosa was employed, and fluorescence imaging was performed using E-CQD, and the imaging effects of the E-CQDs under varied excitation irradiation towards C. pyrenoidosa cells were depicted in Figure 6. As shown in Figures 6a,b, there was no difference in the morphology of the cells under bright field conditions. No fluorescence of C. pyrenoidosa in the absence of E-CQDs was observed under the UV, blue light or green light illumination (Figure 6c,e,g). Obviously, Upon treatment with E-CQDs, Chlorella exhibited blue fluorescence under UV excitation within the wavelength range of 327–383 nm (Figure 6d) and green fluorescence under blue excitation within the range of 460–500 nm (Figure 6f). Similar to C. pyrenoidosa in the absence of E-CQDs (Figure 6g), no fluorescence was observed under green excitation within the range of 541–551 nm (Figure 6h). E-CQDs treatment didn’t affect the growth of Chlorella cells. Moreover, the observed fluorescence was found to be located in the whole cell, which was different from the finding that internalized nitrogen-doped carbon dots were concentrated in the nucleus (Zhang et al., 2020b). These findings suggested that E-CQDs may bioaccumulate within Chlorella cells, which implies that E-CQDs hold practical value as imaging agents for live-cell studies and have the potential to serve as a probe for in vivo bioimaging applications.

The environmentally friendly synthesis of CQDs using various biological materials was compared (Table 1). Blancas et al. (2024) carried out synthesis of CQDs by Hydrothermal method using of Malva sylvestris flower. It required about 24 h for the synthesis, and showed small particle with a 5 nm size. The CQDs form M. sylvestris flower could grow crystalline nanostructures as growth seeds. Wang et al. (2018) developed a novel approach for CQDs fabrication by the use of bamboo biomass. It was heated for 24 h at 400 °C. Comparing with these CQDs reported, the method of preparing E-CQDs using E. gracilis powder as the raw material offers several advantages. It facilitates the acquisition of raw materials, simplifies pretreatment, and streamlines preparation procedures. The average particle size of the resulting E-CQDs was comparable to that of CQDs prepared using rice straw and Chlorella vulgaris as raw materials (Kaur et al., 2024; Thakur et al., 2024). Besides, CQDs from rice straw (Kaur et al., 2024), lemon and onion juices (Slewa, 2024), onion extract (Ghosh Dastidar et al., 2021),wild cherry shrub (Shahabadi et al., 2024), and C. vulgaris (Thakur et al., 2024) exhibited the potential application in abatement of microbes and organic pollutants, antifungal films, fluorescence turn-on probes, biosensor, and photocatalytic reduction and antimicrobial activity, respectively. In this study, E-CQDs have dual-functions that include antibacterial effects and bioimaging applications, which indicated that the nanomatrials could serve as next-generation antibacterial materials and nanotheranostics. However, the antibacterial effects of E-CQDs is relatively low, and the fabrication of elements-doped like N, silicon (Si), cadmium sulphide, cerium oxide, and silver) carbon dots (N-CDs) may be investigated in future, which can increase the activities of E-CQDs. Interestingly, polysaccharides found in marine environments can serve as carbon-rich precursors for synthesizing CQDs. Marine polysaccharides have a distinct advantage over other CQD precursors because they contain multiple heteroatoms, including N, S, and O (Torres et al., 2023). Euglena gracilis can accumulate large amounts of beta-1, 3-glucan paramylon, a polysaccharide (Lee et al., 2024). Therefore, E. gracilis polysaccharides may also possess the potential of CQDs with applications in various fields, including biomedicine (e.g., drug delivery, bioimaging, and biosensing), photocatalysis, water quality monitoring, and the food industry. Sensing, photocatalysis, diagnostics and therapy of E-CQDs could be also studied in the next step.