Enhanced green fluorescent protein-mediated synthesis of biocompatible graphene
© Gurunathan et al.; licensee BioMed Central Ltd. 2014
Received: 7 August 2014
Accepted: 26 September 2014
Published: 3 October 2014
Graphene is the 2D form of carbon that exists as a single layer of atoms arranged in a honeycomb lattice and has attracted great interest in the last decade in view of its physical, chemical, electrical, elastic, thermal, and biocompatible properties. The objective of this study was to synthesize an environmentally friendly and simple methodology for the preparation of graphene using a recombinant enhanced green fluorescent protein (EGFP).
The successful reduction of GO to graphene was confirmed using UV-vis spectroscopy, and FT-IR. DLS and SEM were employed to demonstrate the particle size and surface morphology of GO and EGFP-rGO. The results from Raman spectroscopy suggest the removal of oxygen-containing functional groups from the surface of GO and formation of graphene with defects. The biocompatibility analysis of GO and EGFP-rGO in human embryonic kidney (HEK) 293 cells suggests that GO induces significant concentration-dependent cell toxicity in HEK cells, whereas graphene exerts no adverse effects on HEK cells even at a higher concentration (100µg/mL).
Altogether, our findings suggest that recombinant EGFP can be used as a reducing and stabilizing agent for the preparation of biocompatible graphene. The novelty and originality of this work is that it describes a safe, simple, and environmentally friendly method for the production of graphene using recombinant enhanced green fluorescent protein. Furthermore, the synthesized graphene shows excellent biocompatibility with HEK cells; therefore, biologically synthesized graphene can be used for biomedical applications. To the best of our knowledge, this is the first and novel report describing the synthesis of graphene using recombinant EGFP.
Graphene has a two-dimensional (2-D) nanostructure with a single layer of carbon atoms and has attracted much interest in recent years because of its unique mechanical, thermal, catalytic, electronic, optical, and biological properties -. Graphene and graphene-based materials have been widely used in several applications including bio-sensing , antibacterial compositions -, drug delivery , tissue scaffolds , catalysis , and energy storage . The production of graphene in large quantities using an environmentally friendly approach is essential but also a significant challenge .
Several methods have been established for the synthesis of graphene and its derivatives, including exfoliation of graphite (Gt) , flash reduction , hydrothermal dehydration , mechanical exfoliation , epitaxial growth , photocatalysis , and photodegradation . Although several methods are available for the preparation of graphene, solution-based chemical reduction of graphene oxide (GO) to graphene is considered one of the most efficient methods for low-cost and large-scale production of graphene . Reduction of GO by chemical methods seems to be promising, because of the low cost and potential for large-scale production. Such methods are also appropriate for chemical modification and subsequent processing. However, in chemical methods, the use of hydrazine and hydrazine derivatives as strong reducing agents for the formation of graphene can be toxic or explosive, resulting in challenges for large-scale production. The resulting graphene also possesses very limited solubility or even irreversible agglomeration during preparation in water and most organic solvents unless capping reagents are used owing to the strong -π-π stacking tendency between rGO sheets ,. To overcome the aggregation and solubility problems, several polymers or surfactants have been used, such as poly(N-vinyl-2-pyrrolidone) , poly(sodium-4-styrene sulfonate) , poly(allylamine) , and potassium hydroxide . Recently, Akhavan et al.  demonstrated a possible route for inexpensive mass production of high-quality graphene sheets from natural and industrial carbonaceous wastes.
The toxicity of GO and graphene has been studied in various cell types such as neuronal cells , lung epithelial cells , fibroblasts , primary mouse embryonic fibroblast cells , and cancer cells , and the results vary across cell and material types.
Surface modification of graphene has been reported to alter its toxicity , with reduced GO and carboxylated graphene reported to be less toxic than GO or native graphene . Single-layer GO sheets were found to be internalized and sequestered in cytoplasmic, membrane-bound vacuoles in human lung epithelial cells and fibroblasts, with toxicity induced at concentrations above 20µg/mL after 24 h ,. Sanchez et al.  reported that graphene-family nanomaterials (GFNs) can be either benign or toxic to cells, and that the biological responses depend on layer number, lateral size, stiffness, hydrophobicity, surface functionalization, and concentration. In addition, the biocompatibility and cytotoxicity depend on the type of reducing agent used for the functionalization of GO.
Graphene has been used as a possible biocompatible nanocarrier for delivering drugs  and also as a functional biomaterial. Sun et al.  reported that non-toxic PEGylated nano-graphene oxide could deliver water-insoluble cancer drugs. Fan et al.  showed that graphene/chitosan composites were biocompatible in L929 cells and that the absence of metallic impurities in graphene sheets makes them potential candidates as scaffolds for tissue engineering. Furthermore, Chen et al.  reported that graphene oxide (GO)/ultra-high-molecular-weight polyethylene (GO/UHMWPE) composites showed remarkably enhanced hardness and slightly improved yield strength compared with pure UHMWPE. The addition of small amounts of GO did not affect the attachment and proliferation of MC3T3-E1 osteoblasts cultured on GO/UHMWPE composite surfaces, indicating its excellent biocompatibility. Akhavan et al.  reported size-dependent cyto- and genotoxic effects of reduced graphene oxide nanoplatelets (rGONPs) rGONPs on cells. A cell viability test showed significant cell death on treatment with 1.0µg/mL rGONPs with an average lateral dimension (ALD) of 11±4 nm, whereas rGO sheets an ALD of 3.8±0.4µm exhibited a significant cytotoxic effect only at the high concentration of 100µg/mL after 1 h of exposure time. Akhavan et al.  demonstrated the size-dependent cytotoxic and genotoxic effects of reduced graphene oxide nanoplatelets on human mesenchymal stem cells (hMSCs). Furthermore, Akhavan et al.  used ginseng extract-reduced GO to differentiate stem cells. Park et al.  used graphene-as a substrate to promote human neural stem cell adhesion and differentiation into neurons. Lee et al.  reported that the strong non-covalent binding ability of graphene allows it to act as a pre-concentration platform for osteogenic inducers, which accelerate the differentiation of mesenchymal stem cells (MSCs) growing on it toward the osteogenic lineage. Akhavan et al.  used graphene nanogrids as two-dimensional selective templates for accelerated differentiation of human MSCs (hMSCs) isolated from umbilical cord blood into osteogenic lineages. The biocompatible and hydrophilic graphene nanogrids showed high actin cytoskeleton expression coinciding with the patterns of the nanogrids. Akhavan and Ghaderi  introduced a reduced graphene oxide (rGO)/TiO2 heterojunction film as a biocompatible flash photo stimulator for the effective differentiation of hNSCs into neurons. Graphene nanogrids on a SiO2 matrix containing TiO2 nanoparticles (NPs) were also applied as a photocatalytic stimulator to accelerate the differentiation of human neural stem cells (hNSCs) into two-dimensional neural networks .
Several environmentally friendly methods have been developed using various biomolecules such as ascorbic acid , amino acids , glucose , and bovine serum albumin  as reducing agents or stabilizers. In addition, microorganisms have also used to reduce GO, including Shewanella, Escherichia coli,, Pseudomonas aeruginosa, Bacillus marisflavi, and Ganoderma spp . Some purified proteins have also been used for synthesis of graphene, such as melatonin , l-glutathione , and humanin . Recently, the synthesis of graphene has been increased significantly because of the wide range of resources and availability of simple, cost-effective, and environmentally friendly approaches. The major problem encountered during the synthesis of nanoparticles using biomass is the isolation and purification of the nanoparticles from the biomass, which requires many downstream processing steps including sonication and ultracentrifugation to attain maximum yield . Moreover, endotoxin may be present in the nanoparticles, which may limit the use of the nanoparticles in medical applications . Therefore, this study attempted to use a recombinant protein.
Recombinant enhanced green fluorescent protein (EGFP) (Gene Bank Accession no. U57607) is a protein composed of 293 amino acid residues (32.7 kDa) that has an isoelectric point of 6.2 and exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. EGFP has been widely used as a biological reporter to identify tissue and cells with target gene expression ,. Previous studies showed no obvious detrimental effects of EGFP and no toxicity, i.e., it is biologically inert ,. In addition, EGFP was selected here as a reducing and stabilizing agent for synthesis of graphene because it is a natural protein from the jellyfish Aequorea victoria and has been proven to be an excellent biological reporter . Thus, without any other toxic reagents added, the raw material and reaction products are all environmentally friendly, which should increase the efficiency and large-scale synthesis of graphene. Additionally, EGFP contains five cysteine amino acid residues, each containing a thiol group that can be oxidized to form the disulfide derivative cysteine, which functions as a nucleophile . Protons have high binding affinity to oxygen-containing groups, such as hydroxyl and epoxide groups on GO, resulting in the formation of H2O molecules ,. The unique chemical structure of EGFP makes it not only an ideal reducing agent but also an effective capping agent. Therefore, we addressed the following objectives: first, the development of a simple, dependable, and environmentally friendly approach for synthesis of graphene using recombinant EGFP; second, the characterization of GO and EGFP-reduced GO; and finally, the evaluation of cellular responses of GO and EGFP-rGO in human embryonic kidney 293 cells.
Results and discussion
Synthesis and characterization of EGFP-rGO
The reduction of GO was confirmed using UV-vis absorption spectroscopy. As shown in Figure1, the absorption peak of the GO dispersion was located at 230 nm with a shoulder peak at about 300 nm, which was consistent with previous reports ,,. After the reduction process, the peak was red-shifted to 258 nm and the absorbance was increased dramatically in the entire spectral region. This result suggests that GO was reduced by EGFP and that the aromatic structure of graphene may be restored. Further evidence showed that the UV-vis absorption spectrum of GO was characterized by the π-π* of the C=C plasmon peak at approximately 230 nm and a shoulder at approximately 300 nm that is often attributed to n-π* transitions of the carbonyl groups ,. With reduction by EGFP, the plasmon peak gradually red-shifted to approximately 258 nm, indicating the restoration of sp2 carbon and possible rearrangement of atoms . Similar trends were also observed for the reduction of GO by L-ascorbic acid ,, L-cysteine , melatonin , heparin , dopamine , and humanin .
FTIR spectra of GO and EGFP-rGO
XRD analysis of GO and EGFP-rGO
Size distribution analysis of GO and EGFP-rGO
Surface properties of GO and EGFP-rGO
Surface morphology analysis of GO and EGFP-rGO by SEM
Raman spectroscopy analysis of GO and EGFP-rGO
The major effects of deoxygenation are the restoration of the sp2 network and the introduction of small and isolated aromatic domains, and these effects are responsible for the observed increase in the ID/IG ratio in rGO ,,,. Wang et al.  suggested that the G band is broadened and shifted upward to 1,595 cm?1, and the increased intensity of the D band at 1,350 cm-1 could be attributed to the significant decrease in the size of the in-plane sp2 domains resulting from oxidation and ultrasonic exfoliation, in addition to the partially ordered graphite crystal structure of graphene nanosheets. The Raman spectra of graphene-based materials also show a two-dimensional (2D) band that is sensitive to the stacking of graphene sheets. It is well known that the two-phonon (2D) Raman scattering of graphene-based materials is useful to differentiate monolayer graphene from multilayer graphene as it is highly sensitive to the stacking of graphene layers ,,. Another characteristic of single-layer graphene is the relatively strong Raman intensity of the 2D band with respect to the G-band . Usually, a Lorentzian peak for the 2D band of monolayer graphene sheets is observed at 2,679 cm-1, whereas this peak is broadened and shifted to a higher wave number in the case of multilayer graphene ,,. We observed the 2D band at 2699 cm-1, which is the same as the previously reported peak position for single-layer graphene ,. Thus, our sample could consist of single-layer graphene flakes.
It should be noted that this ratio is higher than those reported for rGO produced using various reducing agents such as L-cysteine , dextran , baker's yeast , DTT ,, and NaBH4. The Raman spectroscopy analyses described here agree with those of previous studies that used various biomolecules and organisms to reduce GO to graphene, such as L-cysteine , Baker's yeast , heparin , Escherichia coli, P. aeruginosa, Humanin , Ganoderma spp , and Ginkgo biloba.
Biocompatibility of GO and EGFP-rGO
Effect of EGFP-rGO on LDH leakage
Effects of EGFP-rGO on oxidative stress
Effect of EGFP-rGO on cell morphology
Commonly, the reduction of GO using chemical reducing agents is harmful to human health and the environment, and aggregation is another problem that occurs during the reduction process. Here, we show the synthesis of biocompatible graphene using recombinant EGFP. EGFP is one of the most widely used tools in biology because of its stability and lack of toxicity. In the present study, we explored the potential application of EGFP for a different purpose other than the tagging usually reported in the literature. We have developed a simple, dependable, and environmentally friendly method for the fabrication of reduced GO. Our findings suggest that GO induced significant concentration-dependent decreases in the viability of HEK cells, whereas graphene exerted no toxic effects on HEK cells at a concentration of 100 µg/mL. Therefore, it is concluded that the use of a biological substrate in a simple and environmentally friendly approach for synthesis of graphene resulted in significant deoxygenation of suspended GO suspensions, thus providing a suitable substitute for chemical reducing agents and potentially enabling biomedical applications of graphene-based materials. This work may provide additional insight into graphene synthesis.
Materials and methods
Gt powder, NaOH, KMnO4, NaNO3 anhydrous ethanol, 98% H2SO4, 36% HCl, and 30% H2O2 aqueous solution were purchased from Sigma-Aldrich (St Louis, MO, USA). Penicillin-streptomycin solution, trypsin-ethylenediaminetetraacetic acid solution, Dulbecco's Modified Eagle Medium (DMEM), and 1% antibiotic-antimycotic solution were obtained from Gibco (Life Technologies, Carlsbad, CA, USA). Fetal bovine serum and the in vitro toxicology assay kit were purchased from Sigma-Aldrich. Enhanced green fluorescent protein was purchased from Bio-vision (Cat.No. 4999-100; Milpitas, California, USA).
Synthesis of GO
GO was synthesized as described previously ,. In a typical synthesis process, natural Gt powder (2 g) was added to cooled (0°C) H2SO4 (350 mL), and then KMnO4 (8 g) and NaNO3 (1 g) were added gradually while stirring. The mixture was transferred to a 40°C water bath and stirred for 60 min. Deionized water (250 mL) was slowly added and the temperature was increased to 98°C. The mixture was maintained at 98°C for a further 30 minutes and the reaction was terminated by the addition of deionized water (500 mL) and 30% H2O2 solution (40 mL). The color of the mixture changed to brilliant yellow, indicating the oxidation of pristine Gt to Gt oxide. The mixture was then filtered and washed with diluted HCl to remove metal ions. Finally, the product was washed repeatedly with distilled water until pH 7.0 was achieved, and the synthesized Gt oxide was further sonicated by ultrasonication for 30 min.
Preparation of EGFP-rGO
Reduction of GO was performed as described previously , with suitable modifications. Using GO as a precursor, EGFP-rGO was prepared using EGFP as both a reducing agent and a stabilizer. In a typical procedure, reduced GO (rGO) was obtained from the reaction of EGFP with GO. A mixed aqueous solution containing EGFP (100 µg/mL) and GO (1 mg/mL) was ultrasonicated for 15 min, and the mixture was maintained at 40°C for 1 h. The mixture was then cooled to room temperature and ultrasonicated for a further 15 min. After being vigorously stirred for 5 min, the mixture was stirred in a water bath (90°C) for 1 h. The resulting stable black dispersion was then centrifuged and washed with water three times. A homogenous EGFP-rGO suspension was obtained without aggregation. Finally, the obtained EGFP-rGO sheets were redispersed in water before further use.
Characterization of GO and EGFP-rGO
GO and EGFP-rGO were characterized according to methods described previously . UV-visible spectra were recorded using a WPA Biowave II spectrophotometer (Biochrom, Cambridge, UK). The particle sizes of the GO and EGFP-rGO dispersions were measured using a Zetasizer Nano ZS90 instrument ( Malvern Instruments, Worcestershire, UK). X-ray diffraction (XRD) analyses were performed in a Bruker D8 DISCOVER X-ray diffractometer (Bruker AXS GmBH, Karlsruhe, Germany). The X-ray source was 3 kW with a Cu target, and high-resolution XRD patterns were measured using a scintillation counter (»=1.5406°A). The XRD was run at 40 kV and 40 mA, and samples were recorded at 2θ values between 5° and 80°. The dried powder of GO and EGFP-rGO was diluted with potassium bromide and the Fourier transform infrared spectroscopy (FTIR) (Perkin Elmer Inc., USA) and spectrum GX spectrometry were recorded within the range of 500-4000 cm_1. A JSM-6700 F semi-in-lens field emission scanning electron microscope was used to acquire SEM images. The solid samples were transferred to a carbon tape held in an SEM sample holder, and then the analyses were performed at an average working distance of 6 mm. Raman spectra of GO and EGFP-rGO were measured using a WITEC Alpha300 laser with a wavelength of 532 nm. Calibration was initially performed using an internal silicon reference at 500 cm-1 and gave a peak position resolution of less than 1 cm-1. The spectra were measured from 500 to 4500 cm-1. All samples were deposited onto glass slides in powdered form without using any solvent.
Cell culture and exposure of cells to GO and EGFP-rGO
Human embryonic kidney 293 cells were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin in a humidified incubator maintained at 37°C and 5% CO2. At approximately 75% confluence, cells were harvested using 0.25% trypsin and subcultured in 75 cm2 flasks, 6-well plates, or 96-well plates depending on the intended use. Cells were allowed to attach to the substratum for 24 h prior to treatment. The medium was replaced three times per week, and cells were passaged at subconfluency. Cells were prepared in 100µL aliquots at a density of 1×105/mL and plated in 96-well plates. After the cells were cultured for 24 h, the medium was replaced with medium containing GO or EGFP-rGO at different concentrations (0-100 µg/mL). After incubation for an additional 24 h, cells were analyzed for viability, lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) generation. Cells not exposed to GO or EGFP-rGO served as the control. Further, morphology of cells treated with GO or EGFP-rGO or untreated was examined using an OLYMPUS IX71 microscope (Japan) using appropriate filter sets.
The WST-8 assay was performed as described previously . Typically, 1 × 104 cells were seeded in a 96-well plate and cultured in DMEM supplemented with 10% FBS at 37°C under 5% CO2. After 24 h, the cells were washed with 100µL of serum-free DMEM two times and incubated with 100µL of different concentrations of GO or EGFP-rGO suspensions in serum-free DMEM. After 24 h of exposure, the cells were washed twice with serum-free DMEM, and 15µL of WST-8 solution was added to each well containing 100 x00B5;L of serum-free DMEM. After 1 h of incubation at 37°C under 5% CO2, 80 °L of the mixture was transferred to another 96-well plate because residual GO or EGFP-rGO can affect the absorbance values at 450 nm. The absorbance of the mixture solutions was measured at 450 nm using a micro plate reader. Cell-free control experiments were performed to determine whether GO and EGFP-rGO react directly with the WST-8 reagents. Typically, 100 µL of GO or EGFP-rGO suspensions with different concentrations (0-100 µg/mL) were added to a 96-well plate and 10 µL of WST-8 reagent solution was added to each well; the mixture was incubated at 37°C under 5% CO2 for 1 h. After incubation, the GO or EGFP-rGO was centrifuged and 100 µL of the supernatant was transferred to another 96-well plate. The optical density was measured at 450 nm.
The cell membrane integrity of human embryonic kidney 293 cells was evaluated by determining the activity of lactate dehydrogenase (LDH) leaking out of the cells according to the manufacturer's instructions (in vitro toxicology assay kit, TOX7, Sigma, USA) and also as described previously . Briefly, the cells were exposed to various concentrations of GO and EGFP-rGO (0-100 µg/mL) for 24 h, and then 100 µL per well of each cell-free supernatant was transferred in triplicate into wells in a 96-well plate, and 100 µL of the LDH assay reaction mixture was added to each well. After 3 h of incubation under standard conditions, the optical density of the color generated was determined at a wavelength of 490 nm using a micro plate reader.
Determination of ROS
ROS were estimated according to a method described previously . Intracellular ROS were measured based on the intracellular peroxide-dependent oxidation of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes, USA) to form the fluorescent compound 2',7'-dichlorofluorescein (DCF), as previously described. Cells were seeded onto 24-well plates at a density of 5 ×104 cells per well and cultured for 24 h. After washing twice with PBS, fresh medium containing different concentrations of GO or EGFP-rGO (0 100 µg/mL) was added and the cells were incubated for 24 h. The cells were then supplemented with 20 µM DCFH-DA, and incubation continued for 30 min at 37°C. The cells were rinsed with PBS, 2 mL of PBS was added to each well, and the fluorescence intensity was determined using a spectrofluorometer (Gemini EM) with excitation at 485 nm and emission at 530 nm.
All assays were carried out in triplicate and the experiments were repeated at least three times. The results are presented as means ±SD. All experimental data were compared using the Student's t test. A p value less than 0.05 was considered statistically significant.
This work was supported by the KU-Research Professor Program of Konkuk University. Dr Sangiliyandi Gurunathan was supported by a Konkuk University KU-Full-time Professorship. This work was also supported by the Woo Jang-Choon project (PJ007849).
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