Fabrication of Nontoxic Reduced Graphene Oxide Protein

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Fabrication of Non-toxic reduced Graphene Oxide Protein Nanoframework as Sustained Antimicrobial Coating for Biomedical Application Priyadarshani Choudhary, Thanusu Parandhaman, Baskaran Ramalingam, Natarajan Duraipandy, Manikantan Syamala Kiran, and Sujoy K. Das ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

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ACS Applied Materials & Interfaces

Fabrication of Non-toxic reduced Graphene Oxide Protein Nano-framework as Sustained Antimicrobial Coating for Biomedical Application Priyadarshani Choudhary,a,b Thanusu Parandhaman,a,b Baskaran Ramalingam,a Natarajan Duraipandy,a,b Manikantan Syamala Kiran,a,b and Sujoy K Dasa,b,* a

Biological Materials Laboratory, Council of Scientific and Industrial Research (CSIR)-Central Leather Reserach Institute (CLRI), Chennai-600020, India, and bAcademy of Scientific and Innovative Research (AcSIR), New Delhi-110001, India.

Abstract. Bacterial colonization on medical devices is a major concern in the healthcare industry. In the present study we report synthesis of environmental sustainable reduced graphene oxide (rGO) at large scale through biosynthetic route and its potential application for antibacterial coating on medical devices. HRTEM image depicts formation of graphene nanosheet, while DLS and ζ potential studies reveal that in aqueous medium the average hydrodynamic size and surface charge of rGO are 4410 ± 116 nm and -25.2 ± 3.2 mV, respectively. The Raman, FTIR and XPS data suggest in-situ conjugation of protein with rGO. The as-synthesized rGO protein nano-framework exhibits dose-dependent antibacterial activity and potential of killing of 94% of Escherichia coli when treated with 80 µg/mL of rGO for 4 h. The hemolytic and cytotoxicity studies demonstrate that rGO protein nano-framework is highly biocompatible at the same concentration showing significant antimicrobial properties. The rGO coated on glass surface obtained through covalent bonding exhibits potent antibacterial activity. Antibacterial mechanism further demonstrates that rGO-protein nano-framework in dispersed state (rGO solution) exerts bactericidal effect through physical disruption accompanied by ROS mediated biochemical responses. The rGO subsequently enters into cytoplasm through damaged membrane causes metabolic imbalance in the cells. In sharp contrast, physical damage of the cell membrane is the dominant antibacterial mechanism of rGO in the immobilized state (rGO coated glass). The obtained results help indepth understanding of the antibacterial mechanism of the biosynthesized rGO and a novel way to develop non-toxic antibacterial coating on medical devices to prevent bacterial infection. Keywords: Pleurotus sajor-caju, reduced graphene oxide (rGO), biosynthesis, coating, antibacterial activity, hemolysis and cytocompatibility.

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1. Introduction Bacterial colonization on the medical devices including implants poses substantial health risks, requiring frequent replacement.1 Besides causing death to patients this culminates in huge financial loss. It is projected that more than $1.62 billion/year financial burdens will be incurred by 2020 on the healthcare system due to this implant related infection.2 In recent years the surface coating of the medical devices to prevent bacterial colonization is an active research area in biomedical field. Various strategies have been adopted for surface coating using antimicrobial polymers, quaternary ammonium salts, metal nanoparticles or metal ions to avoid bacterial infections.3-6 However, potential toxicity issues due to release of coating material along with simultaneous loss of antimicrobial efficacy are major areas of concern.4 In addition it involves high costs, tedious and intricate preparation process and materials handling problem. Thus coating the devices with new material to prevent microbial infection is highly desirable. Because of high surface area, mechanical strength, electrical conductivity, chemical inertness, and excellent optical and thermal properties graphene (a two dimensional atomic crystal consisting of sp2 carbon atom arranged in a hexagonal lattice) is attracting much interest in biomedical applications.7-12 Recent studies exhibited the antimicrobial activities of graphene,13,14 which inspired to explore the possibility of developing graphene based antimicrobial coating to prevent bacterial infection. The major concern for application of graphene coating on medical devices is the low availability of non-toxic graphene compounded by high production cost. Significant research efforts have been devoted to large scale production of graphene through reduction of graphene oxide (GO), including chemical, electro/photochemical and thermal reduction.15,16 However, use of toxic and corrosive reducing agents like hydrazine hydrate, hydrogen gas, high temperature, and prolonged reaction time raises toxicity, safety and economic issues. Thus, it is highly desirable to develop a new synthetic route for the production of reduced graphene oxide (rGO) under mild environmental conditions. Again, the rGO tends to aggregate due to π-π stacking interaction, which requires chemical modification to improve its stability. The bioinspired or biomimetic approach utilizing bacteria, fungi, algae, sea weed, plant, etc.17-26 has emerged as a highly attractive green chemical methodology for the production of different types of metal nanoparticles like gold, silver, platinum, palladium, copper, etc. In the biosynthetic process the proteins, amphiphilic biopolymers, having both hydrophobic and hydrophilic patches on their surfaces play major roles in the formation and stabilization of metal nanoparticles.20,21,26 Recent 2


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studies showed that fungal cell free proteins extract act as reducing and shape determining agent in the biosynthesis of metal nanoparticles.22,25 The initial success of metal nanoparticle biosynthesis inspired the exploration of microbial synthetic route for preparation of biocompatible rGO for biomedical application. A few recent studies have reported antibacterial activity of chemically synthesized rGO,14,27-29 however, the antimicrobial mechanism has not yet been fully understood. A thorough understanding of antimicrobial mechanism and toxicity of rGO are necessary prerequisites for its application in biomedical field. In the present study, we explored five different microbial species such as Pleurotus sajor-caju (mold), Rhizopus oryzae (fungi), Shewanella oniedensis (bacteria), Shewanella algae (bacteria) and Shewanella putrifaciens (bacteria) for environmental sustainable synthesis of rGO and investigated the potential biomedical application of the synthesized rGO. Of all the various microbial species, the synthesis of rGO at gram scale has been carried out using intracellular protein of P. sajor-caju and the as-synthesized rGO protein nano-framework applied for surface coating to prevent bacterial colonization. In addition, the physicochemical characteristics of the rGO and its effects on antibacterial activity, hemocompatibilty and cytotoxicity are evaluated. To understand that antibacterial mechanism and interfacial interaction with bacterial cells, the interaction of rGOprotein nano-framework with E. coli in dispersed (rGO solution) and immobilized state (rGO coated glass) has been thoroughly studied. We believe that the results obtained provide a novel approach towards potential use of biosynthesized rGO as non-toxic antibacterial coating on medical devices to prevent bacterial infection. 2. Experimental Section Materials Graphite

powder,

sodium

nitrate,

potassium

permanganate,

hydrogen

peroxide,

3-

(Aminopropyl)triethoxysilane (APTES), and all other fine chemicals were purchased from SigmaAldrich and used directly without further purification. Live/Dead viability assay kit was purchased from Invitrogen. All microbiological media and ingredients were purchased from Hi-media and used as stated. Ultrapure Millipore water (18.2 MΩ) was used in the biosynthesis experiments. Microorganisms The Shewanella oneidensis MR-1 (ATCC 700550) was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Luria Bertani agar (3.4 % of Luria Bertani broth 3



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and 1.5 % agar) slants, whereas Shewanella algae (MTCC 10455), Shewanella putrifaciens (MTCC 8104), and Escherichia coli (MTCC 062) were obtained from Institute of Microbial Technology, India and grown in nutrient agar (0.3% beef extract 0.5% peptone and 1.5 % agar) slants. Rhizopus oryzae (MTCC 262) and Pleurotus sajor-caju (MTCC 141) were also obtained from Institute of Microbial Technology, India and cultivated in potato dextrose agar (20% potato extract, 2% dextrose and 1.5% agar) slants. The organisms were stored at 4 °C and sub-cultured at regular intervals of 30 days to maintain the cell viability. Preparation of Biomass and Intracellular Protein Extract S. oneidensis was cultured in fresh LB broth and incubated at 30 °C for 24 h under shaking (120 rpm). S. algae, S. putrifaciens and E. coli were cultured in fresh nutrient media at 37 °C for 24 h in an incubator shaker (120 rpm). R. oryzae and P. sajor-caju were cultured in potato dextrose broth and incubated at 30 °C for 72 h and 28 °C for 240 h, respectively. The cultures following growth were harvested, washed three times with 50 mM phosphate buffer (pH 7.2) and dried by lyophilization. 0.1 g of the dried biomass was then weighed and used for synthesis of rGO. The intracellular protein was isolated from microbial biomass following the protocol as described in the supporting information and the protein concentration was estimated by BCA method.30 The intracellular protein was then used for biosynthesis of rGO as described above. In-situ Biosynthesis of rGO In situ biosynthesis of rGO was carried out by microbial reduction of GO. The aqueous solution of GO was initially prepared from graphite powder using modified Hummer’s method 31 and 10 mL of 1 mg/mL GO solution was taken in a 50 mL conical flask. The bacterial culture or fungal mycelia (0.1 g dry weight basis) were then added to this GO solution and incubated at 30 °C for 24 h under shaking. The synthesized rGO was purified by washing with deionized and double distilled water five times to remove the loosely bound protein from rGO surface. The solution was collected at different time intervals and subsequently characterized for synthesis of rGO. The yield of rGO formation was measured by UV-vis spectrometer and dry weight basis. The synthesis of rGO by intracellular protein extract was carried out to determine the role of protein(s) in the reduction of GO. The experiments were carried out as described above by incubating GO solution (1 mg/mL) in 50 mM phosphate buffer (pH 7.0) containing 0.5 mg/mL protein extract without altering other

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reaction conditions. The large scale (5 L) biosynthesis of rGO was carried out using intracellular protein of P. sajor-caju at optimum reaction conditions as described above. Characterization of rGO The as-synthesized rGO was collected from the reaction medium by centrifugation (12,000 rpm for 15 min), washed several times with deionized and double distilled water and finally sonicated in ultrapure water for 10 min. The UV-vis spectra of the solution were recorded on JASCO UV-vis spectrophotometer (V650 model) in the range of 200−800 nm. The size distribution and surface charge of rGO was determined, respectively by dynamic light scattering (DLS) and Zeta (ξ) potential measurement on Zetasizer (Malvern Zetasizer, UK) equipped with a 633-nm HeNe laser. The X-ray diffraction (XRD) and Thermogravimetric analysis (TGA) of the dried powder samples were recorded on Philips X-ray diffractometer at 0-80° with a Cu Kα source (λ= 1.54 Å) and SDT Q600 TA Instrument (New Castle, USA) from 25 to 800 °C with heating rate of 10 °C/min, respectively. Fourier transform infrared spectra of the samples were recorded on PerkinElmer FTIR spectrometer (FTIR-MB3000) from 500−4000 cm-1 at a resolution of 4 cm-1. The X-ray photoelectron spectroscopy (XPS) and Raman spectra of the samples were measured on Kartos AXIS 165 X-ray photoelectron spectrometer and Bruker RFS 27 FT-Raman Spectrometer, respectively. The morphology of the biosynthesized rGO was studied by high-resolution transmission electron microscopy (HRTEM, JEOL JEM 2010). Atomic force microscopic (AFM) image of rGO coated glass surface was recorded in tapping mode at ambient condition using a multimode AFM, while field emission scanning electron microscopic (FESEM) analysis was investigated by JEOL JSM 6700F FESEM instrument equipped with energy dispersive X-ray analysis (EDAX). Antibacterial Activity The antibacterial activity of the synthesized rGO was studied using E. coli as a model organism. Briefly, freshly grown E. coli cells (106 CFU/mL) were treated separately with different concentration of GO and rGO (0-80 µg/ mL) and incubated at 37 °C under shaking condition for various time intervals (0-4 h). At the end of incubation, 100 µL of the culture was spread over the nutrient agar plate and further incubated for 18 h at 37 °C in static condition. The phosphate buffer saline (PBS) (50 mM, pH = 7.2) was served as negative control, whereas gentamicin was served as positive control. The killing efficacy (%) was determined using following equation: 5



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Antibacterial efficacy (%) = [(Nnegative control ‒ Ntreated)/ (Nnegative control ‒ Npositive control)] × 100 where Nnegative

control,

Npositive

control

and Ntreated are numbers of cells grown on agar plate following

treatment with PBS, gentamicin and graphene material, respectively. Hemolysis Experiment and In-vitro Biocompatibility Study Hemolysis experiment was carried out as described by Devi et al.32 Different concentrations (20-80 µg/mL) of GO and rGO were incubated separately with 50 µL of 5% (v/v) red blood cells (RBC) in 50 mM PBS (pH 7.2) at 37 °C for 1 h with shaking (150 rpm). Triton X-100 and 50 mM PBS (pH 7.2) was served as positive and negative control, respectively. At the end of incubation, the hemolytic activity was measured spectrophotometrically at 540 nm using microplate reader (Tecan Infinite M200) and the percentage of hemolysis was determined using following equation:

where, EX, NC, and PC are absorbance of RBC solution following treatment with rGO, PBS, and Triton X-100, respectively. The biocompatibility of rGO was performed with 3T6 fibroblast cell line using water-soluble tetrazolium

salt,

2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-

tetrazolium monosodium salt (WST-8).33 Briefly, 105 cells/mL in DMSO medium supplemented with 5% fetal bovine serum (FBS) were seeded with gradient concentration of rGO in a 96-well plate and then incubated at 37 °C in a humidified atmosphere containing 5% CO2 in air. After 24 h incubation, cells were washed with serum free medium and stained with 20 µL of cell counting kit (CCK-8) solution containing WST-8 at 37 °C for 1 h. The formation of WST-8 formazan was measured at 450 nm using microplate reader (Tecan Infinite M200). The control experiment was performed under identical condition without addition of any compound. The effect of rGO on the cell viability was determined using formula

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The cell viability of both treated and untreated cells were also studied using Live/Dead assay kit and the fluorescent microscopic images of the cells were recorded on Leica fluorescence microscope (DMIR-B). More detailed experimental procedure is described in Supporting Information. Coating of rGO on Glass Surface The ultrasonically cleaned glass substrate was initially treated with APTES (10 µL of APTES in 20 mL of isopropyl alcohol) for 3 h at 35 °C, washed twice with isopropyl alcohol, and double distilled water and further incubated at 100 °C for 1 h. The amine functionalized glass substrate was cooled down to room temperature and treated with 80 µg/mL of rGO for 24 h. The rGO coated glass surface was washed twice with double distilled water and ethanol to remove loosely bound rGO and finally dried at 60 °C overnight. The stability of the coating was checked by washing the glass surface several times under water. The contact angle of rGO coated glass substrate was measured on Holmarc (HO-IAD-CAM-01B) instrument and compared with the untreated glass substrate. Atomic force microscopic (AFM) image of rGO coated glass surface was recorded in tapping mode at ambient condition using a multimode AFM (NTEGRA Prima), while field emission scanning electron microscopic (FESEM) analysis was investigated by JEOL JSM 6700F FESEM instrument equipped with energy dispersive X-ray analysis (EDAX). Further, the antibacterial activity of the rGO coated glass surface was tested by zone of inhibition study. The uncoated glass surface was taken as a control. Antibacterial Mechanism Measurement of Cell Membrane Damage The alteration of the cell membrane potential of E. coli (106 CFU/mL) upon treatment with rGO (80 µg/mL) was investigated by staining the cells with 5 µL of SYTO 9 and propidium iodide (PI) solution. The gentamicin and PBS treated cells were served as positive and negative control, respectively. The fluorescent microscopic images of the cells were recorded on Leica fluorescence microscope (DMIR-B). The estimation of intra and extracellular protein and nucleic acids in untreated and rGO treated cells was determined by BCA method30 and UV-vis spectrometer, respectively (see Supporting Information for details).

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Cell Size and Surface Charge Measurement rGO (80 µg/mL) treated E. coli (106 CFU/mL) were suspended in 50 mM sterile phosphate buffer (pH 7.2) and surface charge and cell size were recorded on Zetasizer (Malvern Instruments, UK). The phosphate buffer treated cells was served as control. Intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) Concentration The intracellular ROS production and GSH concentration in rGO treated E. coli were measured following the protocols as described earlier.34,35 In brief, E. coli (106 CFU/mL) was initially incubated with 200 µM of H2-DCFDA at 37 °C for 30 min in dark condition followed by addition of rGO (0-80 µg/mL). The cells were further incubated for 4 h under similar condition and the fluorescence spectra of the cell suspension were recorded on a Shimadzu RF-6000 spectrofluorometer at excitation of 503 nm. For intracellular GSH concentration measurement the rGO treated E. coli was lysed with 5% TCA for 15 min on ice and the cell lysate (100 µL) was mixed with 900 µL of Tris-HCl (pH 8.3) and 100 µL of 1 mg/mL o-phthaldialdehyde solution. The reaction mixture was then incubated for 90 min in dark at 30 °C. The fluorescence intensity of the solution was measured on Shimadzu RF-6000 spectrofluorometer with excitation and emission wavelengths of 350 nm and 420 nm, respectively. The concentration of GSH was determined using the standard curve. The phosphate buffer (50 mM, pH 7.2) and H2O2 (1 mM) treated cells were served as negative and positive control, respectively in both experiments. Membrane Depolarization and Lipid Peroxidation Assay The membrane depolarization of E. coli upon treatment with rGO was examined as described by Thiyagarajan et al.36 with little modification. Briefly, E. coli (106 CFU/mL) in HEPES buffer (5.0 mM HEPES, 20 mM glucose, 100 mM KCl pH 7.2) suspensions was incubated with 1 µM DiSC3(5) for 1 h at 37 °C. Different concentration of rGO was added to the cell suspension and fluorescence emission spectra (λEx = 622 nm and λEm = 670 nm) was recorded at different time intervals in a spectrofluorometer (Shimadzu RF-6000 spectrofluorometer). The fluorescent microscopic images of both treated and untreated cells were also recorded on Leica fluorescence microscope (DMIR-B). For lipid peroxidation assay, the rGO treated E. coli was lysed with 10% TCA as described above and 1 mL of the cell lysate was mixed with equal volume of thiobarbituric acid (TBA) reagent (50 mM in glacial acetic acid). The reaction mixture was boiled for 30 min in water bath and cooled down to room temperature for absorbance measurement on UV-vis spectrophotometer at 532 nm. 8


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Tetracycline treated cell was served as positive control, whereas untreated cells served as negative control. Respiratory Chain Dehydrogenase Activity Assay The effect of rGO on respiratory chain enzyme, lactate dehydrogenase activity of E. coli was carried out as described earlier by Das et al.37 100 µL of iodonitrotetrazolium chloride (INT, 50 mg/mL) solution was added to both rGO treated and untreated E. coli cells suspension and incubated at 37 for 60 min in dark. Cells were collected at the end of incubation, and cell pellets were dissolved in 1 mL solutions of acetone and ethanol (1:1 ratio) mixture. The formation of water insoluble iodonitrotetrazolium formazon (INF) was recorded at 490 nm on UV-vis spectrophotometer. The tetracycline treated cell was served as positive control. Interaction of rGO with DNA The genomic DNA was isolated from E. coli after being treated with different concentration of rGO (0–80 µg/mL) for 6 h using phenol/chloroform extraction method (see Supporting Information).38 The isolated DNA samples were then analyzed by 1.0% agarose gel electrophoresis followed by staining with ethidium bromide. In a separate experiment, the isolated genomic DNA was incubated with rGO at different concentration for 1 h at 37 °C and then analyzed by gel electrophoresis as described above. Statistical Analysis. All the experiments were repeated five times and data represent an average of five independent experiments ± SD (standard deviation) shown by error bar. Two-tailed Student’s t test was performed for calculating the statistical significance at p value < 0.05. 3. Results and Discussion Synthesis and Characterization of rGO In situ bioreduction of GO to rGO was carried out through incubation of GO with microbial biomass for 24 h. This leads to gradual color change of GO solution from light brown to black (Figure 1A), whereas, the control experiment without biomass remains light brown. The UV-vis spectrum (Supporting Information Figure S1) shows red shifting of the characteristic absorbance peak of GO from 230 nm to 260 nm in rGO upon the reduction process. Initially different microbial species were screened to explore the synthesis of rGO because of diverse metabolic activity of the microorganisms. It was observed that among all the biomass tested, maximum yield of rGO (Figure 9



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1B) obtained when GO was treated with P. sajor-caju. The role of microbial protein in synthesis of rGO was investigated using intracellular protein of the organisms and results demonstrated that the intracellular protein of P. sajor-caju also produced maximum amount of rGO (data not shown) compared to the other organisms under similar reaction conditions. It is worth to mentioning that highest amount of biomass production and intracellular protein concentration are obtained in P. sajor-caju compared to other species tested in this study (Supporting Information Table S1). Based on the yield of rGO, biomass production and intracellular protein concentration, P. sajor-caju was selected of all other species for biosynthesis of rGO. The kinetics of rGO production (Figure 1C) by intracellular protein of P. sajor-caju revealed that absorption band intensity of rGO increases with time and reaches saturation after 12 h incubation at 30 °C. Adopting this biosynthetic strategy, the synthesis of rGO was scaled up to 5 L volume in a single batch with a yield of 4.2 ± 0.51 g at ambient condition (Supporting Information Figure S2) using intracellular protein. We believe that simple and low cost growth medium for preparation of P. sajor-caju intracellular protein will make this process economically viable for large scale production of rGO. The average hydrodynamic sizes of GO and rGO were measured both in aqueous solution and NB broth by DLS and the result is shown in Table 1. It is noted that the initial average hydrodynamic size of GO is 3352 ± 86 and 3678 ± 105 nm in aqueous solution and NB broth, respectively. However, after reduction of GO with protein, the hydrodynamic sizes of rGO increases to 4410 ± 116 and 4819 ± 221 nm, respectively in aqueous solution and NB broth. Previous studies also reported the higher hydrodynamic size of rGO compared to GO,39-42 which are in good agreement with our observation. Albeit the exact reason for this size variation is not clearly understood, we presume that reduction and subsequent functionalization with protein increases the Brownian motion rate of rGO. Wang et al.40 reported that reduction of GO with heparin increases the average hydrodynamic size of rGO in comparison to the pristine GO. Liao et al33 demonstrated that aggregation of graphene sheet in aqueous solution is the leading cause of its higher hydrodynamic size compared to GO. We therefore, tested the water dispersibility of GO and rGO-protein nano-framework in deionized and double distilled water at different time intervals. Initially, both the samples were sonicated in deionized and double distilled water at a concentration of 0.5 g/mL for 10 min and kept it room temperature (30 °C) for 24 h. The result (Supporting Information Figure S3) shows that rGO has better dispersibility in aqueous solution than the GO and remained disperse even after 24 h. We therefore, suggest that in situ functionalization of the as-synthesized rGO with protein is responsible for high dispersibility. Moreover, the protein conjugation may also plays role for higher 10


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hydrodynamic size of rGO-protein nano-framework compared to the GO precursor. The hydrodynamic size of the particles since depends on the size, surface properties, aggregation, state of dispersion, stability/biodegradability, dissolution, hydration and solvent characteristics; this invites further studies for complete understanding the higher hydrodynamic size of rGO than the GO. The surface charge of GO and rGO was determined by ξ potential analysis, which recorded ξ potential values (Table 1) of GO and rGO as -33 ± 2.73 and -25.2 ± 3.2 mV, respectively at pH 6.0. The increase of ξ potential value of rGO might be due to removal of oxygen containing functional groups in rGO. Functionalization of the rGO with proteins during biosynthesis process might also reduce the surface charge density of rGO. The observed ξ potential of rGO (-25.2 ± 3.2 mV) is close to the accepted values of colloidal stability (±25 mV), indicating high stability of the rGO solution. The crystal structure of the GO and rGO was characterized by XRD. The spectrum of GO (Figure 2A) shows a sharp peak at 2θ value of 11° (001) corresponding to the interplanar distance of 0.83 nm between GO sheets.43 Upon interaction with protein this peak disappears and a new broad peak appears at 23.4° (002), which is very similar to that of graphite. This suggests staking of graphene sheets upon the removal of oxygen containing functional groups.44 TGA analysis was carried out to further assess the reduction of GO and Figure 2B depicts the weight loss profile of both GO and rGO as a function of temperature. The TGA curve of GO shows initial weight loss of 4% with an onset temperature at slightly more than 100 °C due to removal of bound water molecule. The second stage of weight loss of about 23% in the range of 150-250 °C is associated with the decomposition of oxygen containing groups. However, a significant weight loss is observed between 450-600 °C due to the pyrolysis of carbon skeleton.45 A total weight loss of ~70% is recorded at temperature below 800 °C. In contrast, the TGA curve of rGO shows higher thermal stability and a total weight loss of ~35% is recorded at temperature below 800 °C due to deoxygenation of rGO with enhanced van der Waals forces between layers.46 Raman spectra (Figure 2C) of GO and rGO were recorded to understand the structural changes of GO upon reduction process. The characteristic G band related to first-order scattering of E2g photon of sp2 carbon atom appears at 1576 cm-1, while the D band leading to the structural imperfection induced by hydroxyl/ or epoxide group is assigned at 1338 cm-1 in GO.47 On the other hand, the G band is blue shifted to 1557 cm-1 in rGO owing to recovery of the hexagonal network structure of carbon atoms. At the same time the intensity of D band increases in rGO implying formation of large number of defects in the synthesized rGO, which mainly results from the mild reducing action of protein. The ratio of ID/IG is often used to estimate 11



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the defects in graphite materials. The ID/IG ratio in rGO is found to be 1.21, which is higher in comparison to GO (0.9). The increase in ID/IG ratio in rGO is attributed to the formation of large amounts of defects in as-prepared rGO. Thus, removal of oxide functional groups attached to the GO surface leads to the formation of defects in rGO48. The in situ protein mediated reduction of GO and subsequent conjugation to form rGO-protein nano-framework was studied using FTIR spectra. The FTIR spectrum of the intracellular protein (Supporting Information Figure S4) shows absorption bands at 1635, 1552, 1394, 1320, 1160, and 1023 cm-1 attributing to the presence of carboxyl, hydroxyl, amine and phosphate groups. These functional groups provides enormous binding sites for conjugation with rGO possibly through covalent, hydrophobic and π–π stacking interactions with sp2 bonded carbon atom of rGO. The IR spectrum (Figure 2D) of GO shows broad band at the region of 3000–3500 cm-1 centered at 3423 cm-1 due to O–H (hydroxyl groups) stretching vibration. The absorption band for C–H stretching vibration is appeared at 2928 cm-1. The strong signal at 1727 cm-1 is ascribed to C=O stretching vibration of carboxylic acid group. Further, the doublet bands at 1618 and 1404 cm-1 are corresponded to the symmetric and asymmertric vibrations of carboxyl O=C-O groups. The characteristic peaks at 1225 and 1048 cm-1 could be attributed to the C–O (epoxy) stretching vibration and C–O (alkoxy) stretching vibration, respectively.49 On the other hand the FTIR spectrum of rGO demonstrates disappearance of absorption bands at 1720 and 1225 cm-1, confirming successful removal of oxygen containing groups from GO after interaction with the intracellular protein. The broad O–H peak of GO is also replaced by a relatively sharp peak of –NH stretching vibration at 3401 cm-1 following reduction of GO by intracellular protein. The disappearance of hydroxyl group with amide group confirms in situ conjugation of protein with rGO, which is corroborated with the observation of Smith et al.50 related to the interaction of interactions of proteins with different forms of graphene. The disappearance of hydroxyl group with amide group confirms in situ conjugation of protein with rGO. The appearance of a new peak at 1556 and 1152 cm-1 corresponding to amine and phosphate groups, respectively further suggests conjugation of protein with graphene sheets. The other peaks in rGO at 1398 and 1042 cm-1 are originated from the O–H deformation, and C–O (alkoxy) stretching, respectively.51 The FTIR analysis thus confirmed successful reduction of GO by intracellular protein and in situ conjugation of protein to form rGO protein nano-framework. The removal of protein(s) from rGO surface either by calcination process or incubation with 2% SDS solution for 24 h at 30 °C reduces the dispersibility of rGO (data not shown); the uncapped rGO (protein free) easily gets aggregated

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and precipitated in aqueous solution. All the studies were therefore, carried out with protein conjugated rGO referred as rGO-protein nano-framework. X-ray photoelectron spectroscopy (XPS) analysis was recorded to understand the chemical composition of GO and rGO. The low resolution XPS survey spectra (Figure 2E) show that both GO and rGO contain core level C1s and O1s peaks, while additional peak of N1s appears in rGO. The figure also reveals that O1s peak intensity is much higher in GO in comparison to the C1s, while reverse is noted in rGO. The core level C1s spectrum (Figure 2F) of GO is deconvoluted into three peaks at 288.8, 286.6, and 284.7 eV, characteristics of C=O (carboxyl), C-O (hydroxyl and epoxide) and C-C (unoxidized graphite carbon skeleton) groups, respectively.52 Following protein induced reduction of GO (Figure 2G), the peak intensities of carbon atoms bonded to oxygen especially that of C–O (hydroxyl and epoxide groups) reduces significantly, suggesting removal of oxygen-containing functional groups. In addition, a new peak characteristic of C–N group appears at 285.5 eV. This indicates the presence of amine groups with graphene. The N1s core levels spectra (data not shown) is further resolved into two different peaks at 400.1 and 401.5 eV corresponding to the imine (-C=N-) and protonated amine functional groups, respectively. The association of amine groups with rGO could be attributed due to electrostatic interaction of amine groups of protein with residual COO- groups on the edges of as-synthesized rGO. The intensity C/O ratio was also calculated to measure the oxygen content in GO and rGO. It was noted that the CC/CO intensity ratio of rGO is higher (2.81) than the CC/CO ratio of GO (0.887). The ‘CC’ refers to the sum of C-C and C=C bonds, whereas the ‘CO’ includes all combinations of carbon and oxygen bonds. The C-N and C-O bonds have also been taken into consideration while determining the ‘CC’ intensity of rGO because of difficulty in separation of C-O and C-N peaks. The result thus confirms significant removal of large fraction of oxygenated groups on GO by protein mediated reduction process. The morphology and crystalline structure of GO and rGO-protein nano-framework have been investigated by HRTEM images. The images ((Figure 2H-I)) depict that both GO and rGO possess sheet like structure with lateral size up to a few microns. The images further reveal the finer structural differentiation between GO and rGO. The GO (Figure 2H) exhibits quite dense and stacked layer structure with lot of wrinkles and crumpled on the surface. The edge of the sheet shows presence of multilayers with thickness of ~100 nm. In sharp contrast, the rGO sheets (Figure 2I) are quite thin and possess less number of wrinkles compared to the GO sheets. The well exfoliated rGO are folded on one edge and mainly consist of few layers with thickness of 6-10 nm, which is an intrinsic feature of rGO. The SAED patterns demonstrate crystalline structure of rGO 13



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with a six fold symmetry (Figure 2I, inset), while GO exhibits an amorphous structure (Figure 2H, inset) owing to the oxygen-containing groups. This further confirms the formation of graphene.53 The results therefore, suggest that the proposed biosynthetic rout is a novel approach for large scale production of highly water dispersible rGO at ambient condition without using any toxic chemical and organic solvent. The conjugation of protein with two dimensional rGO sheets occurs in in situ growth process through covalent bond, hydrophobic, and π–π stacking interactions, and hence improves the functional properties of rGO. Antibacterial Activity of the Biosynthesized rGO The antibacterial activity of the as-prepared rGO against E. coli was evaluated by colony count method. Figure 3A plate photos shows the antibacterial activity of rGO, while Figure 3B demonstrates concentration dependent antibacterial activity of GO and rGO, respectively. It is clearly noticed that killing efficiency increases with increase in concentration of both GO and rGO. E. coli when treated with 20 µg/mL of GO and rGO, 23.6 ± 1.4 and 37.5 ± 2.1% killing efficiency is recorded, respectively. However, the cell death reaches to 79.3 ± 4.7 and 94.2 ± 3.1% respectively, when treated with 80 µg/mL of GO and rGO. The result therefore, demonstrated that rGO exhibits better antibacterial activity (p value

Fabrication of Nontoxic Reduced Graphene Oxide Protein

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