Degradable Carbon Dots with Broad-Spectrum Antibacterial Activity

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Biological and Medical Applications of Materials and Interfaces

Degradable carbon dots with broad-spectrum antibacterial activity Hao Li, Jian Huang, Yuxiang Song, Mengling Zhang, Huibo Wang, Fang Lu, Hui Huang, Yang Liu, Xing Dai, Zonglin Gu, Zaixing Yang, Ruhong Zhou, and Zhenhui Kang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08832 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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ACS Applied Materials & Interfaces

Degradable carbon dots with broad-spectrum antibacterial activity Hao Li §, #, Jian Huang †, #, Yuxiang Song §, Mengling Zhang §, Huibo Wang §, Fang Lu ‡, Hui ┴ Huang §, Yang Liu *, §, Xing Dai ⸸, Zonglin Gu ⸸, Zaixing Yang ⸸, Ruhong Zhou *, ⸸, and

Zhenhui Kang *, § §Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou 215123, China. †School of Biology & Basic Medical Sciences, Soochow University, Suzhou 215123, China. ‡School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing 100029, China. ⸸School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China. ┴Department of Chemistry, Columbia University, New York, NY 10027, USA. KEYWORDS: carbon dots, degradation, broad-spectrum antibacterial, cell wall, gene.

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ABSTRACT: The infection of bacteria and fungus is one of the most challenging global threats to human health. With the recent advancement in nanoscience and nanotechnology, much progress has been achieved in the development of antimicrobial nanomedicine, however, these nano-material based antibacterial agents still suffer from potential biological toxicity, poor degradation and various secondary pollution. Here, we demonstrate the fabrication of low-toxic and degradable carbon dots (CDs) from vitamin C (VC) by one-step electrochemical method. These newly generated CDs display a strong broad-spectrum antibacterial activity and antifungal activity even at low concentrations, as they destroy the bacterial walls during the diffusive entrance, perturb secondary structures of DNA/RNAs of bacteria and fungus, and inhibit important gene expressions, to finally kill the bacteria and fungus. We also show that these wellcharacterized CDs can be completely degraded into CO2, CO and H2O under visible light in air (or at very mild temperature, about 37 ℃).

INTRODUCTION Bacteria and fungus exist in every corner of the world, and their infection is one of the biggest global challenges to human health.1 Notably, the overuse and/or abuse of antibiotics in the longterm lead to the emergence of multiple drug-resistant (MDR) bacteria, which increases the difficulty of infectious diseases therapy. Also, fungus and bacteria can cause widespread plant diseases (blast, sheath blight, etc.), which impede the development process of grain crops and affect economic benefits. Pesticides are used widely for the resistance of plant diseases, while their continuously over-use always leads to severe environmental problems.2-4 Up to now, great efforts have been paid in the development of antibiotics, drugs and antimicrobial materials. Due


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to their unique features such as increased membrane permeability and lack of efflux pump compared with traditional antibiotics, a series of nanoparticles (such as, cationic conjugated polymers,5 peptide hydrogels,6 semiconductor, noble-metal particles,7-11 carbon nanotube graphene sheets15, 16, graphene oxides

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and element-doped carbon nanoparticles

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12-14

) and show

strong antimicrobial activity. However, these antimicrobial nanoparticles always possess the high biological toxicity and hard degradation, causing a series of secondary pollution and threat. For instance, some nanometer materials exhibit significant cytotoxicity to human and animal cells,2224

so much as that they can damage the development of zebrafish embryo.25 For the hard-

degradable nanomaterial, they can alter bacterial community structure in a dose-dependent fashion,

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and influence taxa associated with ecosystem processes of N2 fixation, methane

oxidation, and complex C decomposition. 27 It is highly desirable to develop the bio-safe and degradable nanomaterials with broad-spectrum and high antibacterial property. In recent years, the carbon dots (CDs) have gradually become a rising star in the nanocarbon family, due to their benign, abundant and inexpensive nature. 28 they have inspired extensive studies on them due to their great potential for a wide variety of technical applications.

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Here, we show the

fabrication of low-toxic and degradable carbon dots (CDs) from vitamin C (VC) by one-step electrochemical method. We demonstrate that this kind of CDs can be completely degraded into CO2, CO and H2O by visible light irradiation or at very mild temperature (about 37 ℃) after 20 days. Notably, these CDs show a strong broad-spectrum antibacterial activity and antifungal activity, efficiently inhibiting the growth of Staph. aureus, Bacillus subtilis, Bacillus sp. WL-6, Escherichia coli, the ampicillin-resistant Escherichia coli, Rhizoctonia Solani as well as Pyricularia Grisea. For the bacteria and fungus, the best bacteriostatic concentration was 100 μg·mL−1 and 300 μg·mL−1, respectively. A series of experiments suggest that CDs can enter the


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bacteria by the diffusion, destroy the bacterial wall, bound to the DNA and RNA of bacteria and fungus, inhibit the RsNPs gene expression and finally kill the bacteria and fungus at low concentration. EXPERIMENTAL SECTION Materials & Characterization. All the chemicals and biological reagents were purchased from Sigma-Aldrich, Adamas-beta and Beijing Chemical Reagent (Beijing, China), and were used as received. Milli-Q ultrapure water was used for all experiments. All solid samples were dried under vacuum at 60 ℃ for 24 h before the measurement. The transmission electron microscopy (TEM) and plus high-resolution TEM (HRTEM) images were obtained with a FEI/Philips Tecnai G2F20 TWIN TEM. The scanning electron microscopy (SEM) images of bacteria were obtained with a Zeiss Supra 55. The size of samples was measured by using a dynamic light scattering (DLS) instrument (Zetasizer Nano ZS, ZEN 3690, Malvern). The Fourier Transform Infrared (FT-IR) spectra of the carbon particles were obtained with a Bruker Fourier Transform Infrared Spectrometer (Hyperion). The photoluminescence (PL) study was carried out on a Horiba Jobin Yvon (Fluoro Max-4) Luminescence Spectrometer, while UV-visible spectra were obtained using a Perkin Elmer UV-Vis spectrophotometer (Lambda 750). The X-ray photoelectron spectra (XPS) were measured by a KRATOS Axis ultra DLD X-ray photoelectron spectrometer. The degradation products of CDs were analyzed by the Agilent TD-GC/MS (7890A-5975C). Fluorescence optical microscope (Leica, DM 4000 M) was used for images of the bacteria and fungi. The circular dichroism (CD) spectrum was obtained with a JASCO J-815 spectropolarimeter. The cell imaging graphs were obtained using Laserscanning confocal fluorescence (Leica, TCS-SP5).


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CDs fabrication. All chemicals were used as received without further purification. In typical experiment, 50 g vitamin C (Adamas) was dissolved in the 1 L ultrapure water, and then the two electrodes (graphite rod, 99.99%, Alfa Aesar Co. Ltd.) were applied with 0.1 A direct current (DC) power supply (Scheme S1). After 3 weeks, the solution was filtered with slow-speed quantitative filter paper, and then the product was dialyzed in a 500 Da dialysis bag against deionized water to remove the excess vitamin C. Determination of the fluorescence quantum yields. Quantum yield (QY) was measured according to established procedure by using quinine sulfate in 0.10 M H2SO4 solution as the standard. The absorbance was measured on a Perkin Elmer UV-Vis spectrophotometer (Lambda 750). Absolute values were calculated according to the following equation (1):

Where, Q was the quantum yield, m was the slope of the plot of integrated fluorescence intensity vs absorbance and n is the refractive index. The subscript refers to the reference fluorophore, quinine sulphate solution. In order to minimize re-absorption effects, absorbance in the 1 cm quartz cuvette was kept below 0.15. Cellular toxicity test. The HEK 293 T human normal cell line, human renal proximal tubular epithelial cells (HK-2) and human peritoneal mesothelial cells (HPMC) were cultured in the standard medium at 37 ℃ in 5% CO2. Cells were seeded in a 96-well plate for 24 h. Serial dilutions of CDs with known concentrations were added into cells. After 48 h incubation, the relative viabilities of cell samples were determined by colorimetric 3-(4, 5-dimethylthiazol-2-yl)2, 5-diphenyl tetrazolium bromide (MTT) assays were performed to assess the metabolic activity of cells treated as described above. Cells were lysed with acidulated sodium dodecyl sulfate


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(SDS). Absorbance was measured at 570 nm using microplate reader (Bio-Rad 680, U.S.A.). All measurements were done in triplicate, and at least three independent experiments were carried out. Antibacterial activity test. All utensils were autoclaved at 121 ℃ for 20 min to ensure sterility. Bacteria of Staph. aureus, Bacillus subtilis, Bacillus sp. WL-6, Escherichia coli and ampicillin-resistant Escherichia coli were grown overnight in the Luria-Bertani (LB) liquid medium and harvested at the exponential growth phase via centrifugation. The supernatant was discarded and the bacterial was re-suspended in normal saline. The bacteria concentration was determined by measuring the optical density (OD) at a wavelength of 600 nm. The OD 600 values of bacteria solutions were adjusted to 0.1. Next, the bacteria solutions diluted 100 times and the 10 μL of bacteria solutions daubed the adding different concentrations CDs LB agar plates, respectably. These plates were incubated at 37 ℃ for 48 h. The other experiment, the 20 μL of bacteria solutions (OD600 = 0.1) were added into the 5 tubes of LB liquid medium, respectably. And then the LB liquid medium contained different concentrations CDs. These were incubated at 37 ℃ for 48 h. Finally, the concentrations of bacteria solutions were test by the measuring the OD at a wavelength of 600 nm. The bacterial viability was calculated according to the following equation (2):

Where, V was the bacterial viability, VOD’600 was the value of OD600 of bacteria solution with different concentrations CDs. VOD600 was the value of OD600 of bacteria solution without CDs. Bacterial cell culture. Staph. aureus, Bacillus subtilis, Bacillus sp. WL-6, Escherichia coli and ampicillin-resistant Escherichia coli were cultured in Luria-Bertani (LB) medium under


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shaking (180 rpm) at 37 ℃. The concentration of bacteria was determined by measuring the optical density at 600 nm (OD 600) via UV-vis spectroscopy. Anti-fugus activity test. All utensils were autoclaved at 121 ℃ for 20 min to ensure sterility. Rhizoctonia Solani and Pyricularia Grisea were cultured in 3.8% (m/v) potato agar medium (sigma) and incubated in 28 ℃. Different concentrations of CDs were directly added into the medium. The growth of fungus plaque was estimated by measuring the diameter of plaque. Conjugation of CDs to DNA and RNA. RNA (Ribonucleic Acid from yeast, Yuanye) and double-stranded DNA (-DNA, from E. coli W3350, TOYOBO) were used in ultrapure water. An excess of CDs was added into a 50 μg·mL−1 RNA and DNA solution, respectively. And the mixture solution was incubated for 20 min and then washed 5 times by ultrafiltration with 5 kDa cutoff membrane to remove unconjugated CDs. The collected DNA/CDs and RNA/CDs conjugates were used to measure the dimeter of them. RNA extraction and agarose gel electrophoresis. Total RNA was extracted from 1 mL E. coli bacteria solution (OD 600: 1.0) using trizol reagents (ambion ltd) followed by the product instruction, the ratio bacteria solution to trizol was 1:1. The total RNA was dissolved in 30 μL DEPC water. For electrophoresis analysis, 2 μg total RNA was used and loaded into 1.5% agarose gel, 160 V for 25 min, and photographed. Bacterial morphology study. The morphology of bacteria with and without the CDs treatment was examined by SEM. The solution of bacterium E. coli or Staph. aureus was dropped onto a silicon wafer, and then treated with 2% glutaraldehyde fixation for 3 h at room temperature and gradient dehydration by a series of ethanol solutions (50%, 70%, 90%, 95% and 100%) for 10 min in 5 each step. After complete nitrogen drying, the silicon wafer was coated with ultrathin gold film by sputtering and imaged with SEM (JSM-7600F, JEOL).


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Theoretical method. The designed VC dimmer was optimized at PBE0/6-31G* level.35, 36 To simulate the UV-vis spectrum, excited states were calculated at PBE0/6-311+G* level 35, 37 based on time-dependent density functional theory (TDDFT). The solvent effect (water) was considered by using SMD

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implicit solvent model. All calculations were performed by using

Gaussian 09 program.39 Molecular dynamics simulation. The simulations were performed using the GROMACS software package (version 4.6.6)

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with the AMBER99SB force fields.41 The

initial configuration of model DNA hairpin is obtained from a crystal structure resolved by NMR experiments (PDB code: 2m8y).42 The atomic Linnard-Jones (LJ) parameters of atoms of CDs were established based on the AMBER force field while their atomic charge was calculated by the R.E.D. Tools.43 The VMD software results. The TIP3P water model

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was used to analyze and visualize the simulation

was used for water molecules. The temperature was

maintained at 300 K by using v-rescale thermostat 46 and pressure was kept at 1 atm by applying Parrinello-Rahman barostat.47 The van der Waals (vdW) interactions were calculated with a cutoff distance of 1.2 nm and the long-range electrostatic interactions were treated with PME (Particle Mesh Ewald) method.48 All solute bonds were maintained at their equilibrium values with the LINCS algorithm,49 and the geometry of water was constrained by using the SETTLE algorithm.50 A time step of 2.0-fs was used in the production runs. RESULTS AND DISCUSSION Preparation and characterization of the CDs. In our experiment, the CDs were synthesized by one-step electrochemical treatment of VC aqueous solution shown in scheme S1. The size and morphology of the obtained CDs were studied by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The TEM and high-resolution TEM (HRTEM) images of assynthesized CDs are shown in Figure 1a, which reveals the CDs have average size about 5 nm


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(size distribution shown in Figure S1) and the lattice spacing value of 0.21 nm, which matches the in-plane lattice spacing of graphene (100 facet; Fast Fourier Transform (FFT) shown in the bottom inset Figure 1a). Figure S2 shows the AFM image of CDs, in which, the line scans of three individual CDs reveal that their heights range from 1.03 to 1.11 nm. As shown in Figure 1b, the UV−vis absorption spectrum of CDs aqueous solution (the red curve) reveals two broad absorption peaks at 243 nm and 293 nm originating from the π−π* transitions of the aromatic sp2 domains (C=C, C–C) and the n−π* transition of multi-conjugate C=O and C–O of the CDs, respectively.51-53 While the raw material VC (the black curve) has only one peak at 260 nm, which should be credited to π−π* (C=C, C–C). Figure S3 displays the excitation−emission map of the CDs aqueous solution. With the excitation wavelength ranging from 250 to 600 nm, they exhibit excitation-dependent emission, indicating that their fluorescence processes are dominated by nonuniform emissive states. Furthermore, the maximum excitation of CDs aqueous solution occurs at 390 nm (Figure S4, the black curve) and the optimal emission is at 500 nm (Figure S4, the red curve). Besides, the fluorescence quantum yield (QY) of CDs is about 30% under excitation at 365 nm with lifetime about 2.21 ns (Figures S5 and S6).


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Figure 1. (a) TEM and HRTEM images of CDs. Left figure: low magnification of CDs. Top right figure: high magnification of CDs. Bottom right figure: FFT of the particle in the top right figure. (b) The UV-vis spectra of the vitamin C and CDs. Inset: Digital photographs of the vitamin C and CDs. (c) FTIR spectra of vitamin C and CDs. (d) XPS full scan spectra of CDs. Inside: High-resolution C 1s XPS spectra of CDs. (e) Simulated formation process of CDs. (f) Viability of 293 T cells after 48 h treatment with different concentrations of CDs as calculated from MTT assay.


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The structure, elemental composition and surface state of CDs were confirmed by the Fourier transform infrared spectra (FT-IR) and X-ray photoelectron spectra (XPS). As shown in Figure 1c, CDs (the red curve) and raw material VC (the black curve) have the similar FT-IR spectra, while the C=O bond stretching in CDs is slightly red shift to ~1631 cm-1 when compared with that of VC (C=O at 1668 cm-1 in VC). A detailed analysis of CDs FT-IR spectrum further suggested that CDs possess rich oxygen functional groups, such as, C–O–C, C=O, O–H and COOH groups. The XPS spectra of CDs are shown in Figure 1d. The survey spectrum of CDs indicates that the CDs only consisted of carbon and oxygen elements. The high-resolution spectrum of C 1s exhibits three main peaks (inset of Figure 1d), in which the anterior peak located at 284.8 eV is attributed the bonding structure of sp2 C, while the other two peaks located at 286.3 eV and 288.8 eV for the C–O and C=O bonds, respectively. To further explore the possible structural features of the CDs, we simulated the UV-vis spectra of different polymeric forms of VC dimmers by theoretical calculations based on time-dependent density functional theory (TDDFT) method and compared them with the experimental data. As shown in Figures S7 and S8, the calculated UV absorption peaks (245.07 and 292.79 nm) of the VC dimmer are in good agreement with the experimental values (243 and 293 nm), in which, the absorption peak of 245.07 nm mainly comes from the π−π* electron transition of VC1 (one unit of VC dimmer), and another peak of 292.79 nm for the n−π* electron transition of VC2 (the other unit of VC dimmer). Based on all of above results, a schematic diagram of VC polymerization to form CDs was shown in Figure 1e. Next, the cytotoxicity of the as-synthesized CDs was tested by 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays in 293 T, HK-2 and HPMC cells. As shown in Figures 1f and S9, when the concentration of CDs is even 800


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μg·mL−1, the cell viability is about ~90%, suggesting their low cytotoxicity and good biocompatibility.

Figure 2. (a) TEM image of the CDs (left) and after 120-hour visible illumination (right). (b) Comparison of the initial diameter distributions of CDs with those after 120-hour visible light irradiation as measured by dynamic light scattering (DLS). (Inset: Photo-degradation process of CDs after visible light irradiation for different durations for 0, 60 and 120 hours). CDs: 9 mg·mL-1. (c) The pseudo-first-order kinetic of CDs degradation under visible light irradiation for the different atmospheres (Air, O2 and N2). CDs: 0.3 mg·mL-1. (d) The pseudo-first-order kinetic


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of CDs degradation under different temperatures (4, 25 and 37 ℃). CDs: 0.3 mg·mL-1. Amount of CO and CO2 evolution of CDs after visible light irradiation for different (e) atmospheres (air, N2 and O2), (f) pH and (g) solvents (Phosphate Buffered Saline (PBS), Luria-Bertani medium (LB) and Fetal Bovine Serum (FBS)). The concentration of CDs: 9 mg·mL-1. (h) The scheme of CDs degradation. The degradation of CDs. A series of experiments suggested that CDs can be completely degraded into CO2, CO and H2O under visible light irradiation or at very mild temperature in air. Firstly, as shown in Figure S10, the CDs solution not only absorb 213-400 nm light but also 400600 nm visible light. In other words, the CDs can use the visible light to photo-degrade themselves. Figure 2a shows the TEM of CDs and CDs after 120-hour visible light illumination. Before visible light irradiation, the diameter of CDs was 6-3 nm shown in left part of Figure 2a. After 120 hours of visible light irradiation, the CDs diameter was reduced to 1.8-2.5 nm (right part of Figure 2a). Dynamic light scattering (DLS) measurements were implemented for CDs before and after degradation (shown in Figure 2b), which indicate the obvious decrease in diameter distributions after 120 hours of degradation process. The photographs of degraded CDs solution show an apparent decrease in the color of CDs, indicating a decrease in CDs concentration after the degradation process. The photo-degradation kinetic curve of CDs was investigated by the first-order simplification of Langmuir–Hinshelwood (L–H) kinetics. As shown in Figure 2c, the reaction rate constant k for the photo-degradation of CDs was 0.356×10-1 h-1, 0.530×10-1 h-1and 0.220×10-1 h-1 under the different atmospheres, for Air, O2 and N2, respectively. It indicates that the CDs are easily photo-degraded in O2. In addition, the CDs can also be degraded easily by mild heating. As shown in Figure 2d, the degradation CDs would be greatly accelerated from 4 to 37 ℃ with increasing the temperature, in which, the reaction rate


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constant k of CDs was 0.322×10-3 h-1, 0.661×10-3 h-1 and 0.196×10-2 h-1, respectively. To identify the products resulting from degradation of CDs, Gas Chromatography-Mass Spectrometer (GC-MS) and Gas Chromatography (GC) was performed. As shown in Figure S11, by analyzing the GC-MS total ion chromatograms (TIC) and mass spectrometry of CDs degradation products, it was found that the degradation products contain main six kinds of organic compound, and their molecular formula and possible molecular structure were displayed in the Table S1. Also, the gases were determined by gas chromatography (GC). The data indicate the degradation gases were CO and CO2. And then the CDs solution (0.03 mg·mL-1) was irradiated under the visible light in air for 30 days, its UV-vis spectra is a flat line. This result indicates the CDs the degraded CDs solution don’t contain any organic fragments (Figure S12). Next, a series of degradation experiments were carried out to investigate the influence of different atmospheres, pH and solvents on photo-degradation CDs. As show in Figure 2e, the amount of CO and CO2 evolution of CDs under the O2 atmosphere are higher than that under the N2 atmosphere. So, the O2 was the main factor for the process of photo-degradation CDs. Furthermore, in the air atmosphere, the CO evolution rate is about 0.0083 mol·h-1 under the visible light irradiation. Figures 2f and 2g show the amount of CO and CO2 evolution of CDs after visible light irradiation for different pH and solvent. When the pH of the solution was changed from 1.0 to 11.0, the CO and CO2 can be detected. However, in the gas product, only CO was detected when the pH was 13.0-14.0, which is due to the CO2 products was absorbed by the lye and form the carbonate (Figure 2f). When CDs were dissolved in the different solvents (Phosphate Buffered Saline (PBS), Luria-Bertani medium (LB) and Fetal Bovine Serum (FBS)), they can also be degraded into CO and CO2 easily, which indicated that the CDs are degradable in the organisms (Figure 2g). Furthermore, we also found that the CDs could be degraded into


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CO and CO2 under dark environment for different temperatures (4, 25 and 37 ℃), and the degradation rate of CDs accelerated with the increase of temperature. In addition, we found that the CDs could be degraded into CO under dark environment for 37 ℃ and the CO evolution rate is about 0.0014 mol·h-1 (Figure S13a). As shown in Figure 2e and S13b, we find that the visible light irradiation can promote the CDs to degrade into CO2, CO and H2O. Moreover, when the CDs solution had the hydrogen peroxide (H2O2) under dark environment, the CDs also can be degraded into CO and CO2 (Figure S13b). Base on above results, as shown in Figure 2h, we can conclude that CDs could be degraded step by step, and finally changed into CO, CO2 and H2O completely in present of O2, H2O2, visible light irradiation, or mild heat treatment. In the process of degradation, the amount of produced CO is too lowest to cause the biological toxicity in the atmosphere.

Figure 3 (a-d) Typical photographs of Staph. aureus, Bacillus subtilis, Bacillus sp. WL-6 and Escherichia coli after the treatment with different concentrations CDs (0, 5, 25, 50, 75 and 100


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μg·mL−1) for 24 h. (e) Bacterial viability of Staph. aureus, Bacillus subtilis, Bacillus sp. WL-6 and Escherichia coli evaluated with different concentrations CDs by UV-vis spectroscopy method. Antibacterial activity and antifungal activity of the CDs. In the bacterial inhibition experiments, we found that the as-prepared CDs could effectively inhibit the growth of Grampositive (Staph. Aureus and Bacillus subtilis) and Gram-negative (Bacillus sp. WL-6 and Escherichia coli) bacteria. As shown in Figure 3a, d, the growth of the bacteria depends on the concentration of CDs, with an elevated concentration of CDs to 0, 5, 25, 50, 75 and 100 μg·mL−1, CDs could cause the number of bacteria (Staph. aureus, Bacillus subtilis, Bacillus sp. WL-6 and Escherichia coli) colony gradually decrease. For Bacillus subtilis and Bacillus sp. WL-6, when the concentration of CDs is 50 μg·mL−1, there is no colony on the LB agar plates (Figure 3b, c). But, for Staph. Aureus and Escherichia coli, the best antimicrobial concentration is 100 μg·mL−1 (Figure 3a, d). At the same time, we also measured the bacteria viability (the process of see the experiment part) and found that when the bacteria were incubated with different concentration of CDs in LB liquid medium at 37 ℃ for 48 h, the bacteria viability was decreased from 100% to 0 with increasing the concentration of CDs (Figure 3e). In addition, for the ampicillin-resistant Escherichia coli (drug-resistant bacteria), we test the effect of CDs on the its growth. As shown in Figure S14, the number of ampicillin-resistant Escherichia coli gradually decrease with increasing the concentration of CDs to 0, 5, 25, 50, 75 and 100 μg·mL−1. Furthermore, there is no colony on the LB agar plates when the concentration of CDs is 100 μg·mL-1. The bacteria viability experiment data indicate that the ampicillin-resistant Escherichia coli viability was decreased from 100% to 0 with increasing the concentration of CDs (Figure S14b). These results


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indicate that the CDs possess broad-spectrum anti-bacteria properties and their best antimicrobial concentration is 100 μg·mL−1.

Figure 4. The typical photographs of (a) Rhizoctonia Solani and (b) Pyricularia Grisea after the treatment with different concentrations CDs (0, 37.5, 75, 150, and 300 μg·mL-1) for 24 h and 72 h. The colony diameter of (c) Rhizoctonia Solani and (d) Pyricularia Grisea evaluated with different concentrations CDs under the different times (24-96 h). To investigate whether the CDs have a wide effect on fungus or not, we selected two pathogenic fungus, Rhizoctonia Solani and Pyricularia Grisea, which are the two-main fungus causing rice diseases, rice sheath blight and rice blast. The two fungi were cultured in potato medium with an increased concentration of CDs (0-300 μg·mL−1), as shown in Figure 4a, b, the living status of both the two pathogenic fungus were inhibited, showing a significantly concentration dependent phenotypes. The diameter of the plaques with treaded different CDs


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concentrations under the different times were shown in Figure 4c, d, the experiment data indicate the growth of pathogenic fungus were significantly inhabited when the CDs concentration was 300 μg·mL−1. Furthermore, when the Rhizoctonia Solani was incubated with 300 μg·mL−1 CDs in LB liquid medium at 37 ℃ for 48 h, the fungus colony stop growing. For the Pyricularia Grisea, that was 96 hours.

Figure 5. (a-b) The Confocal laser scanning microscopy (CLSM) images of Staph. aureus (Gram-positive) treated with CDs (25 μg·mL−1) for 1 hour at 4 and 37 ℃. (ex = 405 nm; Emission was collected at 415−550 nm). (c-d) The CLSM images of Escherichia coli (Gramnegative) treated with CDs (25 μg·mL−1) for 1 hour at 4 and 37 ℃. (ex = 405 nm; Emission was collected at 415−550 nm). (e and f) The SEM images of the Staph. aureus (Gram-positive) and Escherichia coli (Gram-negative) after incubated without and with CDs at 60 μg·mL−1 for 12 h.


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(g) The CLSM images of Rhizoctonia solani treated with CDs and Red dot 1 for 30 mins at 37 ℃: (I) bright field (II) CDs (200 μg·mL−1; ex = 405 nm; Emission was collected at 415−550 nm) (III) Red dot 1 (200× in water; ex = 543 nm; Emission was collected at 580−750 nm) (IV) Merge of images (the red traces mark 1 and 2 were nucleus and ruptured nucleus, respectively). Antibacterial and antifungal mechanism of CDs. To understand the cellular mechanism for the antimicrobial action of CDs, we investigated the uptake of these materials into bacteria by utilizing CDs intrinsic fluorescence. Four groups of bacteria (Staph. Aureus (two groups) and Escherichia coli (two groups)) were incubated with CDs at a sublethal dose (25 μg·mL-1) for 1 hour at 4 and 37 ℃, respectively. As shown in Figure 5a-d, after incubating Staph. aureus and Escherichia coli with CDs, the fluorescence image of the CDs within the bacteria are clearly visualized when excited at 405 nm. This result demonstrated efficient internalization of CDs into the bacteria. At the same time, the CDs can mainly enter into the bacteria by diffusion. Then, morphology changes of the bacteria were investigated by scanning electron microscopy (SEM). As a control, the Figure 5e and f show typical SEM images of Staph. aureus (Gram-positive) and Escherichia coli (Gram-negative) without CDs (control), respectively, which exhibit the good preservation of the bacterial surfaces with obvious and coherent cytomembrane structures. The Staph. aureus (Gram-positive) and Escherichia coli (Gram-negative) incubated with CDs shows that the bacterial cells become rough. This phenomenon suggests that CDs can cover the external surface of cells, which might lead to indirect toxicity by biologically isolating them from the growth medium, and consequently the bacterial cells can neither proliferate nor consume nutrients. 54 And then serious damage and leakage of cellular contents exist on some cell walls of Escherichia coli (Gram-negative), while the untreated Escherichia coli have smooth cell wall. The above experiment result indicates that CDs could be used in antibacterial application and the


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antimicrobial mechanism involved the disruption of bacterial cell walls and/or membranes.

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In

addition, to see how the CDs can inhibit the growth of fungus, the hyphal of Rhizoctonia Solani cultured for 4 days in medium containing 150 μg·mL−1 CDs was separated and photographed using confocal microscope, as shown in Figure 5g, the CDs are accumulated in the nucleus of hyphal cells (fungus: Rhizoctonia Solani), and this result was confirmed by a Red dot 1 (nucleus specific dye) staining treatment. At the merged picture, marked with number 2, the nucleus is crashed after treating with CDs, indicating that the CDs can directly entered into nucleus and disrupted the nucleus, hence to inhibit the growth of fungus. For the Rhizoctonia solani, we study the effect of CDs on the expression level of the Rhizoctonia solani non-ribosomal peptide synthetase gene (RsNPs).56 As shown in Figure S15, the RsNPs gene expression level was significantly inhibited by CDs. In the other words, the actinomycete genome encodes various biosynthetic pathways would be influenced by CDs. So, the activity of fungus was inhibited by CDs.

Figure 6. (a) The size distribution of DNA before (DNA) and after the addition of CDs (DNA/CDs). (b) Fluorescence spectra of the CDs (200 μg·mL−1) toward addition of various


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concentrations of DNA in PBS buffer (10 mM, pH = 7.2) under excitation of 380 nm; inset: fluorescence intensity ratio versus the different concentrations of DNA, I0 and I stand for the emission peak intensity of the CDs before and after the addition of DNA. (c) The circular dichroism spectra of DNA treated by CDs with different concentrations. (d) The size distribution of RNA before (RNA) and after the addition of CDs (RNA/CDs). (e) Fluorescence spectra of the CDs (200 μg·mL−1) toward addition of various concentrations of RNA in PBS buffer (10 mM, pH = 7.2) under excitation of 380 nm; inset: fluorescence intensity ratio versus the different concentrations of RNA, I0 and I stand for the emission peak intensity of the CDs before and after the addition of RNA. (f) The agarose gel electrophoresis of RNA (from E. coli) and CDs treated RNA. (line 1: control; line 2-5: different concentration CDs (0, 12, 15, 20 and 30 μg·mL−1) treated RNA. Next, the binding interaction of CDs on the DNA and RNA were studied by Dynamic Light Scattering (DLS), CD spectroscopy, and gel electrophoresis. First, the diameter of DNA and CDs treated DNA (DNA/CDs) were measured by the dynamic light scattering apparatus. As shown in Figure 6a, there is a slight increase of DNA in hydrodynamic diameter from 16 to 25 nm after addition of CDs, implying the enrichment of CDs onto the DNA. Figure 6b displays the fluorescence spectra of CDs toward addition of different concentrations of DNA (from 0.02 to 0.67 mg·mL-1). The increase of the DNA concentration induces a notable addition of 1.3%26.6% in emission intensity of the DNA/CDs. It demonstrates that CDs can bind to DNA by noncovalent bonds. Next, we studied the effect of CDs on the secondary structure of DNA. As shown in Figure 6c, both the intensities of the negative band (245 nm) and positive band (275 nm) decrease significantly, shifting to zero levels. While there is no any significant shift of the peak locations, it is indicated that the CDs connect with the DNA and they affect the secondary


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conformations of DNA. In other words, the CDs are capable to make the structure of DNA loose and lead to the unwinding of DNA double helix structure. For RNA, the DLS results in the Figure 6d displayed a slight increase of RNA in hydrodynamic diameter from 110.1 to 82.1 nm after addition of CDs, implying their enrichment onto RNA. Figure 6e depicts an obvious increase in the fluorescence intensity of the RNA/CDs (1.2%-50.7%) with the increasing concentration of RNA (0.05-1.57 mg·mL-1). The PL spectrum of the CDs (black trace) is the same as the PL spectrum of the RNA/CDs, which suggested the CDs are connected to the RNA by non-covalent bonds. To furthermore study the influence of CDs on RNA structure, we measured the agarose gel electrophoresis of different concentration CDs treated RNA. As shown in Figure 6f, it displayed the electrophoresis strip of the RNA with adding different concentrations CDs. When the adding concentration of CDs is 30 μg·mL−1 the electrophoresis strip of RNA is shallower than the control sample, suggesting the structure of RNA can be influenced by the CDs. The above systematic studies enabled us to explain the antibacterial and antifungal mechanism of CDs as shown in Figure S16. Firstly, the CDs with oxygen-containing groups (C–O and C=O) are adsorbed in the bacteria and fungus cell wall, and then are able to enter them by the diffusion. Secondly, CDs can bound to the DNA and RNA by the noncovalent bonds in the bacteria and fungus, change the DNA (the secondary conformations) and RNA structure, and then the genetic process of bacteria and fungus will be affected. At the same time, the cell wall of bacteria is damaged by the adsorbed CDs. In addition, for the fungus, the CDs inhibit the RsNPs gene expression. Finally, the activity of bacteria and fungus are inhabited and killed by the CDs. We further performed molecular dynamics (MD) simulations to reveal the dynamical process of CDs interacting with DNA along with the underlying molecular mechanisms (Figures S17-


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S18 and Table S2). Figure 7a shows the final configurations of DNA with or without the presence of CDs. Notably, with the presence of CDs, the DNA hairpin displayed a distinctive structural distortion, which is accompanied by an obvious dislocation of the terminal base-pair. Meanwhile, the root mean square deviation (RMSD) of DNA hairpin also quantitatively demonstrated that treatment of CDs destabilized the native structure of DNA (Figure 7b), particularly during the last 40 ns simulation. The mean RMSDs in the last 40 ns were 3.4  0.3 Å and 2.2  0.3 Å with or without the presence of CDs. In essence, CDs induced the structural distortion and local denaturation of the DNA hairpin. To further analyze the molecular origins of the DNA-denaturing power of CDs, the non-bonded interactions energies (including both the van der Waals (vdW) energy and Coulomb energies) and hydrogen bond number between DNA hairpin and CDs were

Figure 7. (a) The original (cyan) and final configurations of DNA hairpin in control (orange) and CDs added (purple) simulations. (b) Time evolutions of the root mean square deviation (RMSD) of DNA heavy atoms in control and CDs added simulations. The embedded figure represents the


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averaged RMSD values for these two simulations over the last 40 ns data. (c) The direct nonbonded interaction energies, including vdW (black curve) and coulomb (red curve) interaction energies, and hydrogen bond number (H-bond, blue curve) formed between CDs and DNA hairpin as the function of simulation time. (d) Some representative configurations were taken from some key time points to show the terminal base-pair denaturation process. The terminal base-pair was shown with sticks, where red, yellow, blue and tan sticks represent oxygen, carbon, nitrogen and phosphorus atoms, respectively. The purple ribbon was the DNA hairpin. The cyan, red and white spheres were the carbon, oxygen and hydrogen atoms of CDs. computed (Figure 7c). It is noteworthy that during the adsorption process, the lowering in the Coulomb energy between CDs and DNA was much profound than vdW energy, suggesting that long-range electrostatic interactions played a significant role in the binding process of CDs to DNA hairpin. Meanwhile, the hydrogen bond number formed between DNA hairpin and CDs also increased steadily, indicating hydrogen-bonding interactions also played a big role for the binding of CDs to DNA-hairpin. Representative snapshots were also depicted in Figure 7d to further elaborate the interference of CDs to the DNA hairpin. As shown in Figure 7d, at t = 35.6 ns, one CD starts to attack a terminal nucleobase of DNA hairpin. Only after 0.5 ns, at t = 36.1 ns, the original native “edge-to-edge” base-pair packing mode of the terminal base-pair was severely interfered and the two nucleobases adopted a wrong “face-to-face” stacking pattern. Meanwhile, the other part of this CD began migration towards the other nucleobase of the terminal base-pair. At t = 162.5 ns, a significant portion of the DNA hairpin were in contact with the CD, causing larger structural distortions. Hereafter, the binding between the CD and the DNA hairpin was so strong that the “binding mode” stayed intact till the end of the simulation. Additional binding modes of other CDs to the DNA hairpin (less destructive) can be found in Figure S19. Overall,


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our simulations seem to suggest that the destructive effects on the DNA secondary structures were initiated at the terminal base-pairs upon the binding of CDs. CONCLUSIONS We demonstrate the fabrication of low-toxic and degradable CDs from vitamin C by one-step electrochemical method. The CDs show a strong broad-spectrum antibacterial activity and antifungal activity, efficiently inhibiting the growth of Staph. aureus, Bacillus subtilis, Bacillus sp. WL-6, Escherichia coli, the ampicillin-resistant Escherichia coli, Rhizoctonia Solani as well as Pyricularia Grisea. A series of experiments suggest that CDs can enter the bacteria by the diffusion, destroy the bacterial wall, bound to the DNA and RNA of bacteria, and then the genetic process of bacteria and fungus will be affected, finally kill the bacteria and fungus at very low concentration. Notably, this kind of CDs can be totally degraded into CO2, CO and H2O under visible light (or at very mild temperature, about 37 ℃) in air after 20 days. Our finding may open a new way for the design and fabrication of next generation antimicrobial materials. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The preparation procedure of CDs. The size distribution, UV-Vis, AFM, and PL of CDs. GCMS total ion chromatograms (TIC) of CDs degradation products. The mechanism of antibacterial on the CDs. The collapsed CDs model building process and the molecular dynamics (MD) simulations between CDs and DNA (PDF) AUTHOR INFORMATION


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Corresponding Author *(L. Y.) E-mail: [email protected]. *(R. H. Z) E-mail: [email protected]. *(Z. H. K) E-mail: [email protected]. Author Contributions # These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by the Collaborative Innovation Center of Suzhou Nano Science and Technology, the National Natural Science Foundation of China (51725204, 51572179, 31671216, 21471106, 21771132, 21501126), the Natural Science Foundation of Jiangsu Province (BK20161216), the China Postdoctoral Science Foundation (2017M611902) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). REFERENCES (1) Levy S. B.; Marshall B. Antibacterial Resistance Worldwide: Causes, Challenges and Responses. Nat. Med. 2004, 10, 122−129. (2) Liu W.; Liu J.; Triplett L.; J Leach. E.; Wang G. L. Novel Insights into Rice Innate Immunity Against Bacterial and Fungal Pathogens. Annu Rev Phytopathol 2014, 52, 213−241.


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Degradable Carbon Dots with Broad-Spectrum Antibacterial Activity

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