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Amino-Functionalized Graphene Oxide for the Capture and Photothermal Inhibition of Bacteria Lin Mei, Chunlei Lin, Fengyi Cao, Dehong Yang, Xu Jia, Sijie Hu, Xiaomei Miao, and Pengfei Wu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00348 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 27, 2019

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ACS Applied Nano Materials

Amino-Functionalized Graphene Oxide for the Capture and Photothermal Inhibition of Bacteria

Lin Mei, †* Chunlei Lin†, Fengyi Cao†, Dehong Yang†, Xu Jia†, Sijie Hu‡, Xiaomei Miao‡, Pengfei Wu‡

†School

of Materials and Chemical Engineering and ‡College of International

Education, Zhongyuan University of Technology, Zhengzhou, 450007

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ACS Applied Nano Materials 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

ABSTRACT Amino-functionalized graphene oxide (GO-NH2) was prepared for enhanced antibacterial properties through excellent photothermal efficiency. This nanosheets could easily target to the bacterial surface through the electrostatic attraction. Upon white light irradiation, GO-NH2 with superior antibacterial efficacy could inhibit the growth of gram-negative and gram-positive bacteria, resulting in the increase to 32times as against the antibacterial activity of GO alone. Through studies of antibacterial mechanism, it was found that the nanosheets could destroy the cytomembrane of bacterial, resulting in the cytoplasmic leakage. Moreover, the analysis of cell proliferation indicated that the nanosheets had good biocompatibility. This work indicated that graphene oxide could be used as a new nanostructured carbon material to construct antimicrobial agent for photothermal therapy in biomedical field.

Keywords: Graphene oxide; Antibacterial activity; Photothermal efficiency; Antibacterial mechanism; Surface modification; Cytomembrane permeability

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INTRODUCTION Infections by multidrug-resistant bacterial are potential threat to human health.[1] In the United States, the mortality caused by the infection of multidrug-resistant bacterial is 6.5%, leading to about 23,000 deaths every year.[2] Unfortunately, the development of new antibiotics cannot catch up with the increase of drug-resistant bacteria. Moreover, antibiotic treatment of drug-resistant bacterial infections will further lead to the rapid increase of drug-resistant strains. Therefore, the development of highly efficient and lowly resistant antimicrobial materials is urgently required. A wide range of antimicrobial materials, including antimicrobial peptides, polymers, and silver-based compounds (including silver salts and silver nanoparticles), have been used to effectively prevent proliferation of microbes and protect the public health in daily life. Antimicrobial peptides are important innate immune molecules against several pathogenic bacteria. However, due to proteases degradation, their half-lives are short in vivo.[3,4] Polymers can resolve the problems of antimicrobial peptides by bioinspired synthetic technology.[5,6] Recently, self-assembled cationic polymeric nanoparticles with better antimicrobial properties can efficiently interact with the bacterial cytomembrane via the increased cationic charges and local mass.[7] However, the synthetic approach of these polymers is relatively complex and high cost.[8,9] Silver with broad-spectrum antimicrobial activity can inhibit the growth of the bacteria, including several antibiotic resistant bacterial species, resulting in damage of bacterial cytomembrane and chaos of basic metabolic functions of the bacteria. However, these compounds can lead to the delivery of massive silver to wounds and surrounding tissue, 3



causing tissue toxicity and impaired wound healing. There is a popular supposition that physically damaging the bacterial cytomembrane is an alternative method against the potential bacterial resistance.[10] Recently, twodimensional nanomaterials, such as graphene,[11] MoS2,[12] and black phosphorus,[13] have attracted increasing attention because of their excellent optical, electrical, and thermal properties. Among them, graphene nanosheets with high mobility and high carrier concentration has received great attention in various fields and applications, such as biomedicine, supercapacitors, nanoelectronics, biosensors and conductive thin films.[14-16] Graphene oxide (GO) with excellent water-solubility exhibits membrane stress-mediated antibacterial activity, resulting in physical damages on cytomembrane by sharp edges of graphene nanosheets and the leakage of intracellular substance.[17] To improve the antimicrobial activity, the surface modification was used as an effective approach via oxygen-containing groups of GO. Musico et al found that polyvinyl-Ncarbazole modified GO nanocomposite presented outstanding antibacterial properties without obvious cytotoxicity to mammalian cells.[18] Tian et al reported a new multifunctional antibacterial nanocomposite by growing both Fe3O4 nanoparticles and silver nanoparticles on the surface of GO as recyclable and synergistic mode against the bacteria.[19] Bugli et al prepared GO-curcumin composites for drug-resistant bacteria infection treatment.[20,21] However, the antimicrobial action of interaction with bacterial cell membrane of GO-based nanocomposites are dependent on their concentration without through attraction with the bacteria, which limits their further applications. 4


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Recent studies indicated that antibacterial activity can be enhanced via introduction of amide linkages.[22] Surface aminated GO can be achieved by the oxygen-containing groups of GO surface through the amidation reaction, without the damage of graphene structure. Addition of amide groups is expected to increase the local density of the positive charge of GO surface for efficient capture of the bacteria with negatively charged cell membranes via the electrostatic interactions.[23] Then, the bacterial membrane can be direct contacted and damaged by the extremely sharp edges of GO nanosheets.[24,25] Moreover, GO was also used as photothermal agent to further improve antibacterial activity.[26] As far as we know, the use of surface aminated GO as antibacterial materials with photothermal antibacterial efficiency against the bacteria has not yet been reported. In this work, surface amino-functionalized GO (GO-NH2) was designed and synthesized as a new antimicrobial material for efficient capture and effective growth inhibition of the bacteria by white light irradiation. GO-NH2 was obtained by ethanediamine binding to GO with EDC chemistry. The antibacterial activity of the antimicrobial material was studied by a growth inhibitory assay. Furthermore, the antibacterial mechanism of GO-NH2 was also systematically explored. Finally, the cytocompatibility of the antimicrobial material was evaluated using HFL-1 cells. EXPERIMENTAL SECTION Materials Graphite powder, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-phenyl-1-naphthylamine (NPN) and 2-nitrophenyl β-D-galactopyranoside 5



(ONPG) was received from Aladdin Chemical Co., Ltd. (Shanghai, China). Potassium permanganate, hydrogen peroxide, sodium nitrate, potassium peroxydisulfate and ethylenediamine were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sulphuric acid (98%) was obtained from Beijing Chemical Reagent Co., Ltd. (Beijing, China). All of the chemicals were of at least analytical grade. Synthesis and characterization of GO-NH2 GO was prepared by the Hummers method.[27] 20 mL of 4.0 mg/mL GO was bathsonicated for 1 h and then 10 ml of 3.0 mol/L NaOH was added. After bath-sonicated for 3 h, 1.0 mol/L HCl was added to neutralizeand and the solution is centrifugated (10000 rpm for 30 min) and rinsed. Next, 60 mL ultrapure water and 0.5 mL ethylenediamine were added with bath-sonication for 5 min. EDC was then added to reach 5 mmol/L with bath-sonication for 30 min. Afterwards, enough EDC was added to reach 20 mmol/L with stirring for 12 h. After centrifugation at 2000 rpm for 30 min, the supernatant (the final product) was dialyzed (8-14 kDa M.W. cutoff) against ultrapure water for 24 h until further use.[28] Schematic representation of the synthesis of GO-NH2 was as illustrated in Fig. 1.

Fig. 1. Schematic representation of the synthesis of GO-NH2.

The UV-vis absorption spectra of GO and GO-NH2 were recorded on a UV-vis spectrophotometer (SHIMADZU UV-2700). The FTIR spectra of GO and GO-NH2 6


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were measured on a Fourier Transform Infrared Spectrometer (Nicolet iS50). The morphology of the obtained nanosheets was observed using an atomic force microscopy (AFM, NanoManVS). Elemental composition analysis was carried out by X-ray photoelectron spectroscopy (XPS, Axis Ultra DLD). Zeta potential values were determined with Zetasizer (Malvern, Nano ZS90). Bacterial growth inhibitory assay The bacteria were grown overnight in Luria-Bertani (LB) broth at 37 °C and subsequently diluted with LB broth. The fast-growing bacterial suspension was mixed with an equal volume of 2-fold diluted GO-NH2 (or GO) solution. After white light (159 mW/cm2) irradiation for 10 min, the mixture was incubated at 37 °C for 8 h. The growth inhibition of the bacterial cells was assessed by measuring an optical density at 600 nm (OD600) using UV-vis spectroscopy. Each assay was carried out in triplicate. Outer membrane permeabilization assay E. coli suspension was washed with phosphate buffer solution (PBS, 0.01 mol/L, pH 7.4) three times by centrifugation at 6000 rpm for 5 min. The remaining bacteria were resuspended in 1.5 mL of PBS. 50 μL of 1.0 mg/mL NPN was added into 1.5 mL of bacterial suspension (OD600 = 0.4). Then GO-NH2 solution with different concentration was pipetted into the above suspension with white light (159 mW/cm2) irradiation. The fluorescence intensity was determined immediately by fluorescence spectrophotometer (Hitachi, F-7100), with an excitation wavelength of 350 nm and an emission wavelength of 370 - 550 nm, respectively. The control assay was carried out by adding PBS instead of GO-NH2. 7



Inner membrane permeabilization assay The sample concentration for this study was used according to the test of bactericidal activity. Upon white light (159 mW/cm2) irradiation, 150 μL of 10 mg/mL ONPG and GO-NH2 with different final concentration were added into the E. coli suspension (OD600 = 0.4). The mixed solutions were determined by monitoring the change in OD420 using an UV-vis spectrophotometer with different times. The control assay was performed by adding PBS instead of GO-NH2. Preparation of bacterial sample for SEM The bacterial suspension (OD600 = 0.4) was washed with PBS and resuspended in 1.5 mL PBS. Then, 200 µL of 1.0 mg/mL GO-NH2 (or GO) was added and irradiated with the white light (159 mW/cm2) for 2 min. Free GO-NH2 (or GO) was removed by centrifuging at 6000 rpm for 5 min, washing by PBS three times and resuspending in 0.5 mL PBS. The treated bacterial were placed on the poly-ɛ-lysine coated glass slide and fixed with 2.5% glutaraldehyde overnight. After removing the excess glutaraldehyde solution, the remaining bacterial was dehydrated by ethanol aqueous solutions with different concentration (30, 50, 70, 90, 95, 100% v/v) for 10 min successively and dried in a freeze dryer. The sample was glued to the aluminum stud with double-sided adhesive conductive tape. After treated with platinum spraying, the bacterial morphology was observed using a field emission SEM (FEI Quanta 250 FEG). Cytotoxicity test The HFL-1 cells (human fetal lung fibroblast-1 cells) were offered from Henan University of Chinese Medicine (Zhengzhou, China). The cells were cultured in F-12K 8


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medium supplemented with 10% fetal bovine serum in 5% CO2/95% air at 37 °C. The cells were seeded into 96-well plates at a density of 104 cells per well and further incubated for 24 h. The GO-NH2 solution with different concentrations was diluted with culture medium. Then the medium in the wells was replaced with the pre-prepared sample solution. The irradiation with the white light (159 mW/cm2) for 10 min was performed for irradiation group. After incubation for 24 h, 10 µL of 5% MTT solution was added into each well for a further 4 h. Then the medium containing MTT was removed and 100 µL of DMSO was added into each well. After shaking for 15 min, the OD545 value was measured using a microplate reader. The untreated cells were used as positive control. RESULTS AND DISCUSSION Characterization of GO-NH2 From AFM image (Fig. 2), the obtained GO-NH2 possessed flat-sheet morphology with the existence of wrinkles. According to the corresponding height profiles (Fig. 2 inset), an average thickness was 1.36 ± 0.06 nm, indicative of monolayer nanosheets.

Fig. 2. A representative AFM image of GO-NH2 and the corresponding height profile (inset).

IR spectrum assay was used to confirm ethanediamine binding to GO surface (Fig. 3). In spectrum of GO-NH2, the peaks at 3447, 3383 and 1608 cm-1 can be assigned to 9



the N–H antisymmetric stretching, symmetric stretching, and bending vibrations, respectively. Moreover, the peak of C=O stretching vibration shifted from 1727 to 1651 cm-1 due to the appearance of amide linkage (an electron-withdrawing group) on GO sheet, indicating the successful conjugation of ethylenediamine to GO via amide linkage.

Fig. 3. FTIR spectra of GO and GO-NH2.

Further evidence for the formation of GO-NH2 was supplied from the XPS spectrum of GO and GO-NH2. From the spectrum of GO-NH2 in Fig. 4, the peak at 399.85 eV was attributed to N comparing with that of GO, indicating the presence of N-containing functional groups. High resolution N1s peaks in the spectrum of GO-NH2 (Fig. 4 inset) revealed that it consisted of two components with binding energies at 399.4 and 400.4 eV, which were assigned to -CONH- and -CH2-NH2, respectively.[29] These results are consistent with that of IR spectrum.

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Fig. 4. XPS spectrum and curve fitting of the N1s spectrum (inset) of GO-NH2.

UV-vis absorption spectra of GO and GO-NH2 were shown in Fig. S1. Comparing with the spectrum of GO, that of GO-NH2 shows a blue shift from 229 to 203 nm. The color of GO (A) and GO-NH2 (B) are deep yellow and black via a pictorial representation (Fig. S1, inset), respectively. From the surface charge analysis, the zeta potential of GO was found to be −15.8 mV. This negative zeta potential value indicated repulsion between GO and the bacteria. However, the zeta potential of GO-NH2 was 4.20 mV. These nanosheets with positive charge could enhance their adsorption to negatively charged bacterial membranes by electrostatic interaction.[23] From the results of IR spectrum, XPS, UV-vis spectrum and surface charge analysis, it can be confirmed that ethanediamine (amide groups) has been successfully grafted to the GO surface. Study of photothermal efficiency The observed temperature rise reflects the efficiency of photothermal energy conversion from the nanosheets to the solution. The sample concentration for this study was used according to the references.[30,31] Fig. 5 represents the photothermal efficiency 11



of GO-NH2 with three different concentrations (0, 0.10 and 0.25 mg/mL) upon white light (159 mW/cm2, 10 min) irradiation as a function of time. 0.25 mg/mL GO-NH2 had the change of temperature from about 20.5 to 81.4 °C in 10 min, which was higher than that of 0.10 mg/mL from about 21.0 to 55.5 °C. Compared with photothermal property of GO (Fig. S2), GO-NH2 exhibited better photothermal conversion efficiency with the same concentration. These results clearly demonstrate that the GO-NH2 solution with excellent photothermal effects has promising in antibacterial applications.

Fig. 5. Heating curves of 0, 0.10, and 0.25 mg/mL GO-NH2 under white light irradiation (159 mW/cm2).

Study of bactericidal activity To verify whether the GO-NH2 can interact with the bacteria, 200 μL 2.0 mg/mL GO-NH2 were added to freshly prepared bacteria dispersion in PBS. After incubation for 10 min, the black precipitant was appeared when GO-NH2 was added (Fig. 6A). Then the black precipitant was plated on LB agar plates. After 8 h incubation, the representative images of LB agar plates showed that the bacterial colonies cannot be 12


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observed. (Fig. S3) The negatively charged GO with low concentration was hard to damage the negatively charged cell membrane of the bacteria. However, the GO-NH2 with positive charge can enhance their adsorption to the surface of bacteria via electrostatic interactions and directly contact with the bacteria by the extremely sharp edges.[32]

Fig. 6. (A) Representative photos of the bacteria interaction with GO-NH2; (B) Growth inhibition of S. aureus and E. coli in the presence of GO-NH2 with concentration range from 0 to 128 μg/mL.

To evaluate the bactericidal activity of GO-NH2, the bacterial growth inhibitory assay was performed. In this assay, the bacterial suspension was mixed with an equal volume of 2-fold diluted GO-NH2 solution. Therefore, the concentrations were integral multiple. From Fig. 6B, the minimal inhibitory concentrations for GO-NH2 were about 32 and 16 μg/mL against S. aureus and E. coli under irradiation, respectively. When compared with that of GO (Fig. S4), the antibacterial activity against S. aureus and E. coli were increased up to 16 and 32-fold, respectively. As showed in heating curves of the nanosheets (Fig. 5), the temperature increase at MIC was mild. However, the 13



photothermal effect was not the main antibacterial mechanism. The antibacterial mechanism is the combined effects of photothermal effects and the edge of the GONH2. From the zone of inhibition test, the similar results were obtained (Fig. S5). It was reported that the antibacterial activity of GO was impacted in LB medium due to the adsorption of certain LB components on GO basal planes.[33,34] However, positively charged GO-NH2 were inclined to adsorb to the bacteria with more negative charge via electrostatic interaction rather than the protein in LB broth.[35] Therefore, GO-NH2 can exhibit enhanced antibacterial activity in LB broth. Moreover, it was found that the antibacterial activity against E. coli was much higher than that of S. aureus. The isoelectric point of E. coli was lower than that of S. aureus.[36,37] Thus, positively charged GO-NH2 were easily to adsorb to E. coli with more negative charge, result in stronger antibacterial activity. Study of bactericidal mechanism The membrane structure of gram-negative bacteria is different from that of grampositive bacteria. The former has both the outer membrane and the cytoplasmic membrane, while the latter lacks the outer membrane. Therefore, the gram-negative bacteria are selected for the study of outer membrane permeabilization by the hydrophobic fluorescent probe NPN with weak fluorescence in aqueous environments and strong fluorescence in hydrophobic environments.[38,39] When GO-NH2 disorganized outer membrane of bacterial cell (such as E. coli), NPN could partition into the outer membrane, causing the increase of NPN fluorescence intensity. The outer membrane is on the bacterial surface and easily to destroy. So, the GO-NH2 14


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concentration was lower. As shown in Fig. 7A, the increase of NPN fluorescent intensity in E. coli suspensions was almost independent of the nanosheets concentration and the interaction time. Whereas, the control group was unchanged obviously. These results indicated that GO-NH2 could permeabilizate the cytomembrane and destroy the integrality of bacterial cell. The cytoplasm of E. coli contains β-galactosidase, which can interact with ONPG to produce ONP (measuring OD420 by UV-vis spectroscopy).[40] Therefore, the permeabilization of the inner membrane (cytoplasmic membrane) of E. coli was studied by adding ONPG and GO-NH2 to the bacterial suspension. When the inner membrane was damaged, cytoplasmic β-galactosidase would leak out and interact with extracellular ONPG. From Fig. 7B, the increase of ONP concentration in E. coli suspensions was almost independent of GO-NH2 concentration and the interaction time, while the control group was unchanged obviously. This result indicated that GO-NH2 could disintegrate the bacterial inner membrane, result in leakage of cytoplasm.

Fig. 7. Fluorescence intensity of NPN (A) and absorption of ONP (B) at different times and concentrations.

The morphological change of the bacteria before and after interaction with GO-NH2 15



were investigated using SEM observations.[41,42] As showed in Fig. 8A&C, untreated S. aureus and E. coli with smooth surface and integrity of membrane structure were observed . The bacterial cytomembrane are deformed mildly after treatment with 118 µg/mL GO. In contrast, the morphology of the treated bacteria is altered significantly after incubation with 118 µg/mL GO-NH2 for 2.0 min (Fig. 8B&D). Serious damage to the cell walls of S. aureus was occurred with cell collapsing and membrane folding (Fig. 8B). Furthermore, the cytomembrane of E. coli was lysis with the leakage of large cytoplasmic components (Fig. 8D). Similar SEM images of various bacteria had been also observed in the bacteria treated with GO.[43,44] However, it should be noted that similar damaged condition of the bacterial cytomembrane was obtained with shorter interact time and lower dosage by aminated GO in this antimicrobial study, which is obviously different from the former SEM studies in the absence of white light irradiation. When the positively charged GO-NH2 combined with negatively charged bacterial cytomembrane by electrostatic interaction, the combined effects of photothermal effects and the edge of the GO-NH2 could enhance the antibacterial activity. It could disrupt the bacterial cytoplasmic membrane, leading to leakage of cytoplasm. Then the damaged cytomembrane subsequently caused the loss of membrane function and structural integrity, resulting in bacterial death.

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Fig. 8. SEM images of S. aureus (A and B) and E. coli (C and D) before (A and C) and after (B and D) interaction with GO-NH2 for 2 min.

Cell viability The cytotoxicity of GO-NH2 towards HFL-1 cells was studied using the MTT method. As showed in Fig. 9, the viability of HFL-1 cells after treatment with the nanosheets at different concentrations (from 16 to 512 μg/mL) without light irradiation was different between that of light irradiation. Without light irradiation, the cell viability of GO-NH2 was over 80% at concentrations ≤256 μg/mL, which was 8 and 16 times higher than the minimal inhibitory concentration of S. aureus and E. coli, respectively. With white light (159 mW/cm2) irradiation for 10 min, the cell viability of the nanosheets was over 80% at concentrations ≤128 μg/mL, indicating the appearance of light-induced cytotoxicity. Therefore, with light irradiation, good cytocompatibility and efficient antibacterial activity of GO-NH2 can be simultaneously obtained by keeping the concentration of 32 - 128 μg/mL.

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Fig. 9. Cell viability after incubation as a function of GO-NH2 concentrations determined by MTT assay.

CONCLUSION Antibacterial activity of amino-functionalized graphene oxide on gram-positive and gram-negative bacteria was investigated using photothermal property of GO. The advantages of the present method are as follows: (1) This nanosheets exhibits efficient capturing capacity of the bacteria through the electrostatic attraction; (2) Upon white light irradiation, the nanosheets with effective photothermal ability can rapid inhibit the bacterial growth, resulting in up to 32-fold increase in the antibacterial activity; and (3) This antibacterial strategy via dual physically damage (photothermal efficiency and the extremely sharp edges of the nanosheets) does not induce bacterial resistance. Through studies of the permeabilization of the outer and inner membrane and SEM observations, this nanosheets could destroy the bacterial cytomembrane, result in leakage of the cytoplasm. This antimicrobial material with better cytocompatibility could be used as a potential method to control the pathogenic bacterial infection.

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ASSOCIATED CONTENT Supporting Information Procedures of zone of inhibition test. Fig. S1 showing UV-vis spectra of GO and GONH2, and a photograph of GO and GO-NH2; Fig. S2. Heating curves of 0, 0.10, and 0.25 mg/mL GO under white light irradiation at a power density of 159 mW/cm2; Fig. S3. Representative images of LB agar plates after the black precipitant was plated on for 8 h incubation; Fig. S4. showing growth inhibition of S. aureus and E. coli in the presence of GO with concentration range from 0 to 512 μg/mL; Fig. S5 showing inhibition zones of GO and GO-NH2 against S. aureus and E. coli; Fig. S6. SEM images of S. aureus after being treated with GO for 2 min; Fig. S7. SEM images of E. coli being treated with GO for 2 min.

AUTHOR INFORMATION Corresponding Author * Tel.: +86 371 62506163. E-mail: [email protected] (Lin Mei)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 51703259 & 51703260) and Program for Interdisciplinary Direction Team in Zhongyuan University of Technology, China. 19



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Fig. 1. Schematic representation of the synthesis of GO-NH2. 338x254mm (300 x 300 DPI)



Fig. 2. A representative AFM image of GO-NH2 and the corresponding height profile (inset). 59x41mm (300 x 300 DPI)


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Fig. 3. FTIR spectra of GO and GO-NH2. 220x161mm (300 x 300 DPI)



Fig. 4. Wide XPS spectrum and curve fitting of the N1s spectrum (inset) of GO-NH2. 199x152mm (300 x 300 DPI)


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Fig. 5. Heating curves of 0, 0.10, and 0.25 mg/mL GO-NH2 under white light irradiation (159 mW/cm2). 214x194mm (300 x 300 DPI)



Fig. 6. (A) Representative photos of the bacteria interaction with GO-NH2; (B) Growth inhibition of S. aureus and E. coli in the presence of GO-NH2 with concentration range from 0 to 128 μg/mL. 478x269mm (300 x 300 DPI)


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Fig. 7. Fluorescence intensity of NPN (A) and absorption of ONP (B) at different times and concentrations. 338x123mm (300 x 300 DPI)



Fig. 8. SEM images of S. aureus (A and B) and E. coli (C and D) before (A and C) and after (B and D) interaction with GO-NH2 for 2 min. 173x150mm (300 x 300 DPI)


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Fig. 9. Cell viability after incubation as a function of GO-NH2 concentrations determined by MTT assay. 229x176mm (300 x 300 DPI)


Amino-Functionalized Graphene Oxide for the ... - ACS Publications

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