Metabolic Mechanism Investigation of Antibacterial Active Cysteine

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Metabolic Mechanism Investigation of Antibacterial Active Cysteine Conjugated Gold Nanoclusters in Escherichia Coli Ting-Kuang Chang, Tsai-Mu Cheng, Hsueh-Liang Chu, Shih-Hua Tan, Jui-Chi Kuo, Po-Hsuan Hsu, Chen-Yen Su, Hui-Min Chen, Chi-Ming Lee, and Tsung-Rong Kuo ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b03048 • Publication Date (Web): 18 Aug 2019 Downloaded from pubs.acs.org on August 19, 2019

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ACS Sustainable Chemistry & Engineering

Metabolic Mechanism Investigation of Antibacterial Active Cysteine Conjugated Gold Nanoclusters in Escherichia Coli

Ting-Kuang Chang,1,# Tsai-Mu Cheng,2,# Hsueh-Liang Chu,2 Shih-Hua Tan,1 Jui-Chi Kuo,1 Po-Hsuan Hsu,1 Chen-Yen Su,1 Hui-Min Chen,3 Chi-Ming Lee,4 and Tsung-Rong Kuo5,6*

1School

of Biomedical Engineering, College of Biomedical Engineering, Taipei

Medical University, 250 Wuxing St., Taipei 11031, Taiwan 2Graduate

Institute of Translational Medicine, College of Medicine and Technology,

Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan 3Department

of Anatomy and Cell Biology School of Medicine, College of Medicine,

Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan 4TMU

Core Facility Center, Taipei Medical University, Taipei 11031, Taiwan

5Graduate

Institute of Nanomedicine and Medical Engineering, College of

Biomedical Engineering, Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan 6International

Ph.D. Program in Biomedical Engineering, College of Biomedical

Engineering, Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan

Author Contributions #

Ting-Kuang Chang, and Tsai-Mu Cheng contributed equally to this work.

Corresponding Author *[email protected]

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Abstract Elucidating the metabolic mechanism of gold nanoclusters (AuNCs) in bacteria will play a pivotal role in bacterial detection and inhibition. A facile method to investigate the metabolic mechanism of AuNCs is demonstrated in this work. The bacterial nutrition of cysteine conjugated with gold nanoclusters (Cys-AuNCs) were successfully synthesized with orange-red fluorescence, high water solubility and superior biocompatibility by a one-pot green synthesis. The suggested metabolic process was that Cys-AuNCs were metabolized by Escherichia coli (E. coli), as verified through a decrease in the fluorescence intensity that was clearly detected at 30 min, indicating the breakage of cysteine on Cys-AuNCs, which was further confirmed via X-ray photoelectron spectroscopy (XPS), which was used to observe the size decrease in Cys-AuNCs after being metabolized. The metabolic kinetics of Cys-AuNCs were determined by fitting the change in the fluorescence of Cys-AuNCs as a function of incubation time with E. coli, in which the rate constant could be a useful indicator for detecting different bacteria. In addition, the death of E. coli was characterized by an increase in intracellular reactive oxygen species (ROS) through metabolism. After the metabolism of cysteine on Cys-AuNCs by E. coli, significant intracellular ROS generation was induced by the AuNCs which killed the bacterium due to its lack of the ROS scavenger, cysteine. Our work provides a potential rapid method for bacterial detection and inhibition.

Keyword: metabolism; cysteine; gold nanoclusters; antibacterial; kinetics; fluorescence; reactive oxygen species


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Introduction Recent advancements in biomedical sciences have revealed the important roles of nanomaterials due to their unique physical and chemical properties.1-9 Among various nanomaterials, gold nanoclusters (AuNCs) which can reach up to hundreds of gold atoms in size have been intensively developed as biomedical applications, due to their sensing and imaging capabilities.10-13 With different surface ligands and core sizes, AuNCs have exhibited broad fluorescent spectra in the range from ultraviolet to near-infrared with long lifetimes and large Stokes shifts.14 Moreover, with surface modifications via antibodies, peptides, polymers, and small molecules, AuNCs have been commonly applied as fluorescent probes for sensing and imaging because of their high biocompatibility, superior photostability, and excellent specificity. For example, target-specific glucose-conjugated AuNCs have been utilized as a fluorescent probe to detect glucose metabolism in glucose transporter-overexpressing U-87 MG cancer cells.15 Cyclodextrin-capped AuNCs have been used as a label-free fluorescent nano-sensor for specific detection of heavy metal ions such as Co2+ in aqueous solutions and in mammalian cells.16 After being decorated with a cell-penetrating oligoarginine peptide, AuNCs exhibited increased cellular uptake and a capability for intracellular imaging of highly reactive oxygen species (ROS) in living cells and zebraﬁsh.17 With high sensitivity, -lactoglobulin-stabilized AuNCs served as a sensor for detecting and quantifying Hg2+ in beverages, urine, and serum and were also explored as a cell and animal imaging agent.18 Compared to traditional organic fluorescent dyes, fluorescent AuNCs provide sharper images, longer periods of imaging time, and more-diverse biomedical applications. Advances in the preparation of fluorescent AuNCs with different functionalities have brought great potential for the development of highly sensitive techniques for sensing and imaging applications.



Microbes such as Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella, Clostridrium botulism, and Escherichia coli can cause fever, headaches, diarrhea, kidney failure, and even death.19-24 An outbreak of E. coli infection was recently reported due to contaminated romaine lettuce in North America in 2018. Rapid detection and quantification of microbes are important in assessing infection risks associated with contaminated food and water. Traditional assays for microbial pathogen identification include gram staining, culture, and biochemical methods, which are time-consuming and labor intensive. Currently, traditional assays have changed to molecular methods, including polymerase chain reaction amplification and enzyme-linked immunosorbent assay, which save time and feature easy sample preparation.25-27 However, due to the use of antibodies, molecular methods are relatively expensive compared to traditional assays. Recently nanomaterial-based fluorescent approaches have attracted much attention because of their low cost and facile process for detecting microbes. For example, multicolored fluorescence resonance energy transfer silica nanoparticles conjugated with monoclonal antibodies specific for pathogenic bacterial species including E. coli, Sal. typhimurium, and Sta. aureus have been applied for simultaneous and sensitive detection of multiple bacterial targets within 30 min.28 Fluorescent CdSe/ZnS core/shell quantum dots coupled with a 60-base aptamer were proven to specifically detect Bacillus thuringiensis spores at a concentration of about 1000 colony forming units (CFU)/mL.29 The peptide-bound Listeria monocytogenes on a glass surface was directly detected by labeling with highly fluorescent mercaptopropionic acid-capped AuNCs.30 Among different types of nanomaterials, AuNCs have emerged as the most important fluorescent probes for sensing and imaging microbes. However, after incubation with microbes, there is still a lack of information on the detailed mechanisms of metabolism of AuNCs.


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Antibiotics such as penicillins, cephalosporins, aminoglycosides, tetracyclines, macrolides, and fluoroquinolones have long been used to treat diseases caused by microbes.31-34 However, antibiotic-resistant bacteria caused by the overuse of antibiotics and various side effects associated with inappropriate antibiotic therapies have become serious issues in recent years.35-39 Nowadays, developing alternative approaches for treating antibiotic-resistant bacteria is still a major challenge for scientists.40 Recent advancements have applied AuNCs with different surface modifications as nanomaterial-based antimicrobial agents. For example, mannose-capped AuNCs were found to be efficient antibacterial agents for selectively inhibiting the growth of E. coli by inducing agglutination.41 Mercaptohexanoic acid-protected AuNCs exhibited interactions with both gram-positive and gram-negative bacteria to induce a metabolic imbalance in bacterial cells after internalization of the AuNCs, which resulted in increases in intracellular ROS production which consequently killed the bacteria.42 Quaternary ammonium-capped AuNCs with the capability for selectively targeting multidrug-resistant gram-positive bacteria including methicillin-resistant Sta. aureus and vancomycin-resistant Enterococcus spp. have been utilized to kill bacteria through combinations of physicochemical mechanisms such as disruption of bacterial cell membranes, catalytic generation of ROS, and disturbance of intracellular metabolic pathways.43 The intensive development of AuNCs as nanomaterial-based antimicrobial agents has significantly impacted the battle against bacteria. However, to the best of our knowledge, there is still a paucity of detailed information on the overall metabolic processes of AuNC digestion by bacteria. Therefore, investigating AuNCs conjugated with surface ligands metabolized by bacteria is the most important task in understanding the metabolic mechanisms of AuNCs in bacteria. With a better understanding of metabolic mechanisms, AuNCs can be fine-tuned to be highly



efficient nanomaterial-based antimicrobial agents in the near future. In this work, to investigate the metabolism of AuNCs in bacteria, cysteine was selected as a surface ligand to prepare AuNCs because the nutrition of cysteine can be metabolized by bacteria. Cysteine-conjugated gold nanoclusters (Cys-AuNCs) were synthesized using one-pot green synthesis. The water-soluble and biocompatible Cys-AuNCs were exploited as a fluorescent probe in E. coli. Fluorescence changes of Cys-AuNCs were utilized to investigate their metabolic kinetics in E. coli. Structural changes of Cys-AuNCs were characterized before and after incubation with E. coli by X-ray photoelectron spectroscopy (XPS). Moreover, to investigate the cause of death of E. coli, fluorescent images of E. coli were examined before and after incubation with Cys-AuNCs. ROS generation was also measured to demonstrate the cause of death of E. coli induced by Cys-AuNCs.

Experimental Section Materials Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. 96-Well plates were purchased from Falcon. SYTOX green and Hoechst 33342 were purchased from Thermo Fisher Scientific. 2’,7’-Dichlorofluorescein diacetate (DCFDA) was purchased from Sigma-Aldrich. L-cysteine (>99%) and gold(III) chloride (99%) were purchased from Acros.

Preparation of Cysteine-Conjugated Gold Nanoclusters One-pot green synthesis was applied to prepare Cys-AuNCs; 3 mL of a cysteine aqueous solution (50 mM) was added to 3 mL of a HAuCl4 aqueous solution (10 mM) in a round-bottomed flask. The round-bottomed flask containing the cysteine and HAuCl4 solutions was placed in a water bath (40 °C) with vigorous stirring. At first,


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the color of the reactant solution changed from yellow to milky. The reactant solution was further reacted in the water bath (40 °C) with protection from light under vigorous stirring for 24 h. After reacting 24 h, a milky solution containing Cys-AuNCs was obtained. For purification, the Cys-AuNC solution was then centrifuged at 15,000 rpm for 10 min. The supernatant was slowly removed without disturbing the precipitate. Afterwards, the precipitate containing the Cys-AuNCs was redispersed in 6 mL of deionized water by sonification. The purification process was repeated three times to remove any unconjugated cysteine. After purification, the Cys-AuNC solution was stored at 4 °C for further experiments.

Cell Viability Assay of Cys-AuNCs Vero cells were cultured in DMEM with 10% (v/v) FBS. Cell viability assays of a Vero cell line after treatment with Cys-AuNCs were carried out with a resazurin dye reduction assay. Vero cells (104 cells/well) were cultured in 96-well plates for 24 h and then washed twice with a phosphate-buffered saline (PBS) solution. Afterward, Vero cells in 96-well plates were treated with Cys-AuNCs solutions containing DMEM (Cys-AuNCs concentrations of 360, 180, 90, 45, 22.5, 11.25, and 5.625 μg/mL). An equal volume of water as the Cys-AuNCs solution was added to DMEM for the control experiments. After incubation of Cys-AuNCs for 24 h, the cell viability assay consisted of resazurin (at a final concentration of 0.02 mg/mL) being added to each well of the 96-well plates and incubated for 4 h. Values of the absorbance at 570 nm and 600 nm were detected by a plate reader. The cell viability assays were replicated eight times.

E. Coli Culture E. coli was cultured in LB medium in a shaker at 200 rpm under 37 °C. LB



medium was prepared by adding 25 g of LB Broth (Miller) to 1000 g of sterilized water. For metabolic experiments, E. coli (1 mL, 8 x 107 CFU/mL) was first added to a round-bottomed tube. The CFU value of bacterial solution was calculated based on the optical density at a wavelength of 600 nm (OD600). For E. coli, OD 600 of 1.0 = 8 x 108 CFU/mL. Afterward, the solution of E. coli was centrifuged at 4000 rpm for 5 min. After removal of the supernatant, 3 mL of Cys-AuNCs (0.36 mg/mL) was added to the round-bottomed tube with precipitation of E. coli. In the Cys-AuNC solution (0.36 mg/mL), the concentration of cysteine on the surface of Cys-AuNCs was calculated to be ~1 mM. The solution of Cys-AuNCs and E. coli was then incubated in a shaker at 200 rpm and 37 °C. After different incubation times, the solution of Cys-AuNCs and E. coli was measured with a fluorescence spectrometer.

Analysis of E. Coli Death by Fluorescence Imaging For fluorescence imaging, SYTOX green nucleic acid stain is an excellent green-fluorescent nuclear and chromosome counterstain that is impermeant to live cells and live bacteria, making it a useful indicator of dead cells and dead bacteria within a population. In this work, SYTOX green (5 μM) was added to E. coli solutions, and they were then incubated on a shaker at 200 rpm and 37 °C for 15 min (while being protected from light). Afterwards, the E. coli solution with SYTOX green was centrifuged at 10000 rpm for 2 min. After removing the supernatant, the precipitate of E. coli was redispersed in sterilized water. The washing process was repeated three times. Sequentially, the bacteria were seeded onto coverslips and then fixed with 4% paraformaldehyde. Hoechst 33342 solution (1 μg/mL) was also added to the coverslips. Bacteria on the coverslips were observed under a fluorescence microscope (Leica DM1000). For better image contrast, the SYTOX green channel was assigned a false green color, and the Hoechst 33342 channel was assigned a false


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blue color.

Analysis of Intracellular ROS Production by E. Coli To analyze ROS production, DCFDA dye was used to examine intracellular ROS concentrations. DCFDA is as a cell-permeable fluorogenic probe used to quantify ROS. DCFDA is rapidly de-esterified in cells and bacteria to form the fluorescent 2',7'-dichlorofluorescein (DCF) with excitation/emission spectra of 488/525 nm. In this study, DCFDA (10 μM) and Hoechst 33342 (1 μg/mL) were added to E. coli treated with a Cys-AuNC solution (1 mL, 0.36 mg/mL) and then incubated in a shaker at 200 rpm and 37 °C for 15 min. Afterwards, the E. coli solution with DCFDA was centrifuged at 10000 rpm for 2 min. After removing the supernatant, the precipitate of E. coli was redispersed in sterilized water. The washing process was repeated three times. The concentration of DCF was measured with a fluorescence spectrometer at excitation/emission wavelengths of 488/525 nm. The fluorescence intensity of DCF indicated the amount of ROS production. The fluorescence intensity of Hoechst 33342 with excitation/emission spectra of 350/461 nm indicated the total amount of bacteria. With different bacterial numbers in different experiments, the amount of ROS was normalized to the total bacterial number. The relative ROS level was calculated by normalizing the ROS level between the experimental group and the water group.

Results and Discussion Optical and Structural Characterizations of Cys-AuNCs Optical properties of Cys-AuNCs were measured on a UV-Vis absorption spectrometer (Jasco V-770) and fluorescence spectrometer (Jasco FP-8500). As shown in Figure 1a, the UV-Vis absorption spectra revealed the disappearance of the



surface plasmon absorption of gold nanoparticles because Cys-AuNCs exhibited high gold oxidation states which resulted in a lack of free electrons to generate coherent oscillations.44-45 Furthermore, with an excitation wavelength of 360 nm, the fluorescence spectra of Cys-AuNCs showed a maximum fluorescence intensity at 570 nm. The extra shoulder observed at 408 nm was due to the Raman signal of deionized water (see Supporting Information of Figure S1). The fluorescence quantum yield of Cys-AuNCs was measured to be 7.2% by integrating the sphere (Jasco ILF-835). The fluorescence of AuNCs was demonstrated from ligand-metal charge transfer between thiolate and AuNCs.46-48 The average size and shape of Cys-AuNCs were characterized by transmission electron microscopy (TEM, Hitachi HT-7700). Cys-AuNCs revealed a roughly spherical shape as shown in the TEM image of Figure 1b. HRTEM image of Cys-AuNCs was shown as Figure S2 in the supporting information. The average size of Cys-AuNCs was calculated to be 4.1 nm based on 100 nanoclusters in the TEM image of Figure 1b. A histogram of the size distribution of Cys-AuNCs and its Gaussian fitting curve are shown in Supporting Information as Figure S3. Compared to the size of Cys-AuNCs calculated by TEM image, the dynamic light scattering size of Cys-AuNCs was also obtained to be 4.5 nm using Malvern/Zetasizer Nano ZS. Fourier-transform infrared (FTIR) spectroscopy (Thermo Scientific Nicolet iS10) was used to examine cysteine before and after conjugation with the AuNCs. In the FTIR spectra of Figure 1c, cysteine exhibited characteristic IR bands including asymmetric stretching of COO- (1590 cm-1), symmetric stretching of COO- (1400 cm-1), bending vibrations of NH (1540 cm-1), stretching vibrations of NH (2356 cm-1), and a broad band of stretching of NH3+ (~3000 cm-1). Moreover, weak stretching of SH (2550 cm-1) of cysteine was observed before conjugation with the AuNCs. After conjugation of cysteine with the AuNCs, the FTIR spectrum of Cys-AuNCs revealed a slight shift compared to the FTIR spectrum of cysteine. The


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slight shift of the FTIR spectrum of Cys-AuNCs can be ascribed to a change in the dipole moment of cysteine after it was conjugated onto the surface of AuNCs with a high electron density.49 More importantly, the stretching of SH (2550 cm-1) of cysteine had disappeared from the FTIR spectrum of Cys-AuNCs due to the formation of Au-S covalent bonds between cysteine and AuNCs.50-52 Furthermore, the value of zeta potential of Cys-AuNCs was measured to be -25.2 mV. The molecular formula of Cys-AuNCs was determined to be Au18Cys15 by an electrospray ionization mass spectrometric (ESI-MS) analysis as shown in Figure S4. Overall, UV-Vis absorption spectra, fluorescence spectra, TEM images, FTIR spectra, zeta potential measurement and ESI-MS analysis confirmed that fluorescent Cys-AuNCs were successfully prepared by one-pot green synthesis.

Cell Viability Assay For applications as a fluorescent probe and antimicrobial agent, Cys-AuNCs have to be biocompatible to achieve bench-to-bed translation. To demonstrate their safe utilization, cell viabilities of Cys-AuNCs were investigated in a Vero cell line with a resazurin dye reduction assay. Water-soluble Cys-AuNCs of various concentrations (360, 180, 90, 45, 22.5, 11.25, and 5.625 g/mL) were respectively examined to investigate their cytotoxicities. As shown in Figure 2, the resazurin dye reduction assay revealed high cell viabilities (>80%) for Cys-AuNCs. To further confirm the cytotoxicity of Cys-AuNCs, SYTOX green dye was applied to stain dead Vero cells, and Hoechst 33342, a cell-permeant nuclear stain, was applied to stain all Vero cells (both alive and dead). After incubation with Cys-AuNCs for 2 h, there was no significant cell death of Vero cells as shown in fluorescence images of Figure S5. Results of the cell viability assay indicated that biocompatible Cys-AuNCs could be applied as a promising fluorescent probe and antimicrobial agent.



Metabolism of Cys-AuNCs by E. Coli To investigate the metabolism of fluorescent Cys-AuNCs, they were incubated with E. coli, and then changes in the fluorescence of Cys-AuNCs were detected after different incubation times. In the fluorescence spectra of Figure 3, the spectra of solutions composed by Cys-AuNCs and E. coli were integrated by three fluorescence signals including the Raman signal of water, fluorescence from the reduced form of nicotinamide adenine dinucleotide (NADH), and fluorescence from the Cys-AuNCs. By utilizing software for peak separation (Systat PeakFit 4.12), the spectra of Figure 3 were respectively separated into three peaks with different incubation times as shown in Figure S6. The spectra of Peak 1 at 408 nm were attributed to the Raman signal of water. Integrated areas under the spectra of the Raman signal of water revealed no significant changes at different incubation times. The spectra of Peak 2 located at 440 nm were contributed by a metabolite of NADH produced by E. coli.53 The intensities of Peak 2 were increased because NADH was produced and increased during the growth of E. coli after incubation with Cys-AuNCs from 0 to 90 min. Fluorescence spectra of cysteine (1 mM), sterilized water incubated with E. coli for 0 min, sterilized water incubated with E. coli for 90 min, cysteine (1 mM) incubated with E. coli for 0 min, and cysteine (1 mM) incubated with E. coli for 90 min were respectively provided in supporting information (Figure S7). As shown in Figure S7, fluorescence of NADH was increased during growth of E. coli with incubation of cysteine. After incubation with Cys-AuNCs for 120 min, the decrease in the intensity of Peak 2 was attributed to the death of E. coli. More importantly, Peak 3 located at 570 nm was contributed by fluorescent Cys-AuNCs. The fluorescence intensities of Cys-AuNCs decreased after incubation with E. coli from 0 to 120 min as shown in Figure S6. Decreases in the fluorescence intensities of Cys-AuNCs were ascribed to the


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metabolism of Cys-AuNCs by E. coli. The inset in Figure 3 showed the orange-red fluorescence coming from Cys-AuNCs at the beginning of incubation by E. coli, and then after incubation with E. coli for 120 min, the orange-red fluorescence had disappeared. The results showed that the fluorescence change of Cys-AuNCs caused by E. coli could be a promising fluorescent probe for microbial detection.

Metabolic Kinetics of Cys-AuNCs by E. Coli To further study the metabolic kinetics of Cys-AuNCs by E. coli, the integrated areas (At) under fluorescence spectra of Peak 3 in Figure S6 at different incubation times were calculated as the references of fluorescence intensities for Cys-AuNCs (Figure 4a). The obtained At was plotted as a function of different incubation times (t) as shown in Figure 4b. At decreased as t increased within 90 min and reached a minimum of At = 89,493, at t = 90 min. The metabolic rate of Cys-AuNCs by E. coli was calculated to be nearly constant (dAt/dt = k, zero-order kinetics) within 90 min. In zero-order kinetics, the value of the rate constant, k, was -707.4. However, as t exceeded 90 min, At revealed similar values because most of Cys-AuNCs had been metabolized by E. coli. The remaining value of At was very likely due to NADH produced by E. coli.54 The overall results in Figure 4 suggest that the fitted line of zero-order kinetics was the most suitable kinetics to reveal the mechanism of Cys-AuNC metabolism by E. coli. More importantly, in the equation of zero-order kinetics, the rate constant, k, could be a potential indicator for detection of different bacteria.55

XPS Analyses of Cys-AuNCs Before and After Incubation with E. Coli To characterize the metabolic mechanism, XPS was utilized to analyze Cys-AuNCs before and after incubation with E. coli. Before incubation with E. coli,



in the XPS spectra of Figure 5, the peaks of Au(4f5/2) and Au(4f7/2) for Cys-AuNCs were located at 87.8 and 84.2 eV, respectively. After incubation with E. coli for 120 min, the peaks of Au(4f5/2) and Au(4f7/2) for Cys-AuNCs were located at 88.7 and 85.0 eV, respectively. In comparison with bulk gold (87.4 (4f5/2) and 84.0 (4f7/2) eV), before incubation with E. coli, the XPS peaks of Au(4f) for Cys-AuNCs exhibited a positive shift because of the decrease in core gold atoms of AuNCs. Moreover, the positive shift in the XPS spectra also indicated that Cys-AuNCs had lots of Au(I).56 After incubation with E. coli for 120 min, the XPS peaks of Au(4f) for Cys-AuNCs obviously revealed a more-positive shift compared to that of Cys-AuNCs before incubation with E. coli. The result of the more-positive XPS shift indicated that after metabolism by E. coli, the size of Cys-AuNCs decreased, and also high oxidation states of gold were produced.57 Investigation of the Death of E. Coli To investigate E. coli death, fluorescence images of E. coli were examined after incubation with Cys-AuNCs for 120 min. As shown in fluorescence images of Figure 6, the total number of E. coli incubated with Cys-AuNCs for 120 min was greater than the total number of E. coli incubated in water for 120 min. The result indicated that E. coli can mobilize the cysteine on the surface of Cys-AuNCs for its growth. Moreover, after incubation in water, no significant E. coli death was observed in the fluorescence image. In comparison with incubation of Cys-AuNCs, drastic death of E. coli was revealed in the fluorescence image corresponding to a decrease in the fluorescence intensity of NADH. Many nanomaterial-based antimicrobial agents were demonstrated to kill bacteria due to their capability to increase ROS production.58 Therefore, ROS generation was investigated in E. coli after incubation with Cys-AuNCs. TEM image of Cys-AuNCs in E. coli was provided as Figure S8 to


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prove that Cys-AuNCs could be metabolized in E. coli. In Figure 7, Cys-AuNCs induced a 2.1-fold increase in the intracellular ROS production compared to that of control experiments in water. The increase in intracellular ROS was the major factor causing the death of E. coli after it had metabolized Cys-AuNCs. The death of E. coli indicated that after most of the ROS scavenger, cysteine, on the surface of Cys-AuNCs had been metabolized by E. coli, a significant increase of ROS was caused by AuNCs which killed E. coli due to a lack of the ROS scavenger.59 Overall, the death of E. coli demonstrated that Cys-AuNCs could be a potential antimicrobial agent.

Conclusions In conclusion, fluorescent Cys-AuNCs were successfully prepared with high water solubility and superior biocompatibility by one-pot green synthesis. Cys-AuNCs were proven to be a fluorescent probe to investigate their metabolic mechanism in E. coli. The metabolic kinetics of Cys-AuNCs in E. coli were systematically analyzed based on changes in the fluorescence intensity. The fitted curve of At versus t indicated that the metabolic kinetics of Cys-AuNCs by E. coli was zero-order kinetics. After metabolism by E. coli, the XPS peaks of Au(4f) for Cys-AuNCs exhibited a positive shift due to a decrease in the size of Cys-AuNCs and an increase in high oxidation states of gold. Moreover, the death of E. coli was expressed by an increase in intracellular ROS via metabolism of Cys-AuNCs by E. coli. Overall, our studies showed that Cys-AuNCs could be a practical fluorescent probe for bacterial detection and a potential antimicrobial agent.



Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Raman signal of deionized water, HRTEM image of Cys-AuNCs, histogram of nanocluster size distribution of Cys-AuNCs and Gaussian fitting, ESI-MS spectra of Cys-AuNCs, fluorescence images of Cys-AuNCs incubation with Vero cells, peak separation of Figure 3, fluorescence spectra of cysteine (1 mM), sterilized water incubated with E. coli for 0 min, sterilized water incubated with E. coli for 90 min, cysteine (1 mM) incubated with E. coli for 0 min, and cysteine (1 mM) incubated with E. coli for 90 min, and TEM image of Cys-AuNCs in E. coli.

Notes The authors declare no competing financial interests.

Acknowledgements We would like to acknowledge Ms. Yuan-Chin Hsiung for her excellent technical support at the TMU Core Facility Center. This work was supported by grants from the Ministry of Science and Technology (MOST 107-2113-M-038-004 and MOST 108-2113-M-038-003) and Taipei Medical University, Taiwan.


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Figures and Captions

Figure 1. (a) UV-Vis absorption spectra (black) and fluorescence spectra (red) of Cys-AuNCs. (b) TEM image of Cys-AuNCs. (c) FTIR spectra of cysteine (black) and Cys-AuNCs (red).

Figure 2. Assessment of cell viability by resazurin dye reduction assay with the range of 360~5.625 μg/mL of Cys-AuNCs in Vero cells.



Figure 3. Fluorescence spectra of Cys-AuNCs incubated with E. coli for different incubation times (0, 30, 60, 90, and 120 min). The fluorescence spectra were obtained with an excitation wavelength of 360 nm. The three arrows indicate the peak center of the Raman signal from water (408 nm), fluorescence from NADH (440 nm), and fluorescence from Cys-AuNCs (570 nm). The inset showed the orange-red fluorescence coming from Cys-AuNCs at the beginning (0 min) of incubation with E. coli (right) under irradiation of a hand-held UV lamp. And then the orange-red fluorescence had disappeared after Cys-AuNCs were incubated with E. coli for 120 min (left).


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Figure 4. (a) Fluorescence spectra of Cys-AuNCs incubated with E. coli at different incubation times. (b) Areas under the fluorescence spectra at different incubation times (At) of Cys-AuNCs were calculated. And then At was plotted as a function of different incubation times (t). The fitted curve of At versus t was simulated on the basis of zero-order kinetics. The value of R2 for the fitted curve is 0.9912.

Figure 5. XPS spectra of Cys-AuNCs. Before incubation with E. coli, the peaks of Au(4f5/2) and Au(4f7/2) were respectively located at 87.8 and 84.2 eV for Cys-AuNCs. After incubation with E. coli for 120 min, the peaks of Au(4f5/2) and Au(4f7/2) were respectively located at 88.7 and 85.0 eV for Cys-AuNCs.



Figure 6. Fluorescence images of Cys-AuNCs incubated with E. coli for 120 min. The blue and green pseudocolors represent the fluorescent signals of total E. coli (stained with Hoechst 33342) and dead E. coli (stained with SYTOX green), respectively. In the control experiments, E. coli were cultured with only water. The scale bars are 100 μm.

Figure 7. After incubation of Cys-AuNCs with E. coli for 120 min, intracellular ROS production was induced by AuNCs. The relative ROS level was normalized with the ROS concentration to the number of E. coli after incubation for 120 min. In the control experiments, the relative ROS level of E. coli treated with water was set to 1.0.


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References 1.

Grimsdale, A. C.; Mullen, K., The Chemistry of Organic Nanomaterials. Angew. Chem. Int. Ed. 2005, 44 (35), 5592-5629.

2.

Roduner, E., Size Matters: Why Nanomaterials Are Different. Chem. Soc. Rev. 2006, 35 (7), 583-592.

3.

Ulijn, R. V.; Smith, A. M., Designing Peptide Based Nanomaterials. Chem. Soc. Rev. 2008, 37 (4), 664-675.

4.

Zhang, H.; Liu, H.; Tian, Z.; Lu, D.; Yu, Y.; Cestellos-Blanco, S.; Sakimoto, K. K.; Yang, P., Bacteria Photosensitized by Intracellular Gold Nanoclusters for Solar Fuel Production. Nat. Nanotech. 2018, 13 (10), 900-905.

5.

Sakimoto, K. K.; Zhang, S. J.; Yang, P., Cysteine–Cystine Photoregeneration for Oxygenic Photosynthesis of Acetic Acid from Co2 by a Tandem Inorganic– Biological Hybrid System. Nano Lett. 2016, 16 (9), 5883-5887.

6.

Weng, B.; Lu, K.-Q.; Tang, Z.; Chen, H. M.; Xu, Y.-J., Stabilizing Ultrasmall Au Clusters for Enhanced Photoredox Catalysis. Nat. Commun. 2018, 9 (1), 1543.

7.

Zhang, B.; Pinsky, B. A.; Ananta, J. S.; Zhao, S.; Arulkumar, S.; Wan, H.; Sahoo, M. K.; Abeynayake, J.; Waggoner, J. J.; Hopes, C., Diagnosis of Zika Virus Infection on a Nanotechnology Platform. Nat. Med. 2017, 23 (5), 548-550.

8.

Rao, L.; Meng, Q.-F.; Bu, L.-L.; Cai, B.; Huang, Q.; Sun, Z.-J.; Zhang, W.-F.; Li, A.; Guo, S.-S.; Liu, W., Erythrocyte Membrane-Coated Upconversion Nanoparticles with Minimal Protein Adsorption for Enhanced Tumor Imaging. ACS Appl. Mater. Interfaces 2017, 9 (3), 2159-2168.

9.

Tsai, Y.-C.; Vijayaraghavan, P.; Chiang, W.-H.; Chen, H.-H.; Liu, T.-I.; Shen, M.-Y.; Omoto, A.; Kamimura, M.; Soga, K.; Chiu, H.-C., Targeted Delivery of Functionalized Upconversion Nanoparticles for Externally Triggered



Photothermal/Photodynamic Therapies of Brain Glioblastoma. Theranostics 2018, 8 (5), 1435-1448. 10. Wen, F.; Dong, Y.; Feng, L.; Wang, S.; Zhang, S.; Zhang, X., Horseradish Peroxidase Functionalized Fluorescent Gold Nanoclusters for Hydrogen Peroxide Sensing. Anal. Chem. 2011, 83 (4), 1193-1196. 11. Liu, C.-L.; Wu, H.-T.; Hsiao, Y.-H.; Lai, C.-W.; Shih, C.-W.; Peng, Y.-K.; Tang, K.-C.; Chang, H.-W.; Chien, Y.-C.; Hsiao, J.-K.; Cheng, J.-T.; Chou, P.-T., Insulin-Directed Synthesis of Fluorescent Gold Nanoclusters: Preservation of Insulin Bioactivity and Versatility in Cell Imaging. Angew. Chem. Int. Ed. 2011, 50 (31), 7056-7060. 12. Wu, X.; He, X.; Wang, K.; Xie, C.; Zhou, B.; Qing, Z., Ultrasmall near-Infrared Gold Nanoclusters for Tumor Fluorescence Imaging in Vivo. Nanoscale 2010, 2 (10), 2244-2249. 13. Li, C.-H.; Kuo, T.-R.; Su, H.-J.; Lai, W.-Y.; Yang, P.-C.; Chen, J.-S.; Wang, D.-Y.; Wu, Y.-C.; Chen, C.-C., Fluorescence-Guided Probes of Aptamer-Targeted Gold Nanoparticles with Computed Tomography Imaging Accesses for in Vivo Tumor Resection. Sci. Rep. 2015, 5, 15675. 14. Chen, L. Y.; Wang, C. W.; Yuan, Z.; Chang, H. T., Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging. Anal. Chem. 2015, 87 (1), 216-229. 15. Cheng, T. M.; Chu, H. L.; Lee, Y. C.; Wang, D. Y.; Chang, C. C.; Chung, K. L.; Yen, H. C.; Hsiao, C. W.; Pan, X. Y.; Kuo, T. R.; Chen, C. C., Quantitative Analysis of Glucose Metabolic Cleavage in Glucose Transporters Overexpressed Cancer Cells by Target-Specific Fluorescent Gold Nanoclusters. Anal. Chem. 2018, 90 (6), 3974-3980. 16. Lakkakula, J. R.; Divakaran, D.; Thakur, M.; Kumawat, M. K.; Srivastava, R.,


Page 22 of 29

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cyclodextrin-Stabilized Gold Nanoclusters for Bioimaging and Selective Label-Free Intracellular Sensing of Co2+Ions. Sens. Actuators B Chem. 2018, 262, 270-281. 17. Xie, Y.; Xianyu, Y.; Wang, N.; Yan, Z.; Liu, Y.; Zhu, K.; Hatzakis, N. S.; Jiang, X., Functionalized Gold Nanoclusters Identify Highly Reactive Oxygen Species in Living Organisms. Adv. Funct. Mater. 2018, 28 (14), 1702026. 18. Zang, J.; Li, C.; Zhou, K.; Dong, H.; Chen, B.; Wang, F.; Zhao, G., Nanomolar Hg2+Detection Using Β-Lactoglobulin-Stabilized Fluorescent Gold Nanoclusters in Beverage and Biological Media. Anal. Chem. 2016, 88 (20), 10275-10283. 19. Besser, R. E.; Lett, S. M.; Weber, J. T.; Doyle, M. P.; Barrett, T. J.; Wells, J. G.; Griffin, P. M., An Outbreak of Diarrhea and Hemolytic Uremic Syndrome from Escherichia Coli O157:H7 in Fresh-Pressed Apple Cider. JAMA-J. Am. Med. Assoc. 1993, 269 (17), 2217-2220. 20. Frank, C.; Werber, D.; Cramer, J. P.; Askar, M.; Faber, M.; An Der Heiden, M.; Bernard, H.; Fruth, A.; Prager, R.; Spode, A.; Wadl, M.; Zoufaly, A.; Jordan, S.; Kemper, M. J.; Follin, P.; Müller, L.; King, L. A.; Rosner, B.; Buchholz, U.; Stark, K.; Krause, G., Epidemic Profile of Shiga-Toxin-Producing Escherichia Coli O104:H4 Outbreak in Germany. N. Engl. J. Med. 2011, 365 (19), 1771-1780. 21. Hennessy, T. W.; Hedberg, C. W.; Slutsker, L.; White, K. E.; Besser-Wiek, J. M.; Moen, M. E.; Feldman, J.; Coleman, W. W.; Edmonson, L. M.; MacDonald, K. L.; Osterholm, M. T.; Belongia, E.; Boxrud, D.; Boyer, W.; Danila, R.; Korlath, J.; Leano, F.; Mills, W.; Soler, J.; Sullivan, M.; Deling, M.; Geisen, P.; Kontz, C.; Elfering, K.; Krueger, W.; Masso, T.; Mitchell, M. F.; Vought, K.; Duran, A.; Harrell, F.; Jirele, K.; Krivitsky, A.; Manresa, H.; Mars, R.; Nierman, M.; Schwab, A.; Sedzielarz, F.; Tillman, F.; Wagner, D.; Wieneke, D.; Price, C., A



National Outbreak of Salmonella Enteritidis Infections from Ice Cream. N. Engl. J. Med. 1996, 334 (20), 1281-1286. 22. Mølbak, K.; Baggesen, D. L.; Aarestrup, F. M.; Ebbesen, J. M.; Engberg, J.; Frydendahl, K.; Gerner-Smidt, P.; Petersen, A. M.; Wegener, H. C., An Outbreak of Multidrug-Resistant, Quinolone-Resistant Salmonella Enterica Serotype Typhimurium Dt104. N. Engl. J. Med. 1999, 341 (19), 1420-1425. 23. DePaola, A.; Kaysner, C. A.; Bowers, J.; Cook, D. W., Environmental Investigations of Vibrio Parahaemolyticus in Oysters after Outbreaks in Washington, Texas, and New York (1997 and 1998). Appl. Environ. Microbiol. 2000, 66 (11), 4649-4654. 24. McLaughlin, J. B.; DePaola, A.; Bopp, C. A.; Martinek, K. A.; Napolilli, N. P.; Allison, C. G.; Murray, S. L.; Thompson, E. C.; Bird, M. M.; Middaugh, J. P., Outbreak of Vibrio Parahaemolyticus Gastroenteritis Associated with Alaskan Oysters. N. Engl. J. Med. 2005, 353 (14), 1463-1470. 25. Gutierrez, R.; Garcia, T.; Gonzalez, I.; Sanz, B.; Hernandez, P. E.; Martin, R., A Quantitative Pcr-Elisa for the Rapid Enumeration of Bacteria in Refrigerated Raw Milk. J. Appl. Microbiol. 1997, 83 (4), 518-523. 26. Ofek, I.; Courtney, H. S.; Schifferli, D. M.; Beachey, E. H., Enzyme-Linked Immunosorbent Assay for Adherence of Bacteria to Animal Cells. J. Clin. Microbiol. 1986, 24 (4), 512-516. 27. Tamminen, M.; Joutsjoki, T.; Sjoblom, M.; Joutsen, M.; Palva, A.; Ryhanen, E. L.; Joutsjoki, V., Screening of Lactic Acid Bacteria from Fermented Vegetables by Carbohydrate Profiling and Pcr-Elisa. Lett. Appl. Microbiol. 2004, 39 (5), 439-444. 28. Wang, L.; Zhao, W.; O'Donoghu, M. B.; Tan, W., Fluorescent Nanoparticles for Multiplexed Bacteria Monitoring. Bioconjugate Chem. 2007, 18 (2), 297-301.


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


29. Ikanovic, M.; Rudzinski, W. E.; Bruno, J. G.; Allman, A.; Carrillo, M. P.; Dwarakanath, S.; Bhahdigadi, S.; Rao, P.; Kiel, J. L.; Andrews, C. J., Fluorescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus Thuringiensis Spores. J. Fluoresc. 2007, 17 (2), 193-199. 30. Hossein-Nejad-Ariani, H.; Kim, T.; Kaur, K., Peptide-Based Biosensor Utilizing Fluorescent Gold Nanoclusters for Detection of Listeria Monocytogenes. ACS Appl. Nano Mater. 2018, 1 (7), 3389-3397. 31. Kocaoglu, O.; Calvo, R. A.; Sham, L. T.; Cozy, L. M.; Lanning, B. R.; Francis, S.; Winkler, M. E.; Kearns, D. B.; Carlson, E. E., Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell Division. ACS Chem. Biol. 2012, 7 (10), 1746-1753. 32. McDougal, L. K.; Thornsberry, C., The Role of Β-Lactamase in Staphylococcal Resistance to Penicillinase-Resistant Penicillins and Cephalosporins. J. Clin. Microbiol. 1986, 23 (5), 832-839. 33. Vakulenko, S. B.; Mobashery, S., Versatility of Aminoglycosides and Prospects for Their Future. Clin. Microbiol. Rev. 2003, 16 (3), 430-450. 34. Gossen, M.; Freundlieb, S.; Bender, G.; Müller, G.; Hillen, W.; Bujard, H., Transcriptional Activation by Tetracyclines in Mammalian Cells. Science 1995, 268 (5218), 1766-1769. 35. Stewart, P. S.; Costerton, J. W., Antibiotic Resistance of Bacteria in Biofilms. Lancet 2001, 358 (9276), 135-138. 36. Neu, H. C., The Crisis in Antibiotic Resistance. Science 1992, 257 (5073), 1064-1073. 37. Classen, D. C.; Evans, R. S.; Pestotnik, S. L.; Horn, S. D.; Menlove, R. L.; Burke, J. P., The Timing of Prophylactic Administration of Antibiotics and the Risk of Surgical-Wound Infection. N. Engl. J. Med. 1992, 326 (5), 281-286.



38. Davies, J., Inactivation of Antibiotics and the Dissemination of Resistance Genes. Science 1994, 264 (5157), 375-382. 39. Singh, R.; Sripada, L.; Singh, R., Side Effects of Antibiotics During Bacterial Infection: Mitochondria, the Main Target in Host Cell. Mitochondrion 2014, 16, 50-54. 40. Smith, P. A.; Koehler, M. F.; Girgis, H. S.; Yan, D.; Chen, Y.; Chen, Y.; Crawford, J. J.; Durk, M. R.; Higuchi, R. I.; Kang, J., Optimized Arylomycins Are a New Class of Gram-Negative Antibiotics. Nature 2018, 561 (7722), 189-194. 41. Tseng, Y.-T.; Chang, H.-T.; Chen, C.-T.; Chen, C.-H.; Huang, C.-C., Preparation of Highly Luminescent Mannose–Gold Nanodots for Detection and Inhibition of Growth of Escherichia Coli. Biosens. Bioelectron. 2011, 27 (1), 95-100. 42. Zheng, K.; Setyawati, M. I.; Leong, D. T.; Xie, J., Antimicrobial Gold Nanoclusters. ACS nano 2017, 11 (7), 6904-6910. 43. Xie, Y.; Liu, Y.; Yang, J.; Liu, Y.; Hu, F.; Zhu, K.; Jiang, X., Gold Nanoclusters for Targeting Methicillin‐Resistant Staphylococcus Aureus in Vivo. Angew. Chem. Int. Ed. 2018, 57 (15), 3958-3962. 44. Shang, L.; Dong, S.; Nienhaus, G. U., Ultra-Small Fluorescent Metal Nanoclusters: Synthesis and Biological Applications. Nano Today 2011, 6 (4), 401-418. 45. Muhammed, M. A. H.; Verma, P. K.; Pal, S. K.; Kumar, R. A.; Paul, S.; Omkumar, R. V.; Pradeep, T., Bright, Nir‐Emitting Au23 from Au25: Characterization and Applications Including Biolabeling. Chem. Eur. J. 2009, 15 (39), 10110-10120. 46. Wu, Z.; Jin, R., On the Ligand’s Role in the Fluorescence of Gold Nanoclusters. Nano Lett. 2010, 10 (7), 2568-2573.


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


47. Luo, Z.; Yuan, X.; Yu, Y.; Zhang, Q.; Leong, D. T.; Lee, J. Y.; Xie, J., From Aggregation-Induced Emission of Au (I)–Thiolate Complexes to Ultrabright Au (0)@ Au (I)–Thiolate Core–Shell Nanoclusters. J. Am. Chem. Soc. 2012, 134 (40), 16662-16670. 48. Zheng, J.; Zhou, C.; Yu, M.; Liu, J., Different Sized Luminescent Gold Nanoparticles. Nanoscale 2012, 4 (14), 4073-4083. 49. Ma, X.; Guo, Q.; Xie, Y.; Ma, H., Green Chemistry for the Preparation of L-Cysteine Functionalized Silver Nanoflowers. Chem. Phys. Lett. 2016, 652, 148-151. 50. Xie, J.; Zheng, Y.; Ying, J. Y., Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters. J. Am. Chem. Soc. 2009, 131 (3), 888-889. 51. Tang, Q.; Hu, G.; Fung, V.; Jiang, D.-e., Insights into Interfaces, Stability, Electronic Properties, and Catalytic Activities of Atomically Precise Metal Nanoclusters from First Principles. Acc. Chem. Res. 2018, 51, 2793-2802. 52. Nieto-Ortega, B. n.; Bürgi, T., Vibrational Properties of Thiolate-Protected Gold Nanoclusters. Acc. Chem. Res. 2018, 51, 2811-2819. 53. Bulycheva, E. V.; Korotkova, E. I.; Voronova, O. A.; Kustova, A.; Petrova, E. V., Fluorescence Analysis of E. Coli Bacteria in Water. Procedia Chemistry 2014, 10, 179-183. 54. Forward, J. M.; Bohmann, D.; Fackler Jr, J. P.; Staples, R. J., Luminescence Studies of Gold (I) Thiolate Complexes. Inorg. Chem. 1995, 34 (25), 6330-6336. 55. Sekowska, A.; Kung, H.-F.; Danchin, A., Sulfur Metabolism in Escherichia Coli and Related Bacteria: Facts and Fiction. J. Mol. Microbiol. Biotechnol. 2000, 2 (2), 145-177. 56. Negishi, Y.; Nobusada, K.; Tsukuda, T., Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold (I)− Thiolate Complexes and



Thiolate-Protected Gold Nanocrystals. J. Am. Chem. Soc. 2005, 127 (14), 5261-5270. 57. Ishida, Y.; Akita, I.; Sumi, T.; Matsubara, M.; Yonezawa, T., Thiolate–Protected Gold Nanoparticles Via Physical Approach: Unusual Structural and Photophysical Characteristics. Sci. Rep. 2016, 6, 29928. 58. Fang, F. C., Antimicrobial Reactive Oxygen and Nitrogen Species: Concepts and Controversies. Nat. Rev. Microbiol. 2004, 2 (10), 820-832. 59. Sakimoto, K. K.; Wong, A. B.; Yang, P., Self-Photosensitization of Nonphotosynthetic Bacteria for Solar-to-Chemical Production. Science 2016, 351 (6268), 74-77.


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For Table of Contents Use Only

The Cys-AuNCs have been developed as a potential fluorescent probe for bacterial detection and inhibition in a short period of time.


Metabolic Mechanism Investigation of Antibacterial Active Cysteine

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