The chemicals for synthesizing the carbon dots (glucosamine HCl (GlcNH2·HCl, CAS# 66–84–2), 4,7,10-trioxa-1,13-tridecanediamine (TTDDA, CAS#4246–51–9), β-alanine (C3H7NO2, CAS# 107–95–9), glucose (C6H12O6, CAS# 50–99–7), L-arginine (C6H14N4O2, CAS# 74–79–3), citric acid (C6H8O7, CAS# 5949–29–1), and polyethyleneimine (PEI) [(C37H24O6N2)n, CAS# 71–2353] were purchased from VWR. Components for the preparation of growth media for these bacteria such as bacteriological agar, yeast extract, tryptone, NaCl, proteose peptone, potassium phosphate dibasic, magnesium sulfate heptahydrate, glycerol, Bacto Peptone, and sterile water were also ordered from VWR or Fisher Scientific.

The carbon dot was prepared according to a study conducted by Hill et al. (2016). Glucosamine hydrochloride (1.00 g, 4.63 mmol) was dissolved in distilled H2O (20 ml) and stirred to complete dissolution in a 250-ml conical flask. 4,7,10-trioxa-1,13-tridecanediamine (TTDDA) (1.11 ml, 5.09 mmol) was added to the sugar solution and agitated to ensure homogeneity. The conical flask was then placed in a domestic microwave 700 W, and the solution was heated for 3 min at 700 W (full power). A viscous brown residue was obtained and dissolved in distilled H2O (10 ml) and centrifuged for 3 min at 8500 rpm. The solution was purified by dialysis method for 4 days using Spectra/Por^® seven dialysis membrane tubes of 11.5 mm diameter. The dialysis water was refreshed every 2 h for the first 8 h and then once a day for the remaining 4 days. Then the purified CDs were further sterilized by passing the solution through a filter of 0.45 µm pore size. Other carbon dots including COOH-FCDs (glucosamine + b-alanine), N-CDs (glucose + L-arginine), and PEI-CDs (citric acid + PEI) were also synthesized according to the studies conducted by Hill et al. (2016), Cao et al. (2018), and Wu et al. (2018), respectively (Supplementary Table S1).

Six bacterial strains belonging to different taxonomic groups were used, namely, E. coli (DH5α), Pectobacterium carotovorum (Ecc7), Agrobacterium tumefaciens (EHA101), Agrobacterium rhizogenes (K599), Pseudomonas syringae, and Salmonella enterica subsp. enterica serovar Typhimurium 13311) (Figure 1). Agrobacterium and Pseudomonas were cultured in the YEP medium and King’s medium B, respectively. Pectobacterium, E. coli, and Salmonella strains were grown in the LB medium. Agrobacterium, Pectobacterium, and Pseudomonas were grown at 28°C, while E. coli and Salmonella were grown at 37°C. The OD (600 nm) was adjusted at 0.5 by subculturing the overnight grown cells and harvesting the cells at the logarithmic growth phase 5 ml of culture was centrifuged, and the cells were washed three times with sterile distilled water and eluted in 1 ml of 15% glycerol 50 µl of cells were further aliquoted to 0.65-ml tubes and stored at a −80°C freezer for future use.

FTIR analyses were performed to confirm the functional groups of CDs. The ATR–FTIR analysis of freeze-dried CDs was carried out using a Perkin Elmer Frontier Infrared spectrometer equipped with a liquid N₂ cooled MCT-A (Mercury cadmium telluride) detector and an optic compartment purged with CO₂- and H₂O-free air delivered by a Balston–Parker air purger. The FTIR spectra of the CDs were recorded between 600 and 3800 cm⁻¹. Furthermore, photoluminescence properties such as absorbance, emission, and excitation wavelength of the CDs were recorded using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT). A Zetasizer nano ZS (Malvern Panalytical Inc., Westborough MA) was used to measure the electrostatic charges carried by CDs. For the confocal microscopy, 5 µl of NH2-FCDs (15 µg/µl) and 50 µl of cells (OD₆₀₀ = 0.5) were incubated for 15 min and 1 h, respectively. The cells were centrifuged and washed three times with sterile water to remove LB and carbon dots outside of the bacteria. Earlier reports indicated that NH2-FCDs synthesized through this approach are in 2–10 nm particle size range (Hill et al., 2016).

The agar plate well-diffusion method was utilized to examine the antimicrobial activity of CDs diluted at different concentrations on Gram-negative bacteria (Balouiri et al., 2016). The agar plates were inoculated by spreading 50 µl bacterial suspension (OD₆₀₀ = 0.5) using sterile glass beads. The experiment consisted of three replicates for each treatment. A well of 0.6 mm diameter was cut on the inoculated plates using a cork borer (back of sterile 20–200 µl pipette tips can also be used instead). Then, 80 µl of CD solution was added into each well and allowed to diffuse overnight at 28°C or 37°C depending upon the organism. To determine the minimum incubation time for complete inhibition of bacterial cells, we mixed 50 µl of cells harvested at an OD₆₀₀ of 0.5 with 5 µl of freshly prepared CDs and incubated the solution for different time intervals ranging from 1 to 24 h. The incubation time at which there is complete inhibition of cells was noted.

The diameter of the inhibition zone for each bacterium was tabulated and analyzed using R software (V3.6.3). The mean of the treatments was calculated, and the post hoc analysis was carried out using the Tukey HSD test to compare mean difference between the treatments.

Fourier-transform infrared (FT-IR) spectroscopy was carried out to characterize the chemical functional groups on the NH2-FCDs. Thus, the prepared CDs showed peaks at 870.2 cm-1(C-O) of double olefinic bonds in single vinyl (C=CH2), 1461 cm-1 (C≡N) of the nitrile group, 1616 cm-1 (C=O) of the ketone group, 2867 cm-1 (C=OH) of the aldehyde group, and 3255 cm-1 (-OH) of the hydroxyl (Nandiyanto et al., 2019) (Figure 2A). The photoluminescence properties showed an absorbance profile maximum of 280 nm which lies between t 200 and 450 nm range—a characteristic value for the CDs synthesized using a bottom-up approach (Figure 2). The defined peak of 280 nm could be attributed to π–π* transition of aromatic/alkenyl C=C bonds or C≡N bonds (Tan et al., 2016). The fluorescence emission profile showed an emission band at 450 nm when excited at 365 nm. This emission band at 450 nm is typical for carbon dots, which gives blue fluorescence (Carbonaro et al., 2018) (Figure 2B). The electrokinetic potential, commonly known as the zeta potential, of the NH2-FCDs was measured using a Malvern Zetasizer Nano-ZS ZEN 3600 (Figure 2C). The zeta potential value of NH2-FCDs was found to be (10.50 ± 0.3) mV. The positive charge is the result of primary amines derived from TTDA linker (Hill et al., 2016) (Figure 2C). The physical properties of other carbon dots were also measured as before and are presented in supplementary files (Supplementary Figures S1–S3).

As indicated in Figure 3, the CDs are interacting with the bacterial membrane. For sensitive bacteria (e.g., Pseudomonas), no single colony was observed after 1 h of incubation, confirming complete dissolution of cell membranes in these bacteria. Under the microscope, CDs were found interacting all over the bacterial cell surfaces, indicating their adherence to the cell membranes even after multiple washing steps (Figure 3, Supplementary Figures S4, S5).

In an initial experiment, the antimicrobial properties of these four carbon dots were measured to identify the variation between these carbon dots in their toxicity to different bacterial strains. Except PEI-CDs that had no antimicrobial effects on these bacterial strains (Supplementary Figure S6), other carbon dots displayed some degree of bacterial growth inhibition at different concentrations (Figure 4, Supplementary Figures S7, S8). The agar well diffusion method was used to determine the minimum inhibitory concentration (MIC) of NH2-FCDs with different concentration of CDs from 0.5, 1, 5 to 10 mg/ml. The MIC was different depending on the species. Pseudomonas growth was inhibited at 0.5 mg/ml (Figures 4, 5), whereas for Agrobacterium, Salmonella, Pectobacterium, and E. coli at least 5 mg/ml was needed to observe growth inhibition. The growth inhibition is dependent on the concentration of carbon dots, and more growth inhibition was observed at higher concentration of CDs. These inhibitory concentrations are far more than that reported in the earlier reports for nanoparticles used as antimicrobial agents. For example, the MIC for AgNPs against different bacteria (E. coli, Pseudomonas syringae, and Staphylococcus aureus) was reported as low as 4, 2, and 4 μg/ml (Gondil et al., 2019). However, the MIC of CDs synthesized in our laboratory was as high as 0.5 mg/ml for Pseudomonas and 5 mg/ml for other bacteria. Heavy metal–based nanoparticles are known to be toxic to the bacterial and human cells. Thus, even small concentration of these nanomaterials was effective against bacterial growth. Other studies using carbon dots synthesized from 2,2′-(ethylenedioxy)bis(ethylamine) (EDA) also reported lower MIC (64 μg/ml) on E. coli cells (Dong et al., 2017). However, it should be noted that the number of E. coli cells that they have used (1 × 10⁶) is 400x smaller than that used in our study (OD600 = 0.5 which equals to 4 × 10⁸ number of cells).

In another experiment, both synthesized CDs and their precursors were tested against six bacterial species to determine if the antimicrobial effects are unique to the carbon dots or their precursors. The result indicated that the antimicrobial capacity was enhanced in NH2-FCDs compared to the precursor solution (Figures 6, 7). Similar results were also observed in COOH-CDs and N-CDs as well (Supplementary Figures S9, S10). NH2-FCDs were more effective in E. coli, Salmonella, Pectobacterium, and Agrobacterium. However, both solution and NH2-FCDs were found to be equally effective against Pseudomonas.

An experiment was setup to determine the minimum incubation time required for complete inhibition of bacterial growth. 50 µl of bacterial culture harvested at the log phase was incubated at different time intervals with 5 µl (15.0 mg/ml) of freshly synthesized CDs. Depending on the species, different bacterial strains required different incubation times for complete inhibitions by NH2-FCDs (Figure 8). The complete inhibition was observed in E. coli after 16 h of incubation with CDs, and for Salmonella, 24 h was necessary for complete inhibition. While 2 h incubation time was required for Pectobacterium carotovorum, other bacteria, including A. tumefaciens, A. rhizogenes, and Pseudomonas syringae were more sensitive to the CDs and required as low as 1 h of incubation for complete inhibition. This experiment was also conducted for other CDs as well (Supplementary Figures S11, S12) in which E. coli and Salmonella displayed the highest resistance against these CDs.