Four types of carbon dots (CDs) were synthesized from different precursors and their physical properties and gene delivery competencies for individual plasmid sizes were studied at distinct incubation times.

Materials: chemicals for carbon dots synthesis (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# 5,949-29–1), and polyethyleneimine (PEI) [(C₃₇H₂₄O₆N₂)_n, CAS# 71-2,353] were purchased from VWR. Other components for the preparation of growth media such as bacteriological agar, yeast extract, tryptone, NaCl, proteose peptone, potassium phosphate dibasic, magnesium sulphate heptahydrate, glycerol, bactopeptone, and sterile water were ordered from VWR or Fisher Scientifics.

Experimental setup: Amine-coated carbon dots were prepared according to Hill et al. (2016). Glucosamine hydrochloride (1.00 g, 4.63 mmol) was dissolved in distilled H₂O (20 ml) and stirred to achieve complete dissolution in a 250 ml conical flask. 4,7,10-trioxa-1,13-tridecanediamine (TTDDA) (1.11 ml, 5.09 mmol) was then added to the sugar solution and agitated to ensure homogeneity. The conical flask was placed in a domestic microwave (Model#JES2251SJ02, GE Appliance, Canada), and heated for 3 min at 700 W power. The obtained viscous brown residue was dissolved in 10 ml distilled H₂O and centrifuged for 3 min at 8,500 rpm. The solution was then purified by tube dialysis for 4 days using Spectra/Por® 7 Dialysis Membrane with 11.5 mm diameter. Dialysis water was refreshed every 2 h for the first 8 h, and once a day for the remaining 4 days. The final purified CDs were further sterilized by passing the solution through 0.22 µm filter.

Experimental setup: Carboxylated carbon dots were prepared according to Hill et al. (2016). Glucosamine hydrochloride (1.00 g, 4.63 mmol) was dissolved in distilled H₂O (20 ml) and stirred to achieve complete dissolution in a 250 ml conical flask. β-alanine (0.45 g, 5.09 mmol) was then added to the sugar solution and agitated to ensure homogeneity. The conical flask was then placed in a domestic microwave and heated for 3 min at 700 W power. A viscous brown residue was obtained and dissolved in distilled H₂O (10 ml) and centrifuged for 3 min at 8,500 rpm. The solution was purified by tube dialysis for 4 days using Spectra/Por^® 7 Dialysis Membrane with 11.5 mm diameter. The dialysis water was refreshed every 2 h for the first 8 h, then once a day for the remaining 4 days. Then, the purified CDs were further sterilized by passing the solution through a filter with 0.22 µm pore size. Other carbon dots including NH2-FCDs (Glucosamine + TTDDA), N-CDs (Glucose + L-arginine), and PEI-CDs (Citric acid + PEI) were also synthesized according to Hill et al. (2016), Cao et al. (2018), and Wu et al. (2018), respectively

FTIR analyses were used to confirm the presence of functional groups on the carbon dots. The attenuated Total Reflection-FTIR analysis of the 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 optics 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 3,800 cm⁻¹. Furthermore, photoluminescence properties such as absorbance, emission, and excitation wavelength of the CDs were recorded using Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT). Zetasizer nano zs (Malvern Panalytical Inc., Westborough, MA) was used to measure the electrostatic charges carried by CDs.

Four plasmids of different sizes were used for this experiment including pPSU1 (Addgene #89439, 10,000 bp), pPSU2 (Addgene #89566, 7,750 bp, gifts from Song Tan), CAS9-pMDC123 (Addgene #59184, 13,658 bp, gift from Robert Stupar), and pU6-pegRNA-GG-acceptor (Addgene # 132,777, 3,004 bp, gift from David Liu). One day old bacterial culture was thawed on ice and 2 μg of plasmid was added to each tube. Five μl of each CD (10 mg/ml) was added to each tube and the reaction mixture was incubated for different time intervals (5, 15, 30, 60 min, and 4 h) under white light in the fume hood. Then, the cells were plated on LB agar supplemented with ampicillin (100 mg/ml) in three replicates per treatment and grown overnight at 37°C. The following day, delivery efficiency was confirmed by counting the number of colonies in each plate (in case of pU6-pegRNA-GG-acceptor plasmid the colonies are in red color due to the presence of RFP reporter gene). Identity of colonies and positive confirmation was carried out using specific primers to each plasmid and PCR on five randomly selected colonies.

Each single colony was diluted in 10 μL of double-distilled water and 1 μL of the diluted mixture was used for colony PCR, using M13 forward and M13 reverse universal primers. The DNA was amplified in a C1000 touch thermal cycler (Bio-Rad, Hercules, California, United States), programmed for one cycle of 95°C for 2 min, followed by 35 cycles of 95°C for 10 s, 55°C annealing temperature for 45 s, and 72°C of elongation for 30 s, followed by a final extension at 72°C for 10 min. 10 µL of PCR product was loaded on a 1% agarose gel, ran for 40 min at 100 V, and visualized and documented under ultraviolet light in Gel-Doc XR + system (Bio-Rad, Hercules, California, United States).

The number of transformed colonies was counted using ImageJ software and analyzed in R software (V3.6.3). The statistical analysis was used to compare the variation in the number of colonies at different time intervals and plasmid sizes. Post hoc analysis was carried out using the TukeyHSD test to compare the mean difference between time intervals, plasmid sizes, and effect of light and temperature on gene delivery.

FTIR analyses were performed to confirm the functional groups of CDs. The spectral graph of FTIR is divided into four different regions: 1) the single-bond region (2,500–4,000 cm⁻¹), 2) the triple-bond region (2000–2,500 cm⁻¹), 3) the double-bond region (1,500–2000 cm⁻¹), and 4) the fingerprint region (600–1,500 cm⁻¹).

The FTIR results for NH2-FCDs showed peaks at 870.2 cm⁻¹ (C=O) of double olefinic bonds in single vinyl (C=CH2), 1,461 cm⁻¹ (C=N) of Nitrile group, 1,616 cm⁻¹ (C=O) of ketone group, 2,867 cm⁻¹ (C=OH) of Aldehyde group, and 3,255 cm⁻¹ (-OH) of hydroxyl group (Nandiyanto et al., 2019) (Supplementary Figure S1). In the COOH-FCDs, distinct FTIR peaks were obtained at 3,340 cm⁻¹ indicative of various amino and alcohol groups (Supplementary Figure S2). Similarly, peaks were observed around 1715 cm⁻¹ of ketone (C=O) stretching vibrations, 1,627 cm⁻¹ of amide groups and its surface passivation, 1,570 cm⁻¹ showing strong N-H bending vibration peaks. The skeletal C-C vibrations of the residual carbohydrates were seen around 1,039 cm⁻¹ to 1,231 cm⁻¹. In L-Arginine and glucose (N-CDs), the peaks were observed at 3,903 cm⁻¹ indicating the presence of hydroxyl ions, amino groups, and hydrate molecules (Supplementary Figure S3). Likewise, peaks were observed around 1,594 cm⁻¹ indicating the presence of carbonyl group (C=C), amino group (C=N), and various aromatic rings. These aromatic rings are usually followed by the existence of weak-to-moderate absorption in the area 3,100 cm⁻¹. In Citric acid and polyethyleneimine, the FTIR spectra showed an N-H bending peak at 1,500 cm⁻¹, and O stretching vibration at 1700 cm⁻¹ (C=O) indicating the ketone groups, characteristic absorption bands of O–H and N–H stretching vibrations at 3,367 indicating the characteristics of O-H and N-H stretching, which is a characteristic of citric acid and PEI. The other peak was observed at 2,889 indicating the presence of carbonyl C=C and nitrile C=N and various aromatic stretching. Similarly, peak band at 1,073 represents cyclohexane ring vibration (Supplementary Figure S4).

Carbon-based quantum dots (CDs) are nanosized autofluorescent particles with a high quantum yield (QY). The photoluminescence properties for all the CDs used in this study showed an absorbance profile maximum of 280–285 nm, which fits in the range of 200-450 nm specific to the CDs synthesized using the bottom-up approach (Supplementary Figures S1–S4). 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).

Zeta potential was measured using Malvern Zetasizer Nano ZS instrument (Supplementary Figures S1–S4) which was similar to earlier reports for these CDs.

DNA delivery of the CDs was measured by incubating these CDs (Citric Acid + PEI, 100 µg; L-arginine and glucose, 66 µg; NH2-FCDs, 75 µg and COOH-FCDs, 50 µg) with E. coli cells in the presence of 2 μg plasmid DNA. Of all the CDs used in this study, only COOH-FCDs were able to deliver plasmid DNA into the E. coli cells (Figure 1).

To determine the optimum time for E. coli transformation, we incubated the mixture of COOH-CDs, plasmid, and E. coli cells for five different time periods including 5 min, 15 min, 30 min, 1 h, and 4 h. After the overnight culture, the number of colonies were counted using ImageJ software. As shown in Figure 2, the highest number of colonies was observed at 30 min of incubation throughout the replications; however, it was not significantly different from 60 min incubation (Figure 3). Shorter incubation times resulted in less colonies while no colonies were observed after 4 h incubation with COOH-CDs.

To determine the maximum plasmid size that can be delivered into E. coli cells, we incubated them with COOH-CDs and four plasmid DNA with sizes ranging from 3 to 13.5 kb. The plasmid sizes were as follows: pU6-pegRNA-GG-Acceptor (RFP): 3 kb, pPUS2: 7kb, pPSU1: 10 kb, Cas9-pMDC123: 13.5 kb. Bacterial colonies were counted using ImageJ software. Except for pU6-pegRNA-GG-Acceptor, which can be easily visualized with red color, colony PCR was performed for confirmation of positive colonies in other plasmids (Supplementary Figure S5).

Our results indicate that pU6-pegRNA-GG-Acceptor (RFP) and pPSU2 gave the highest number of colonies (Figures 4, 5). In contrast the number of obtained colonies dropped in pPSU1 with 10 kb plasmid size and no colonies were recovered from Cas9-pMDC123 plasmid that was 13.5 kb in size. This is an indication of plasmid size as a limiting factor in foreign DNA delivery into E. coli cells by COOH-CDs.

An experiment was conducted to study the effect of temperature and light conditions on DNA delivery using COOH-CDs by incubating the mixture of cells (E.coli, OD₆₀₀ = 0.5), plasmid DNA (pU6-pegRNA-GG-Acceptor, 3 Kb), and COOH-CDs at three different temperatures (4°C, 25°C, and 37°C) under light and dark conditions. The mixture was incubated for 30 min under different temperature treatments and plated on LB + Amp100 plates and incubated overnight at 37°C. The number of colonies per plate was counted using ImageJ software and analyzed in R software. Highest number of transformed colonies were obtained when incubation was carried out at 4°C (Figures 6, 7) with no significant variation under light and dark conditions (Figures 6, 8).