One-step synthesis of silver nanoparticles using carbon dots as reducing and stabilizing agents and their antibacterial mechanisms

Accepted Manuscript One-step synthesis of silver nanoparticles using carbon dots as reducing and stabilizing agents and their antibacterial mechanisms Jian-Cheng Jin, Zi-Qiang Xu, Ping Dong, Lu Lai, Jia-Yi Lan, Feng-Lei Jiang, Yi Liu PII: DOI: Reference:

S0008-6223(15)00491-1 http://dx.doi.org/10.1016/j.carbon.2015.05.084 CARBON 9978

To appear in:

Carbon

Received Date: Accepted Date:

22 December 2014 24 May 2015

Please cite this article as: Jin, J-C., Xu, Z-Q., Dong, P., Lai, L., Lan, J-Y., Jiang, F-L., Liu, Y., One-step synthesis of silver nanoparticles using carbon dots as reducing and stabilizing agents and their antibacterial mechanisms, Carbon (2015), doi: http://dx.doi.org/10.1016/j.carbon.2015.05.084

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One-step synthesis of silver nanoparticles using carbon dots as reducing and stabilizing agents and their antibacterial mechanisms Jian-Cheng Jin‡ a, Zi-Qiang Xu‡ a, Ping Dong a, Lu Lai

a,b

, Jia-Yi Lan a, Feng-Lei

Jiang a, Yi Liu* a a

State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for

Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China b

College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou

434023, P. R. China

‡These two authors contributed equally to this work.

*Corresponding author: Tel: 86-27-68756667. Fax: 86-27-68754067. Email address: [email protected]. (Y.

Liu) 1

Abstract: Silver nanoparticles (AgNPs) are potent and broad-spectrum antimicrobial agents. Herein, a novel one-step method has been used to synthesize AgNPs, in which fluorescent carbon dots (CDs) were used as reducing and stabilizing agents. To the best of our knowledge, it has rarely been reported to use CDs as reducing and stabilizing agents at the same time to fabricate AgNPs. Subsequently, their antibacterial activities were investigated. The results demonstrated that the surface property of AgNPs could dominate the stability of AgNPs that lead to the different bactericidal effect. When CDs doped with sulfur, size of the synthesized AgNPs were smaller. These small AgNPs exhibit better stability in culture medium contributed to excellent bactericidal performance, which can completely inhibit the growth of Escherichia coli (E. coli) at a concentration of 150 μM of silver atom. At last, the possible antibacterial mechanisms of AgNPs were proposed as follows: first, AgNPs can easily be absorbed onto the surface of E. coli and affect the permeability and fluidity of the outer membrane; second, AgNPs with smaller size (7.3±1.0, 6.1±0.8 nm) could permeate through the membrane of E. coli to interact with DNA and respiratory chain; third, the release of Ag+ could cause E. coli death.

2

1. Introduction It is widely known that AgNPs can be used as electrodes [1-3], catalyst [4, 5], surface-enhanced Raman scattering (SERS) materials [6, 7], because of its excellent chemical and physical properties. In addition, AgNPs as a new generation of nanoproduct in biomedical applications especially for their excellent antibacterial activities also attract considerable attention [8, 9]. Recently, AgNPs have also been found to be a accelerate agents in wounds and burns healing [10]. However, the mechanisms of their antibacterial activities are still not fully understood. Lok et al. implied that the mode of action of AgNPs against bacteria is similar to Ag+ through proteomic analysis [11]. Kittler et al. studied the kinetic release of Ag+ from AgNPs during storage and showed that toxicity of AgNPs is due to slow dissolution of AgNPs [12]. Liu et al. also observed the same phenomenon [13]. Xiu et al. suggested that the toxicity of AgNPs are not related to specific particle property but due to the silver oxidized by oxygen and subsequent Ag+ release [14]. At the same time, they showed that reactive oxygen species (ROS) is not an important antibacterial mechanism for silver ion and ligands can hinder the toxicity of Ag+ significantly while affect a little on the toxicity of AgNPs [15]. Katherine et al. investigated the interaction between Ag+ and the respiratory chain of E. coli and demonstrated that AgNPs can uncouple the respiratory chain from ATP synthesis [16]. Jung et al. observed changes of cell membrane after treating with Ag+ and speculated that it is the main reason for cell death [17]. Li et al. also found AgNPs can damage cell membrane [18]. Morones et al. found that AgNPs in the range of 1–10 nm can 3

permeate through E. coli [19]. Further, previous study proposed that AgNPs could increase the ROS level [20], inhibit the activity of respiratory enzyme [21], damage DNA and inhibit the DNA duplication [22]. Nevertheless, whether it is a single or synergistic effect could not be confirmed. The shape [23], size [24, 25], surface modification [26], and surface charge [27] especially the stability of AgNPs are vital to their antimicrobial properties. Due to that, many synthesis routes were designed to improve the antibacterial properties of AgNPs. In many cases, AgNPs were synthesized with high energy consuming or using sodium borohydride which could not easily control the reduction process because of its too strong reducing ability. Meanwhile, they are easy to aggregate when using none or weak stabilizers, thus the antibacterial properties become poorer. To solve the problem, some novel stabilizing and reducing agents were eagerly needed. Fluorescent CDs, a new carbon material, can be used in bioimaging [28, 29], biosensing [30], drug delivery [31], photocatalysis [32, 33] et al. Recently, it has been reported that the fluorescence of graphene quantum dots can be enhanced by hydrazine hydrate reduction [34]. Meanwhile, the reduced CDs has been used to prepare gold nanoparticles with the incubation of 24 hours [35]. What’s more, the maximum emission wavelengths of CDs are reversible when treated with sodium borohydride and nitric acid [36]. Therefore, it can conclude that CDs have both reducing and oxidation ability. Furthermore, numerous -COOH and -OH existing on the surface of CDs can coordinate with Ag+ and stabilize AgNPs. Considering this, four types of CDs were used to rapidly synthesize AgNPs in a one-step method. Then, 4

we examined the antibacterial activities of the synthesized AgNPs against E. coli through microcalorimetry and the results showed that two types of them perform excellent antibacterial activities, which can completely inhibit the growth of E. coli at a concentration of 150 μM of silver atom. Finally, the antibacterial mechanisms were studied and important factors were proposed.

2. Experimental Sections 2.1 Materials Sucrose, acetylcholine chloride, mercaptosuccinic acid, N-acetyl-L-cysteine, oleic acid and ether et al. were purchased from Sinopharm Chemical Reagent Co. (China). Silver

nitrate,

peptone,

1,6-diphenyl-1,3,5-hexatriene

(DPH)

and

1,3,5,8-tetramethyl-2,4-bis(alpha-hydroxyethyl) prophine-6,7-dipropionicacid (HP) were purchased from sigma-aldrich. Ultrapure water with 18.2 MΩ·cm (Millipore) was used in all synthesis. 2.2 synthesis of CDs CDs were synthesized according to the method reported by Li [37]. In particular, heating 5 g sucrose and 10 mL oleic acid in a three neck flask at a temperature of 215 °C for 5 min under vigorously stirring, impure CDs was obtained. As for the other three CDs, 4 g sucrose and 3 g acetylcholine chloride, 4 g sucrose and 3 g mercaptosuccinic acid, 4 g sucrose and 3 g N-acetyl-L-cysteine were used, heating at their melting point for 15 min, 6 min, 15 min respectively. After cooling, the supernatant liquid was discarded and the solid product was obtained at the bottom of flask. The precipitate was dispersed with water and then extracted with ether several 5

times in order to remove the remained oleic acid. Finally, the products were freeze-dried and obtained after dialyzed in deionized water using a 1 kDa membrane for 1 day. 2.3 Synthesis of AgNPs Firstly, 100 mL water was boiled to 100 °C, then the obtained CDs (125 μL, 50 mg/mL) were added and boiled for 15 min under stirring. Finally, AgNO3 (5 mL, 3.42 mg/mL) was added subsequently after NH3.H2O (1 mL, 10 %) injected. The reaction was processed for 45 min and cooled to room temperature. Finally, ultrafiltration was conducted with a membrane of 10 kDa in order to remove remained silver nitrate and the AgNPs were obtained. 2.4 Characterization The absorption spectra of the obtained AgNPs were tested on a MAPADA double-beam spectrophotometer at room temperature. The morphology of the CDs and AgNPs were observed by a JEOL 2011 transmission electron microscope at an accelerating voltage of 200 kV. The hydrodynamic diameter and zeta potentials of the AgNPs were measured by Nano-ZS ZEN3600 (Malvern Instruments). X-ray photoelectron spectroscopy (XPS) was carried out on KRATOS XSAM800 X-ray photoelectron spectrometer using Mg as the exciting source. Fourier Transform infrared (FTIR) spectroscopy of the C-dots were recorded by Thermo (USA) FTIR spectrophotometer. 2.5 Detect the concentration of silver atom by inductively coupled plasma atomic emission spectroscopy (ICP-AES) 6

Intrepid XSP Rasial (Thermo, USA) with a concentric model nebulizer and a cinnabar model spray chamber was used for the determination of silver atom concentration of the four types of synthesized AgNPs. 2 μL purified AgNPs was digested by nitric acid in ampulla and the vessel was heated while digestion to almost dryness. Finally, 2 mL deionized water was used to wash the ampulla and the solution was prepared for the experiment of ICP-AES. 2.6 Bacterial culture E. coli (DH 5α) was incubated with sterile LB medium contained of 10 g NaCl, 10 g peptone and 5 g yeast extract per liter at a pH value of 7.2 and temperature of 37 °C. Colony of E. coli grown on LB agar plates was inoculated in 30 mL of fresh medium and grown in a shaking incubator at 37 °C for 12 h. Then, 600 μL of the new bacteria suspension was placed into 30 mL of fresh medium and grown at 37 °C for 10 h. The bacteria were harvested by centrifugation at 5000 g for 5 min and washed twice with sterile phosphate-buffered solution (PBS) buffer (pH=7.4) and resuspended in PBS to an OD 600nm of 0.4. The bacterial concentrations were determined by measuring optical density (OD) at 600 nm (OD of 0.1 corresponds to a concentration of 108 cells per cm3). When conducted the experiments of the membrane property, detected the generation of ROS in E. coli cells, observed morphology of the E. coli cells, the resuspended E. Coli cells were used. 2.7 Microcalorimetry An LKB-2277 isothermal microcalorimeter (Thermometric AB, Sweden) was used to record the heat flow of the E. coli cells during the growth and metabolism. All 7

calorimetric experiments were conducted at a temperature of 37.0 °C. Stopped-flow method was used in this study. In all of the experiments, the flow cell was completely cleaned and sterilized in sequence by pumping 25 mL of aqueous HCl (0.1 M), 25 mL of aqueous NaOH (0.1 M), and 25 mL of sterilized distilled water assisted by an LKB-2132 microperpex peristaltic pump. When a stable baseline was obtained, the cell suspension containing the bacteria and different concentrations of CDs and AgNPs were pumped into the flow cell. When the flow cell (volume, 0.6 mL) was filled, the pump was stopped, the thermal effect versus time associated with microbial activities was recorded by a computer. The rest of bacterial suspension was discarded. Generally, the experiments were stopped when the second peak came to an end. All experimental procedures were performed under aseptic conditions. 2.8 The Stability of AgNPs in culture medium The stability of the synthesized AgNPs in culture medium was determined by the absorption spectra of AgNPs. The control was culture medium, an UV-vis spectroscopy was obtained every 2 min. A new absorption peak indicated the aggregation of the AgNPs. 2.9 The integrity of the outer membrane of E. coli cells The effects of different types of AgNPs on the integrity of outer membrane were studied by examining their sensitization to detergent mediated bacteriolysis. Bacterial cells were treated with or without AgNPs of a concentration of 100 μM for 2 h at a temperature of 37 °C. Then, the mixtures were centrifuged, and the supernatant was removed. The cell pellets were resuspended in the same volume of sterile PBS buffer 8

without AgNPs. Finally, sodium dodecyl sulfate (SDS) (0.1% final concentration) was added, and the OD600 was continuously monitored. 2.10 The fluidity of the E. coli cells membrane Bacterial cells were treated with or without AgNPs of a concentration of 100 μM for 2 h at a temperature of 37 °C. 0.6 μL of the fluorescent membrane probe, 1,6-diphenyl-1,3,5-hexatriene (DPH, 20 mM stock solution in tetrahydrofuran), was added to 3 mL of each cell suspensions and incubated for 60 min in the dark at room temperature to allow probe incorporate into the cytoplasmic membrane. The alteration of E. coli membrane fluidity was investigated by an LS-55 Spectrofluorimeter (Perkin–Elmer Corporation, USA). The excitation and emission wavelengths were 354 and 427 nm, respectively. When probed with HP (3 μL 6 mM stock solution in ethanol), the incubated time was 5 min. The excitation and emission wavelengths were 520 and 626 nm, respectively. 2.10 Microscopic investigation Electron microscopic observation was carried out using a TECNAI G2 transmission electron microscope (TEM) at an accelerating voltage of 100 kV. After completing the exposure period, cells (control and exposed group) were washed with PBS buffer. For TEM examination, 1 μL aliquot of the treated and untreated E. coli cells was put on a carbon coated copper grids. In addition, all ultrathin sections for the TEM were prepared following standard procedures for fixing and embedding of biological samples. 2.11 Generation of ROS detected by Flow cytometry 9

After completing the pretreatment of AgNPs, cells (control and exposed group) were washed with PBS buffer. The obtained cells were incubated with 10 μM DCFH-DA for 30 min in dark. Then, the mixtures were centrifuged and the supernatant was removed. Subsequently, the E. coli cells were resuspended in PBS for flow cytometry experiments.

3. Results 3.1 Synthesis and Characterization of CDs

Prior to obtaining four types of AgNPs, the CDs were synthesized. The four types of CDs were fabricated according to the method reported by Li [37], using sucrose, sucrose and acetylcholine chloride, sucrose and mercaptosuccinic acid, sucrose and N-acetyl-L-cysteine as the starting materials, respectively. The average size of as-prepared CDs were 2.4±0.9, 2.4±1.0, 2.6±0.3, 2.4±0.8 nm, respectively (Figure S1). The functional groups of the four types of CDs were identified by FTIR spectroscopy. The broad peak centered at 3388-3421cm-1 is the stretching vibrations of O–H. The existence of a sharp absorption peak at 1630-1665cm-1 is stretching vibrations of C=O (Figure S2A, B, D). Another sharp peak at 1720-1736 cm-1 associated with COOH stretching vibrations is also observed (Figure S2B, C, D). In addition, the peaks at 1245-1265 cm-1 and 1023-1040 cm–1 correspond to the asymmetric and symmetric stretching vibrations of C-O-C, respectively. Several peaks at 2927-2930 cm-1 and 1400-1414 cm-1 reveal the presence of CH2 and CH3 on 10

the CDs. What’s more, peaks at 1586 cm-1, 1556 cm-1, 1400 cm-1, 1228cm-1 can be assigned to asymmetric stretching vibrations of COO-, bending vibration of N-H, bending vibration of C-H and C-N, stretching vibrationsofr-O-C (Figure S2C, B, D). We also used XPS to investigate the element states of the CDs and the results were shown in Figure S3. All the four types of CDs show two apparent peaks centered at 284.8 and 532.3 eV which can be attributed to C 1s and O 1s. The peak at 399.8 eV associated with the binding energy of N1s is observed for CDs2, indicated that CDs2 is N doped (Figure S3B). In addition, CDs3 and CDs4 show two peaks at 229.0 and 161.3 eV that belongs to S2s and S2p (Figure S3C, D) . What’s more, N1s is also observed for CDs4 (Figure S3D). Combining these information, we can conclude that CDs2, CDs3, CDs4 are N, S, N-S doped, respectively. The results are in good agreement with FTIR. 3.2 Synthesis of AgNPs In order to understand the process of the reaction, the temporal evolution of the UV-vis spectra was recorded. A representative temporal evolution of UV-vis spectra for the synthesis of AgNPs1 was demonstrated in Figure S4A. Within one minute of AgNO3 added to the boiling CDs1, a pronounced color change occurred in suspension from colorless to light yellow and finally became brown-yellow. The inset is the corresponding evolution of absorbance intensity at 402 nm versus time, it is likely that the reaction could be divided into two stages. At stage (I) of the reaction (5-20 min), it could be the growth of AgNPs1, a significant increase in the absorbance intensity was 11

observed. At stage (II) of the reaction (30-60 min), the absorbance intensity decreased a little and then increased slowly and finally inclined to keep stable in company with a slight blue shift of the absorption peak, which perfectly agree with the process of Ostwald ripening. Figure S4B showed the hydrodynamic diameter of AgNPs1 characterized by Dynamic Light Scattering (DLS). At first, the hydrodynamic diameter decreased continuously at 5-20 min which may due to the combined effects of the consumption of CDs1 and the generation of AgNPs1. Because the surface of the water-soluble CDs consist of large amounts of hydrophilic groups such as -OH, -COOH, these hydrophilic groups can dramatically be hydrated and resulting the large hydrodynamic diameter of CDs. Then, the size gradually kept stable in consistent with the result of UV-vis spectroscopy. The as synthesized four types of AgNPs show the characteristic surface plasmon resonance peak at about 402, 409, 410, 404 nm, respectively (Figure S6), demonstrated that four types of AgNPs were successfully synthesized. 3.3 Stability of AgNPs under different pH values The stability of the as synthesized AgNPs1 under different pH values was studied by UV-vis spectra and DLS. In the experiments, the pH values were adjusted by diluted NaOH and HCl solution. As shown in Figure S5A, when the pH value was under 3, the absorbance intensity at 402 nm was obvious lower than that of the pH value between 3 and 12. The same phenomenon was observed when the pH value achieved at 13. Nearly no difference is presented between the pH value of 4 and 12 with respect to the absorbance intensity at 402 nm. Meaning that the synthesized 12

AgNPs show excellent stability property. The results were proved by DLS, as shown in Figure S5B. When the pH values were lower than 3 or higher than 12, the diameters were 9 times larger than of which the pH values from 3 to 12, it might due to the aggregation of AgNPs. Interestingly, there is no obvious difference in the size of the AgNPs1 from the pH value of 4 to 12 compared to the results of pH values of 1, 2 and 13, which confirmed the results of the UV-vis spectroscopy perfectly. 3.4 Characterization of AgNPs Figure 1 shows the TEM photographs of the four types of AgNPs synthesized by four different types of CDs. It can be seen that well-dispersed AgNPs were obtained. The corresponding size distribution of the obtained AgNPs were fitted well with Gaussian distribution (Figure S7) and the average size were 23.4±2.8, 17.2±2.3, 7.3±1.0, 6.1±0.8 nm, respectively.

The surface functional groups and element states of AgNPs were characterized by X-ray photoelectron spectroscopy (XPS). The four types of AgNPs show two apparent peaks centered at 284.8 and 532.3 eV which can attributed to C 1s and O 1s, respectively (Figure S8). Another peak at 161.1 eV associated with S 2p was observed in XPS spectrum of AgNPs4. In addition, the typical Ag 3d XPS spectrum show the binding energy of Ag 3d5/2 and Ag 3d3/2 at 368.1 and 374.1 eV respectively (Figure S9), which are consistent with Ag (0) [38]. Figure S10 show high resolution XPS spectra of C1s for four types of AgNPs. All of the spectrum can be fitted into four types of carbon bond, corresponding to sp2 (C=C) at 284.5-284.6, sp3 (C–C) at 13

285.4-285.6, C–OH at 286.3-286.7, as well as C=O at 287.4-287.9 eV [28]. However, C-S may also exist in AgNPs3 and AgNPs4 for both the binding energy of C-S, C-C are near 285.5 eV [39, 40].It should be noted that XPS only reflects the surface components of AgNPs, the relative contents of the core component cannot be obtained. 3.5 Antibacterial activity study

The as synthesized four types of AgNPs were used to study the antibacterial activity against E. coli by biological microcalorimeter. Microcalorimetry is a useful tool to study the microorganism metabolism for it can easily record the specific growth power-time curves of a microorganism because of its universal, integral, non-destructive and highly sensitive property, which has been proved by our group [41-43]. In this experiment, LKB-2277 Bioactivity Monitor was used to study the effects of AgNPs on the growth metabolism of E. coli. The concentration of the synthesized AgNPs was defined as silver atom concentration determined by ICP-AES. Before investigating the antibacterial activity of AgNPs, we studied the effects of four types of CDs on growth thermogenic curves of E. coli at first. The results were shown in Figure 2, We can see that different concentrations of C-dots (0-200 μg/mL) do not show any toxic effect on the growth of E. coli. Figure 3 showed thermogenic curves of E. coli growth in the presence of different concentrations of the four types of AgNPs. As shown in Figure 3A, when the concentrations of AgNPs1 were controlled lower than 100 μM, it has a little effect on the growth of E. coli. As the concentration increased, the log phase was delayed. A significant lag of log phase and decrease of 14

the maximum heat production was observed while the concentration of AgNPs1 reached at 200 μM. However, the half inhibition level couldn’t reach even at the concentration of 200 μM when treated with AgNPs2 (Figure 3B). In contrast, AgNPs3 and AgNPs4 performed excellent antibacterial activity and the growth of E. coli was totally inhibited when concentration of silver nanoparticles reach at 150 μM (Figure 3C and D). The increase of heat power (P) is exponential during the log phase. The growth thermogenic curves of the log phase correspond to Equation (1): lnPt=lnP0+kt

(1)

where t is the incubation time, Pt is the power output at time t, P0 is the power output at time t =0, and k is the growth rate constant. By using linear regression of lnPt versus t, the growth rate constant k of the microorganism can be obtained. The inhibitory ratio I can directly evaluate the antibacterial activity of AgNPs, which can be calculated through Equation (2): I=(k0−kc)/k0

(2)

where k0 and kc are the growth rate constants for non-treated and AgNPs-treated E. coli. Therefore, the bactericidal property of the AgNPs on the multiplying metabolism of E. coli can be analyzed through the changes of k and I. In addition, the maximum heat power Pm and the heat output Q are also important parameters for the metabolism of a microorganism as they reflect the ability of the microorganism to grow under in vitro environment condition. By analyzing the growth thermogenic curves of E. coli 15

in Figure 3, thermokinetic and thermodynamic parameters about the metabolism of E. coli treated with AgNPs that have good antibacterial activity were obtained (Table 1-3). The Pm decreases as the AgNPs concentration increases and it is dose dependent. The heat production Q of E. coli decreased when AgNPs concentration was low while higher than control when AgNPs concentration was beyond 150 µM. However, it was little affected by AgNPs3 and AgNPs4 (Figure S11). The relationship between the growth rate constant k, suppress ratio I and concentration c were shown in Figure 4. The growth rate constant k clearly decreases as the AgNPs concentration increases, suggesting that AgNPs can inhibit the metabolism of E. coli well. The k-c curves of AgNPs1 seems to be an exponential relationship while the curves are obvious linear relationship for AgNPs3 and AgNPs4. Through Equation 2, the half inhibitory concentration (IC50) can be calculated out. The value of IC50 can reflect the inhibition capability of a compound quantitatively. The smaller the value of IC50, the stronger its inhibitory activity. Therefore, the antibacterial activity of the four types of AgNPs can be compared by its IC50 values. The IC50 values of E. coli for AgNPs1, AgNPs3 and AgNPs4 were 194.42 µM, 81.35 µM and 83.72 µM, which indicate AgNPs3 and AgNPs4 show better performance in bactericidal property. 3.6 Mechanism of bactericidal effect 3.6.1 Stability of AgNPs in culture medium For the mechanism investigation, it is necessary to consider the stability of the 16

four types of AgNPs in culture medium because it could seriously influence the antibacterial activity. The results were shown in Figure 5. On the one hand, the absorbance intensity of AgNPs1 and AgNPs2 at about 400 nm decreases as time goes by. On the other hand, a new absorption peak appeared at 560 and 620 nm for AgNPs1 and AgNPs2 respectively. Dramatically red shift occurred gradually from 560 to 650 nm and from 620 to 680 nm during the monitoring for 54 minutes. It may due to AgNPs1 and AgNPs2 aggregates in culture medium because of salt and nutrients [44, 45]. However, AgNPs3 and AgNPs4 didn’t show new absorption peak meaning that it didn’t aggregate, which could be further proved by TEM photograph of E. coli. Furthermore, the relationship between the new absorption peak and time is a decaying exponential relationship (Figure S12). Herein, we assume that the aggregation extent of AgNPs can be reflected by the absorption peak, the aggregation exacerbate until it reaches an equilibrium. At first, the AgNPs concentration was high and the aggregation speed was fast. Later, the active AgNPs concentration that can be aggregated became low, the aggregation rate decreased. The surface charge of nanoparticles have a great influence on their stability. To identify the stability differences of the four types of AgNPs, their zeta potentials were measured. The results showed that AgNPs3 and AgNPs4 have a value of zeta potential lower than -30 mV, indicating a good stability of AgNPs3 and AgNPs4 while AgNPs1 and AgNPs2 demonstrated a zeta potential slightly higher than -30 mV (Table S1 ). 3.6.2 Effects of AgNPs on membrane properties of E. coli The outer membrane of E. coli is isolated from cells and serve as a barrier to 17

hydrophobic substances and macromolecules [46]. In addition, the process of nutrients move across the bacterial membrane depends on the permeability and fluidity of the outer membrane. Therefore, the bacterial membrane are vital to cell functions. When treated with AgNPs, it firstly interact with the outer membrane of E. coli. Thus, it is necessary to study the effects of AgNPs on the membrane property of E. coli. The destabilization of the outer membrane leads to an increased susceptibility to the bacteriolytic action of amphiphilic molecules, which cannot penetrate an intact outer membrane [47]. In general, 0.1% SDS is almost impermeability to E. coli. However, if the outer membrane is damaged, 0.1% SDS can easily permeate through E. coli. Figure 6 shows that pretreatment of the E. coli with AgNPs sensitized cells to bacteriolysis by 0.1% SDS, as demonstrated by a decrease in the bacterial turbidity (O.D.600). When treated with AgNPs1, the permeability of E. coli dramatically increased. We reasoned that the increase was perhaps caused by the accumulation on somewhere of the outer membrane. AgNPs3 and AgNPs4 show nearly equal effects but weaker than AgNPs1. While AgNP2 exhibits least effect on the outer membrane permeability of E. coli might derive from the high-level aggregation. To evaluate the membrane fluidity, two probes DPH and HP were used to label the membrane. 1,6-diphenyl-1,3,5-hexatriene (DPH) is associated with the lipophilic tails of phospholipids in cytoplasmic membranes with minimal membrane perturbations

for

its

hydrophobic

and

symmetrical property [48].

While

1,3,5,8-tetramethyl-2,4-bis(alpha-hydroxyethyl) prophine-6,7-dipropionicacid (HP) mainly probed on hydrophilic area and acyl group of the membrane [49]. When 18

excited with polarized light, they emit polarized fluorescence. The rotational diffusion of fluorophore is related to fluorescence depolarization and can reflect the membrane fluidity. The higher the polarization value, the lower the membrane fluidity. With the treatment of AgNPs, the membrane fluidity decreased (Figure 7). In details, the fluidity of the hydrophobic area of membrane decreased significantly when treated with AgNPs4. AgNPs1 showed more pronounced changes in decrease of the fluidity of membrane than AgNPs3. As for the fluidity of the hydrophilic area of membrane, significant decrease was observed when treated with AgNPs3. Both AgNPs4 and AgNPs1 didn’t demonstrate obvious effect. Interestingly, AgNPs2 showed little effect on the fluidity of E. coli membrane neither the hydrophobic nor hydrophilic area and it may due to the serious aggregation of AgNPs2. 3.6.3 Microscopic investigation TEM was used to clearly observe the morphology of E. coli when treated with AgNPs. Figure 8 shows the TEM images of E. coli cells treated with AgNPs that have good antibacterial activity. Unexposed to AgNPs, the surface of E. coli are smooth and clear. When treated with AgNPs1, the outer membrane was damaged. It may due to the enrichment of AgNPs1 on some area of E. coli because of aggregation and the ion release. While AgNPs3 and AgNPs4 just absorbed on the surface of the E. coli and no obvious damage to the outer membrane and cell wall was observed. Morones has reported that AgNPs may penetrate inside E. coli when the size of them is 1-10 nm [19]. Fortunately, the average size of AgNPs3 and AgNPs4 are smaller than 10 nm. Considering this, ultrathin sections were made to confirm 19

whether AgNPs can penetrate inside E. coli (Figure 8). Without pretreatment of AgNPs, the E. coli cells have good integrity and the surface are clean. With the treatment of AgNPs1, a few particles are absorbed on the surface of E. coli cells. However, when treated with AgNPs3 and AgNPs4, large amounts of the AgNPs absorbed onto the E. coli cells and parts of AgNPs were inside E. coli where the membrane was damaged. The cellular degradation was also accompanied by electron-translucent cytoplasm and cellular disruption in the damaged cells. 3.6.4 Generation of ROS Many reports illustrated that ROS is the key factor respond for death of cells when treated with AgNPs [50-52]. 2, 7-dichlorodihydrofluorescin diacetate (DCFH-DA) is the most commonly used probe to detect the intracellular ROS level. DCFH-DA itself couldn’t emit fluorescence. Once it goes into cells, it could be hydrolyzed by enzyme in cells to produce DCFH. After oxidized by ROS, it can exhibit strong fluorescence. Flow cytometer was used to detect the cellular ROS level of E. coli. Surprisingly, only parts of DCFH-DA can go into E. coli, it may due to the barrier of cell wall of E. coli. When treated with AgNPs, the uptake of DCFH-DA by E. coli increased especially for AgNPs1.The results of the generation of ROS were shown in Figure 9. AgNPs1 and AgNPs2 induced a higher level of ROS than AgNPs3 and AgNPs4, it may due to their size difference. For the four types of AgNPs in all, the ROS level have a less than about 50 % increase. Comparing to Carlson’s result that AgNPs induced a more than 10-fold increase of ROS levels in macrophages cells [25], it indicated that ROS is not the main factor for the death of E. coli. 20

4. Discussion CDs have been widely studied for its application in bioimaging, biosensors, photocatalysis et al. but their reducibility and capping property have rarely been exploited. The four types of synthesized CDs were used as capping and reducing agents to fabricate well-dispersed AgNPs composites by a one-step method. The reduction process of Ag+ is closely related to pH values. Without ammonia, little AgNPs were produced. It may be attributed to the higher reducing activity of the CDs under high pH values. We investigated the effects of time on the synthesis of AgNPs and found that well-dispersed AgNPs were obtained for about 45 min (Figure1). The stability of the obtained AgNPs1 at different pH values was studied (Figure S5). Nearly no SPR peak was found when pH value was lower than 2, which indicated almost all AgNPs aggregated in solution. The obtained AgNPs have good stability under a wide pH window for its little change from pH value of 3 to 12 illustrated by UV-vis spectra and DLS. It is reported that AgNPs have strong antimicrobial effects as well as low toxicity, good durability, and no drug resistance [53]. However, the exact mechanism of action of AgNPs as an antibacterial agent is not yet fully understood. Many reports explained that the antimicrobial activity of AgNPs on Gram negative bacteria dependents on the concentration of AgNPs, which has been proven by our results. (Figure 3) The stability of the AgNPs is an important factor for the antibacterial activity [54]. AgNPs1 and AgNPs2 aggregates in culture medium might be the main reason for their poor bactericidal performance. AgNPs2 is the most unstable one of the four types of synthesized AgNPs, therefore it has the worst 21

antibacterial activity. AgNPs3 and AgNPs4 are of good stability, so they exhibited good bactericidal property (Figure 4). The stability of AgNPs is closely related to their surface chemistry, which is related to the antibacterial performance. CDs3, CDs4 are S, N-S Co-doped, respectively. Sulfur has a high binding energy to silver. When apply CDs3 and CDs4 to synthesize AgNPs, the sulfur can tightly bind to silver. It may reason the difference in stability for the four types of AgNPs. Therefore, it is necessary to use S-doped CDs to fabricate stable AgNPs in future. AgNPs3 and AgNPs4 have similar surface modification, resulted in their similar stability property in culture medium. Besides, there is little difference in size between AgNPs3 and AgNPs4. Consequently, the IC50 values of E. coli cells for them are nearly the same. When mixed AgNPs and E. coli cells in culture medium, the AgNPs first interact with the salts and protein, forming protein corona [55]. Then, the protein corona capped AgNPs act on E. coli cells. The outer membrane is a barrier for E. coli cells to prevent toxic substance to enter into E. coli cells. As an excellent bactericidal agents, AgNPs can be defined as toxic compounds to E. coli cells. Therefore, it is essential to reveal changes of membrane property of E. coli cells when treated with AgNPs. Previous reports showed that the interaction between silver and the constituents of bacterial membrane caused structural changes and damage to the membranes and intracellular metabolic activity which might be the cause or consequence of cell death [23, 56]. We found that AgNPs1 can significantly enhance the permeability of outer membrane which might be due to the enrichment of AgNPs1 for its aggregation and existed on parts of E. coli membrane according to the TEM results (Figure 8). 22

However, similar phenomenon didn’t occur as for AgNPs2 because of too great extent of the aggregation and different ion release ability. The membrane fluidity of the hydrophilic and hydrophobic area were significantly decreased when treated with AgNPs4 and AgNPs3 respectively for large amounts of them absorbed and accumulated on the surface of E. coli membrane. Siva Kumar et al. proposed that oxygen associates with silver and reacts with the sulfhydryl (-SH) groups on cell wall to form R-S-S-R bonds thus, blocking respiration and causing death of cells [57]. The attachment of both silver ions and nanoparticles to cell wall caused accumulation of envelope protein precursors, which resulted in dissipation of the proton motive force. Silver nanoparticles also exhibited destabilization of the outer membrane and rupture of the plasma membrane, thereby causing depletion of intracellular ATP [11]. Numerous AgNPs3 and AgNPs4 are absorbed on the surface of E. coli cells (Figure 8). They can increase the outer membrane permeability and might easily bond to proteins containing sulfhydryl on membrane to damage the outer membrane. Sondi et al. reported that the action of AgNPs as a bactericide was closely associated with the formation of “pits” in the cell wall of the bacteria. Then, AgNPs accumulated in the bacterial membrane caused a increase in permeability, resulting in cell death [56]. Unfortunately, these “pit” were not obvious observed in our experiment. Morones Reported that AgNPs with the size 1-10 nm can penetrate inside E. coli and release Ag+ lead to cell death [19]. The size of AgNPs3 and AgNPs4 are smaller than 10 nm and ultrathin section experiments were conducted. Obviously, AgNPs1 cannot go into E. coli cells, only a little of them absorbed onto the surface of E. coli cells (Figure 8) 23

because some of them aggregated in PBS buffer. AgNPs3 and AgNPs4 were found to have the ability to permeate through E. coli cells (Figure 8) which may further act on the respiratory chain and result in the inhibition of metabolic activity of E. coli cells causing the death of E. coli cells. However, most of them were just on the surface of E. coli and AgNPs3 and AgNPs4 were just found in damaged E. coli. Thus, we suppose that only if the cell wall of E. coli cells damaged, can AgNPs3 and AgNPs4 enter into. Meanwhile, Ag+ can inevitably release from AgNPs when AgNPs absorb on or enter into E. coli and would cause a series events such as interact with respiratory chain and inhibit cell division leading E. coli death. Release of Ag+ and generation of ROS are the two main mechanisms for AgNPs caused E. coli cells death reported by previous studies [15, 50, 58]. In our experiments, the enhancements of ROS were less than about 50% (Figure 9), which showed little effect compared to other reports [25]. Therefore, ROS release is not the important events in E. coli death. Recently, Xiu et al. pointed out that the Ag+ release from AgNPs contributes to the antimicrobial activity directly by showing a lack of toxicity of AgNPs when tested under strictly anaerobic conditions that preclude Ag atom oxidation and Ag+ release [14], this further verified our results. For these evidence, we speculate that AgNPs absorb onto the surface of E. coli, penetrate inside E. coli, release of Ag+ are the main mechanisms that AgNPs perform excellent antibacterial activity and the schematic diagram is shown in Figure 10. Furthermore, only 150 μM AgNPs ( 16.2 ppm Ag) can completely inhibit E. coli growth. It is much better than the results of Kang [53] (37.6 ppm Ag) and Song [23] (500 ppm Ag). Inspiringly, the 24

as synthesized AgNPs have great stability for over 10 months which satisfy the requirement that AgNPs as a commercial antibacterial agents. Therefore, our AgNPs fabricated by CDs show much potential in antimicrobial systems.

5. Conclusions Well-dispersed four types of kinds of AgNPs have been fabricated through one step reduction process by using fluorescent CDs as reductant and stabilizer agents. The surface property could remarkably influence the stability of AgNPs resulting the different bactericidal property. AgNPs synthesized by CDs containing sulfur have excellent stability property and show better antibacterial performance, which can completely inhibit the growth of E. coli at a concentration of 150 μM of silver atom. We speculate that AgNPs act primarily in three ways against E. coli. Firstly, AgNPs can attach to the surface of the outer cell membrane and disturb its proper function, such as membrane permeability and fluidity. Secondly, AgNPs with small size (7.3±1.0, 6.1±0.8 nm) are able to penetrate inside the bacteria and cause damage by possibly interacting with intracellular substance such as DNA, protein and the respiration chain. Finally, release of the Ag+ could cause E. coli death.

Acknowledgements The authors gratefully acknowledge the financial support from National Science Foundation for Distinguished Young Scholars of China (21225313), National Natural Science Foundation of China (21473125), Natural Science Foundation of Hubei 25

Province (2014CFA003), Fundamental Research Funds for the Central Universities (2042014kf0287) and Large-scale Instrument And Equipment Sharing Foundation of Wuhan University.

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33

Figure 1. TEM photographs of the four types of synthesized AgNPs. Figure 2. Growth thermogenic curves of E. coli treated with CDs. Figure 3. Growth thermogenic curves of E. coli treated with AgNPs. Figure 4. Relationship between k, I and the concentrations of AgNPs. Figure 5. The stability of four AgNPs in culture medium. Figure 6. The outer membrane permeability of E. coli treated without or with 100 μM AgNPs. Figure 7. The membrane fluidity of E. coli treated without or with 100 μM AgNPs. Figure 8. TEM images and ultrathin section of E. coli cells. Figure 9. The ROS level in E. coli detected by Flow cytometer. Figure 10. Schematic of AgNPs synthesis and the mechanisms for their antibacterial activity.

34

Figure 1. TEM photographs of the four types of synthesized AgNPs. A) AgNPs1, B) AgNPs2, C) AgNPs3, D) AgNPs4.

35

Figure 2. Growth thermogenic curves of E. coli treated with CDs. A) CDs1, B) CDs2, C) CDs3, D) CDs4.

36

Figure 3. Growth thermogenic curves of E. coli treated with AgNPs. A) AgNPs1, B) AgNPs2, C) AgNPs3, D) AgNPs4.

37

Figure 4. Relationship between k, I and the concentrations of AgNPs.

38

Figure 5. The stability of four types of AgNPs in culture medium. A) AgNPs1, B) AgNPs2, C) AgNPs3, D) AgNPs4. The black line means AgNPs suspensions in water. The other colors represent AgNPs suspensions in culture medium from t=0 to 2, 4, 6, 8, 16, 24, 32, 48, 56 min.

39

Figure 6. The outer membrane permeability of E. coli treated without or with 100 μM AgNPs.

40

Figure 7. The membrane fluidity of E. coli treated without or with 100 μM AgNPs. A) probed with DPH, B) probed with HP. Error bars represent standard error of the mean. **P < 0.01 versus the control group.

41

Figure 8. TEM images and ultrathin section of E. coli cells. The concentration of AgNPs was 100 μM.

42

Figure 9. The ROS level in E. coli detected by Flow cytometer. The concentration of AgNPs was 100 μM. Error bars represent standard error of the mean. **P < 0.01 versus the control group.

43

Figure 10. Schematic of AgNPs synthesis and the mechanisms for their antibacterial activity. a) AgNPs with smaller size (7.3±1.0 nm, 6.1±0.8 nm) penetrate inside damaged E. coli and then interact with DNA in cytoplasm and respiratory chain on cytoplasm membrane. b) Release of Ag+ from AgNPs that absorbed on surface of the E. coli. c) Release of Ag+ from AgNPs that permeate through damaged E. coli.

44

Table 1. Parameters of E. coli growth at different concentrations of AgNPs1. AgNPs

c(µM)

k(min-1)

Pm(µW)

Q(J)

I(%)

AgNPs1

0

0.03019

29.67

0.329

0

6.25

0.03036

27.73

0.270

-0.56

12.5

0.03050

28.20

0.287

-1.03

25

0.02969

28.00

0.281

1.66

50

0.02910

26.26

0.288

3.61

100

0.02660

26.25

0.289

11.89

150

0.02261

20.60

0.387

25.11

200

0.01362

8.54

0.346

54.89

45

Table 2. Parameters of E. coli growth at different concentrations of AgNPs3. AgNPs

c(µM)

k(min-1)

Pm(µW)

Q(J)

I(%)

AgNPs3

0

0.03057

27.33

0.311

0

6.25

0.03082

28.73

0.299

-0.82

12.5

0.03061

28.30

0.319

-0.13

25

0.02800

28.53

0.305

8.41

50

0.02218

19.94

0.297

27.45

100

0.01110

12.83

0.328

63.69

150

-

0.72

-

100

46

Table 3. Parameters of E. coli growth at different concentrations of AgNPs4. AgNPs

c(µM)

k(min-1)

Pm(µW)

Q(J)

I(%)

AgNPs4

0

0.03097

28.8

0.282

0

6.25

0.02908

27.7

0.298

6.10

12.5

0.02933

28.8

0.292

5.30

25

0.02854

27.1

0.294

7.85

50

0.02621

23.9

0.293

15.37

100

0.01156

6.85

0.277

62.67

150

-

0.65

-

100

47