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Article Cite This: ACS Omega 2019, 4, 387−397
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Bio-Reduced Graphene Oxide as a Nanoscale Antimicrobial Coating for Medical Devices Priyadarshani Choudhary†,‡ and Sujoy K. Das*,†,‡ †
Biological Materials Laboratory, Council of Scientific and Industrial Research (CSIR)-Central Leather Research Institute (CLRI), Chennai 600020, India ‡ Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
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S Supporting Information *
ABSTRACT: Antimicrobial coating on biomedical devices to prevent bacterial colonization is of great interest in healthcare industry nowadays. In this context, graphene nanomaterial has created an excellent opportunity in the biomedical research. The whole cell biomass of Rhizopus oryzae has been explored as a reducing agent for eco-friendly synthesis of reduced graphene oxide (rGO) and minimizing the extensive use of toxic chemicals. The as-synthesized rGO was then characterized by UV−vis spectroscopy, thermo gravimetric analysis, and high-resolution transmission electron microscopy (HRTEM) studies. The hydrodynamic size and surface charge were analyzed by dynamic light scattering and ζ potential studies, and the values were ̀ ± 25 nm and −21.2 ± 5.22 mV, respectively. The HRTEM analysis depicted the ultrathin sheet-like found to be 4350 morphology of rGO. The graphene oxide (GO) and as-prepared rGO were then coated onto the surface of aluminum plate for testing antibacterial properties. The attenuated total reflectance and Raman spectroscopic analyses were recorded to understand the coating of GO and rGO on the surface of aluminum plate, while surface morphology of the coated plates were analyzed by scanning electron and atomic force microscopy. The antibacterial activity was tested against Gram-negative bacteria Escherichia coli for both GO- and rGO-coated plates, which demonstrated excellent antibacterial activity of rGO-coated surface compared with that of the GO-coated plate. Additionally, the rGO-coated plate showed cytocompatibility when tested on 3T6 fibroblast cell line. The obtained result will therefore help to develop graphene-based nontoxic antibacterial coating for biomedical applications. field. Various organic polymers, quaternary ammonium salts, and inorganic (copper, zinc, silver etc.) materials have been explored in recent years as antibacterial coating to overcome the issue of bacterial adhesion and colonization;10−15 however, because of the leaching of metal ions, release of coating materials and self-aggregation of these materials had proven to be harmful for human health and environment.16 Recently graphene, a two-dimensional (2D) atomic crystal consisting of carbon atom arranged in a hexagonal lattice has drawn a major attention because of its versatile property such as large surface area, unique physical and chemical properties, and excellent thermal, mechanical, and optical properties.17 However, the synthesis of graphene involves a top down approach where multilayer of graphite is cleaved to a single layer of graphene via physical, chemical, and mechanical
1. INTRODUCTION Bacterial adhesion and subsequent deposition on several medical instruments and devices including implants, ventilators, catheters, and so forth is a major challenge facing the biomedical industries at present.1−4 The excessive bacterial deposition onto these surfaces has often leads to bacterial colonization and biofilm formation. Sometimes, the bacterial depositions also become the reasons for transmission of several infectious and contaminating diseases3 Further, it causes failure of medical devices, which leads to huge financial loss.5 However, the bacteria adhesion can be minimized by making the surface antiadhesive.1 In context to this, many researchers have designed hydrophobic coating material, which has significantly reduced the bacterial adhesion.6−8 However, still the issue is not completely resolved as recently few reports showed that even superhydrophobic surface fails to stop bacterial adhesion and colonization when bacterial population reaches threshold level.9 Therefore, the development of antibacterial coating is an active research area in biomedical © 2019 American Chemical Society
Received: October 13, 2018 Accepted: December 25, 2018 Published: January 7, 2019 387
DOI: 10.1021/acsomega.8b02787 ACS Omega 2019, 4, 387−397
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Figure 1. Bio-reduction (A) of GO to rGO using microbial biomass of R. oryzae at 30 °C for 24 h under shaking of 120 rpm; color images (B) of biomass extract, GO and rGO; UV−vis spectrum (C) of GO and rGO; TGA spectrum (D) of GO and rGO; HRTEM of GO (E) with the inset SAED pattern of GO; HRTEM of rGO (F) and SAED (G) pattern of rGO.
exfoliation.18−20 In sharp contrast, the graphene synthesis by bottom up approach is achieved by chemical vapor deposition, epitaxial growth, and chemical process.21−23 Among these, although chemical methods have potential for large-scale production in a cost-effective manner, still it is not preferable for large-scale synthesis because of extensive use of toxic chemicals, high temperature, and organic solvents.24,25 Hydrazine and its derivatives are usually used as a strong reducing agent in the formation of graphene;26−29 however, it is highly toxic and difficult to handle. The exploitation of toxic chemicals often restricts the application of graphene in biomedical fields. Hence, it is highly desirable to develop an eco-friendly process to prepare nontoxic, and antibacterial graphene material for biomedical application. Biological synthesis of metal nanoparticles using bacteria,30,31 fungi,32,33 plant,34,35 algae,36,37 sea weed,38 and lichen39 have gained significant importance in recent years as ecofriendly methodology alternative to the chemical and physical processes. It has been reported that Chlorella vulgaris, the green microalga has ability to produce gold, platinum, palladium, ruthenium, rhodium, and iridium nanoparticles,40 over which the cell-free extract efficiently produces gold and silver nanoparticles intracellularly. Similarly, Rhizopus oryzae and Bacillus subtilis have been reported for the synthesis of various sizes and shapes of metal nanoparticles.41−45 Biosynthesis of reduced graphene oxide (rGO) using microbial biomass has been explored recently, and Escherichia coli46 and Bacillus Marisflavi47 have been reported for eco-friendly synthesis of rGO at ambient temperature. In recent years, graphene derivatives [graphene oxide (GO) and rGO] have been explored to inhibit the bacterial growth,
where GO and rGO were reported for effectively inhibiting the growth of E. coli.48 Further, it has been reported that various factors such as size, electronic properties, surface chemical properties, as well as incubation time, concentration, medium, or other external parameters could affect the antimicrobial activity of graphene nanomaterials.49−51 The most of antibacterial activities of graphene derivatives have been studied in the dispersion state. However, there is very limited study on antibacterial activity in the immobilized state particularly as a coating material. In addition, there are only a few reports regarding the antibacterial activity of biosynthesized rGO in immobilized state. Hence, there is enormous scope for studying the antibacterial activity of biosynthesized graphene for the development of nontoxic coating material for biomedical application. In this present study, we explored the eco-friendly synthesis of rGO under ambient condition by whole cell biomass of R. oryzae without using any addition reducing agent or toxic chemical. The R. oryzae has many advantages over other bacteria and viruses because of its less complex structure and also in terms of handling and mass production. R. oryzae is nonpathogenic with high nutritional value and has large amounts of high quality protein. The mycelia of R. oryzae can be obtained by growing the fungus in the easy available low cost growth medium and does not require any special biosafety measure.52 Therefore, biosafety and biocompatibility issues for biosynthesis of rGO using R. oryzaecould be avoided. The assynthesized rGO was then coated onto the surface of aluminum plate and subsequently characterized using various spectroscopic and microscopic analyses. The antibacterial efficacy of the GO- and rGO-coated aluminum plates was 388
DOI: 10.1021/acsomega.8b02787 ACS Omega 2019, 4, 387−397
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tested against E. coli using a colony forming unit (CFU) and Live/Dead assays. The biocompatibility of GO- and rGOcoated plates was also performed using the 3T6 fibroblast cell line. The results obtained from this investigation indicated the feasibility of using rGO as nontoxic antibacterial coating to prevent bacterial colonization on the surface.
proteins could bind with rGO surface, which increased the hydrodynamic size of rGO. Therefore, DLS takes the ligand shell into account the size of the whole conjugate is measured by DLS, resulting in higher size measurement of rGO. Our obtained result is corroborated with previous studies; where Wang et al.58 and Gurunathan et al.46 reported that the average size of rGO increased upon reduction of GO by heparin and E. coli, respectively. It is also possible that the biomass, which is not only acting as reducing agent but also functionalizing the rGO surface, leads to the increased the Brownian motion and ultimately becomes the cause for higher particle size of rGO. The surface charge analysis of GO and rGO further demonstrated that at pH 6.0 the GO and rGO had ξ potential values of −31 ± 3.63 and −21.2 ± 5.22 mV, respectively. The removal of oxygen-containing functional groups could be the possible reason for increased ξ potential value of rGO in comparison to GO. The thermo gravimetric analysis (TGA) analysis was also recorded to study the thermal stability of GO and rGO, which is highly essential to understand the heat stability of the coating material. Figure 1D shows the thermogram pattern and the decomposition temperature of the samples. The TGA curve of GO showed initial weight loss of 4% below 100 °C, which is attributed to the evaporation of absorbed water molecule from GO. The second stage of weight loss of about 25% in the range of 100−200 °C is associated with the decomposition of oxygen containing groups.59 A very sharp weight loss with a higher rate at the temperature range of 400− 600 °C is due to the pyrolysis of the carbon skeleton. A total weight loss of ∼78% was recorded in the entire temperature range of 800 °C. The TGA analysis of rGO demonstrated conspicuous changes in thermogram pattern in comparison to the GO and only 41% of total weight was recorded in the entire temperature range.60 The enhanced van der Waals forces between the deoxygenated layers of rGO might be responsible for higher thermal stability of rGO, which has significant implication in biomedical application of rGO. Finally, high-resolution transmission electron microscopy (HRTEM) image of GO and the synthesized rGO was recorded by drop-casting the respective solution on copper grids. The image (Figure 1E) of GO sample revealed rough, rippled, and folded surface with a sheet-like structure. The multilayer structure is easily visible at edge of the sheet and thickness of the layer was found to be ∼100 nm. On the other hand, the rGO (Figure 1F) sample showed an ultrathin structure with less folding and relatively smooth surface.39 The SAED patterns (Figure 1G) further showed the sixfold symmetry of the crystalline structure of rGO, whereas SAED patterns of GO sample (Figure 1E, inset) revealed amorphous structure because of the oxygen-containing groups. This result therefore, suggested that microbial reduction of GO could provide a novel eco-friendly process for synthesis of rGO by avoiding the use of toxic chemicals or energy intensive physical process. 2.2. Coating of GO and rGO on Aluminum Plate. The surface coating of GO and rGO was carried out through covalent attachment on the amine-functionalized aluminum plates for the development of nontoxic antibacterial coating and is schematically presented in Figure 2A. The photographs of pristine aluminum plates (left panel, Figure 2B) and upon coating with GO (middle panel, Figure 2B) and rGO (right panel, Figure 2B) clearly revealed coating of GO and rGO on the surface. The stability of the coated plate was proved upon
2. RESULT AND DISCUSSIONS 2.1. Synthesis and Characterization of rGO. In the present study, GO was synthesized by modified Hummers and Offeman’s method53 and reduced to rGO using whole cell biomass of R. oryzae as shown in Figure 1A. In the synthesis process, the GO solution (1 mg/mL) was incubated with the biomass of R. oryzae at 37 °C for 24 h under shaking condition. The brown color of GO changed gradually from dark brown to eventually black, indicating the reduction of GO to rGO. The photographs of whole cell biomass of R. oryzae (left panel) and GO solution before (middle panel) and after reduction (right panel) process are shown in Figure 1B. No obvious color change was observed in control experiment performed without R. oryzae mycelia. This indicated that mycelial biomass played a significant role in reduction of GO to rGO. Further, it was found that both GO and rGO are soluble in water, and the difference in appearance is due to the change in structural and physiochemical properties.54 The optical absorption spectra of GO and rGO were recoded to monitor the structural changes in conversion from GO to rGO. As shown in Figure 1C, the GO solution exhibited a sharp absorption peak at 230 nm and a shoulder at 307 nm characteristic of π−π* electronic transitions of C−C aromatic bonds and n−π* transitions of CO bonds, respectively.55 Upon reduction, the absorption band at 230 nm was shifted to 260 nm with disappearance of 307 nm peak,56,57 suggesting rearrangement of π electronic configuration of GO in rGO because of the removal of oxygen containing group.47 Similar features have been observed for the reduction of GO with L-ascorbic acid54 and L-cysteine.55 Particle size and surface charge are among the essential factors that determine the effect of GO and rGO in any biological system for achieving the desired effect in biological field. The particle size and surface charge of GO and rGO solution were determined by dynamic light scattering (DLS) and ζ potential analyses, respectively. DLS analysis provides the hydrodynamic diameter of the GO and rGO as water forms a covering hydrated layer over the surface of the GO and rGO when it is in solution; on the other hand, ζ potential measures the surface charge of the material. In addition, these provide information about state of dispersion, aggregation, and solubility of the materials. The results showed (Table 1) that average size of GO was 3032 ± 41 nm, and after reduction, the average size of rGO increased to 4350 ± 25 nm. This change in size signifies that as-prepared rGO has more colloidal stability, which could increase the Brownian motion rate of rGO after reduction process. It is also possible that during reduction process proteins were secreted from the biomass and the secreted Table 1. Hydrodynamic Size and ζ-Potential of GO and rGO water material
hydrodynamic size (nm)
ζ-potential (mV)
GO rGO
3032 ± 41 4350 ± 25
−31 ± 3.63 −21.2 ± 5.22 389
DOI: 10.1021/acsomega.8b02787 ACS Omega 2019, 4, 387−397
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Figure 2. Schematic presentation (A) of rGO coating on aluminum plate; color image (B); and (C) contact angle of uncoated, GO- and rGO-coated aluminum plate.
washing it under running water for few hours and observed formation of stable coating. The hydrophobic/hydrophilic property of the coated surface was further measured by contact angle of the coated plates. The contact angle of GO- and rGOcoated plates were found to be 65 ± 2.36° and 78 ± 3.12°, respectively, wherein the uncoated aluminum plate showed the contact angle of 24 ± 1.36° (Figure 2C). Thus, the results confirmed that both GO- and rGO-coated surface exhibited higher hydrophobicity as compared to that of the uncoated aluminum surface. Moreover, the rGO-coated surface possessed higher hydrophobicity as compared to the GOcoated and uncoated aluminum surface, indicating that rGOcoated surface have better water repellent characteristic compared with the GO-coated and uncoated aluminum surface. The subsequent changes occur during coating of GO and rGO was further studied using attenuated total reflectance (ATR) spectra using ATR spectrophotometer (JASCO ATR spectrometer), as shown in Figure 3A. The 3-aminopropyl triethoxysilane (APTES)-coated aluminum plate showed characteristic peaks at 3353 and 3285 cm−1 corresponding to −NH2, whereas the peak at 2928 and 2864 cm−1 is attributed to the vibration of −CH2 group occurring because of the alkyl chain of APTES. The peaks at 1659, 1633, and 1321 cm−1 are assigned to −NH, C−N, and CN bonds, respectively,61 whereas the peak at 1054 and 754 cm−1 are the characteristics peaks of Si−O−Si and Si−O−C, respectively.62 Upon GO coating, the GO-coated aluminum plates showed a strong and broad band at 3326 and 3285 cm−1 because of O−H (hydroxyl groups) and −NH2 stretching vibration. The peaks at 2859 and 2930 cm−1 are due to the vibration of −CH2 group. The strong signal at 1727 cm−1 refers to CO (carbonyl bond) stretching vibration of carboxylic acid,46,63 whereas the peaks at 1568 and 1475 cm−1 corresponds to the hydroxyl group. Further, the bands at 1630 and 1310 cm−1 corresponds to the C−N and CN bond, respectively, confirming the role of APTES in functionalization process of GO. The characteristic peaks at 1225 and 1048 cm−1 in GO-coated plates are due to the C−O (epoxy) stretching vibration and the C−O (alkoxy) stretching vibration, respectively.64,65 The spectrum of rGOcoated plates confirmed the reduction of GO by showing
Figure 3. ATR (A) and XRD (B) of uncoated, GO-coated and rGOcoated aluminum plate, respectively; and Raman spectra (C) of GOand rGO-coated aluminum plate.
significant changes in its chemical structure before and after deoxygenation. The characteristic peaks at 3326 and 2982 cm−1 corresponds to −NH2 and −CH2 group, respectively. The peak at 1680 cm−1 signifies CC stretching vibration, which confirmed the removal of oxygen-containing groups and restoration of π-conjugation.66,67 However, the peak at 1618 and 1310 cm−1 corresponds to the C−N and CN bond, respectively, as the role of APTES functionalization process of rGO with the aluminum plate.68 The crystal structure of the GO- and rGO-coated aluminum plate was characterized by X-ray diffraction (XRD) as depicted in Figure 3B. The XRD data not only provide the information about surface coating but also give idea about biomassmediated reduction of GO to rGO. The diffraction peak of the GO-coated plate appeared at 2θ value of 11.07° and 43° associated with (001) and (100) planes, respectively, indicating a 2D structure of exfoliated GO sheets, whereas a relatively broad peak appeared at 2θ value of 26.07° in the rGO-coated plate with the disappearance of a sharp peak of GO. The appearance of a broad peak in rGO and disappearance of diffraction peaks of GO is due to the removal of oxygen-containing functional groups between the layers of graphene and thus signifies the biomass-mediated reduction of GO and formation of few layers of graphene.69−71 On the other hand, the uncoated aluminum plate does not show any characteristic peak. The structural changes of GO- and rGO-coated aluminum plates were further studied using Raman spectra. The Raman spectra have been found to be very sensitive and act as an extensive tool for characterization of carbon-based material, especially CC double bond.58 The typical G and D bands 390
DOI: 10.1021/acsomega.8b02787 ACS Omega 2019, 4, 387−397
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related to the first-order scattering of E2g photon of sp2 carbon atom and A1g symmetry that corresponds to the structural defects were presented in Raman spectra.72 In GO-coated aluminum plate, the G and D bands appeared at 1340 and 1576 cm−1, respectively (Figure 3C), whereas in rGO-coated aluminum plate, these peaks were shifted to 1565 and 1337 cm−1, respectively. The shifting of these peaks indicated that the extensive oxidation of GO leads to the annihilation of sp2 domain. The ID/IG ratio of GO- and rGO-coated plated was also determined to measure the defect and it was found to be 0.96 and 1.17, respectively. The increase in the ID/IG ratio in rGO-coated plate is attributed to the formation of large amounts of defects in rGO in comparison to the GO samples. Thus, the removal of oxide functional groups attached to the GO surface by R. oryzae-mediated reduction leads to the formation of more defects in rGO. It has been reported47 that biomass of B. marsiflavi could be used as a reducing agent, and upon reduction the G-band of rGO was broadened and shifted to around 1608 from 1587 cm−1. Similarly, the D-band shifted to 1395 from 1343 cm−1 and became prominent, suggesting the annihilation of sp2 domains and the formation of defects in the sheets because of massive oxidation. Thus, our obtained results are found to be in good agreement with this study and also supports R. oryzae biomass-mediated reduction of GO. The surface morphology and topology of rGO-coated aluminum plate was then studied using scanning electron microscopy (SEM) and atomic force microscopy (AFM) analyses. The SEM image of uncoated aluminum plate depicted smooth surface (Figure 4A), wherein uniform coating
coated plate also demonstrated similar peaks except N (data not shown). The AFM images (Figure 4D) of GO- and rGOcoated aluminum plate also showed typical hierarchical structure with a lot of nanoscaled flakes adhering on the aluminum surface similar to the surface morphology and topology as observed in SEM micrograph. 2.3. Antibacterial Activity of Coated Surface. The antibacterial activity of GO and rGO in dispersion phase had been studied (Supporting Information Figure S1) against E. coli bacteria and were found to be 80.39 ± 2.01 and 96.6 ± 1.32% respectively. The equivalent amount of GO and rGO solution were then used for coating the aluminum plates. To develop a sustainable antibacterial coating, the bactericidal activity of the GO- and rGO-coated aluminum plates was then tested against E. coli. The coated plates were seeded with bacteria and incubated for 4 h at 37 °C. The bacteria settled on the surface were collected by washing with sterile phosphate buffer and spread over nutrient agar plates with appropriate dilution to count the bacterial colonies grown on the agar plates. The uncoated aluminum plate was served as the control. A drastic reduction in the bacterial colony was observed following exposure of E. coli with GO- and rGO-coated aluminum surface. Only few colonies were observed after exposure to the GO- and rGO-coated aluminum plates (Figure 5B,C), whereas densely populated bacterial colonies were observed upon incubation on uncoated aluminum plate (Figure 5A). It was further noticed that the rGO-coated plate was more effective in killing of bacteria in comparison to the GO-coated surface. Figure 5D shows the bacterial killing efficiency (%) of the coated and uncoated surface. The cells incubated with the GO-coated surface demonstrate 72.47 ± 3.9% cell deaths, whereas in rGO-coated surface, the cell death increased to 93.21 ± 2.86%. The killing efficiency was very negligible when incubated with uncoated aluminum surface. However, there was slight variation in antibacterial activity of GO and rGO in dispersion state and after coating (immobilized state). The GO and rGO exhibited higher antibacterial activity in dispersed phase in comparison to coated materials. The higher antibacterial activity of GO and rGO in the dispersion phase compared with the coated plates might be due to the following reasons. After binding on the cell wall GO and rGO could penetrate into the cells and cause damage to the intracellular organelles apart from physical disruption of the cell wall. On the other hand, the intracellular penetration was restricted in immobilized state, that is, coated pates. In addition the contact with bacteria would be more in the dispersed state of GO and rGO compared with their immobilized phase, as the exposed surface of the coated plates could only interact with the bacterial cells. Therefore, physical disruption of the bacterial cells is only possible reason for antibacterial activity of the coated plates. Carpio et al.73 also reported that PVK−GO nanocomposite has higher antibacterial activity compared to the GO because of high dispersion of PVK−GO nanocomposite. Adding more to this context, Rodrigues et al.74,75 also demonstrated that PVK−SWNT and PVK−GO nanocomposites exhibited higher antibacterial activities compared with the pristine SWNT and GO because of improved dispersion of PVK−SWNT and PVK−GO nanocomposites. Finally, we would like to conclude that although GO and rGO dispersion exhibited higher killing efficiency, and the GO- and rGO-coated plates have sufficient antibacterial activity to prevent bacterial growth on the surface of the aluminum plates and pave the way for the development
Figure 4. SEM images of uncoated (A) and rGO-coated (B) aluminum plate, respectively; EDAX spectrum (C) of rGO-coated aluminum plat; and AFM image (D) of rGO-coated aluminum plate.
of rGO over the surface of aluminum plate characterized by interconnected nanoflakes was observed as shown in Figure 4B. These further revealed rough and rippled surfaces of the coated plates. Figure 4C showed the energy-dispersive X-ray analysis (EDAX) spectrum of the rGO-coated aluminum plate. The C and O peaks confirmed the rGO coating, wherein the N and Al peaks were due to the associated protein of the biomass and aluminum plate, respectively. The EDAX spectrum of GO391
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Figure 5. Plate photograph of bacterial cells after incubation with uncoated (A), GO-coated (B) and rGO-coated (C) aluminum plate. Antibacterial activity of uncoated, GO-coated, and rGO-coated aluminum plate against E. coli (106 CFU/mL) at 37 °C (D) and fluorescence microscopic image of bacterial cells after incubation with uncoated (E), GO-coated (F) and rGO-coated (G) aluminum plate. Determination of ROS production (H) by H2-DCFDA method. Schematic presentation showing antibacterial activity (I) of uncoated and rGO-coated aluminum plate. Data represent an average of five independent experiments, ±standard deviation (SD) shown by error bar. All of the inactivation of rGO are significantly different from GO with p-values of
