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  • BioGraphene Suspensions in Sera
    May 08, 2018 | ACS MATERIAL LLC

    Synthesizing the graphene-bio interface has become an increasingly important step for biological applications. Touted as the material of the millennium, graphene has undergone rapid development focusing on optoelectronic applications for lightweight and ultrafast electronic devices. Properties such as high surface area, light weight, high mechanical strength and fluorescence also make graphene highly attractive for biological and health care applications. Colloidal stability and low toxicity are vital for the implementation of graphene materials for such applications like biosensors, drug delivery and bioelectronics, however, there have been challenges due to hydrophobicity, the presence of metal impurities, oxidative functional groups and extensive heterogeneity. Introducing the use of biological sera has allowed for graphene compatibility with biological protocols and has opened up a whole new and exciting platform for producing high quality graphene for biotechnology.

    Introduction

    Production of graphene for biological applications, in water, is challenging because of the strong hydrophobicity of graphene sheets in water.4  Graphene oxide (GO) made by oxidative methods (e.g., Hummers method)5 makes it more hydrophilic and solubilizes it in water.6 GO is often used as a model system for biological applications.3 The GO is usually modified with PEG,7 proteins,8 DNA,9 or other biomolecules for biological applications.10, 11 However, the presence of metal impurities, oxidative functional groups and extensive heterogeneity render GO unsuitable for a variety of biological studies. Therefore, high quality graphene made in media that are compatible with various biological protocols is urgently required, but not available, until now.

    Direct exfoliation of graphite to graphene in water is achieved with small organic molecules,12 surfactants,13 proteins,8,14,15 and others.16 These exfoliating agents stabilize graphene in water, but majority of them are toxic to most biological systems. On the other hand, biomolecules such as serum proteins are better candidates as exfoliation agents due to their wide spread compatibility with numerous biological systems as well as their high solubility in water. Previously protein-based exfoliation of graphite in a shear reactor has been reported.8 Herein, graphene suspensions made in animal sera for cell and animal model studies are given (Figure 1). The data show that graphene loaded sera reported here are excellent media for this purpose, when compared to graphene produced with surfactants. 

     Fig.1

    Figure 1. Graphene prepared in six different animal sera (Bovine, Chicken, Horse, Human, Porcine and Rabbit) with a kitchen blender and compared with those made by ultrasonication and shear reactor. Low toxicity of graphene in bovine serum against two mammalian cell lines and C. elegans has been demonstrated.

    Serum is a complex mixture of proteins, peptides, amino acids, salts, sugars, and many other small molecules,17 in which the exfoliation of graphite is feasible.8  Graphene in animal serum could improve the biocompatibility of graphene suspensions for  cell culture, and serum could be a good platform to study nanotoxicity of graphene with specific cell lines or animals in vivo.19, 20 Graphene produced in animal sera is expected to have a strong impact on graphene biotechnology, because many animal model studies can be carried out with good compatibility. For example, graphene in mice serum can be used readily for a mice model study, without having to deal with the compatibility issues with the media used for the graphene preparation. Graphene produced in serum (~7 mg/mL, Figure 1) was used to study the toxicity of graphene to human cells and nematodes at a wide range of doses. Human Embryonic Kidney 293T (HEK-293) cell line is a good model system for toxicity studies because of its robustness and high transfection efficiency,21 and it has been used to study cytotoxicity of other carbon materials.22 The toxicity studies were also done with C. elegans (nematodes), as model system.23 A wide range of dose dependent toxicity studies of graphene up to 500 µg mL-1 were feasible in the current studies, because high concentrations of graphene could be produced directly in sera. The scalable production of graphene with high colloidal stability in sera and its low toxicity might catalyze graphene research for biological applications.

      Fig.2a

           Fig.2b

    Figure 2. Raman spectra and TEM images of exfoliated graphene in bovine serum.

    Graphene Suspension in Sera

    Graphene made in animal sera indicate low toxicity in certain cell lines as well as nematodes and low toxicity is due to the adsorption of serum proteins on to the graphene sheets. Graphite is exfoliated in 6 different animal sera, using a kitchen blender, for maximum efficiency of production. Raman spectra and electron microscopy of the graphene/serum samples confirmed the formation of 3-4 layer, submicron size graphene, independent of the type of serum used (Figure 2, Table 1). All samples had 3-4 layers per flake while the flake size varied. The graphene samples were produced at high efficiency, at a rapid rate and required no purification except for the separation of the graphite crystals by light centrifugation.  These samples were then ready for toxicity studies. Graphene in serum was directly transferred to cell culture media without post treatment for toxicity studies. Contrary to many reports, no acute toxicity was detected for human embryonic kidney cells, human lung cancer cells or nematodes (C. elegans) for up to 7 days at various doses (50-500 µg/mL). But prolonged exposure at higher doses (300-500 µg/mL, 10-15 days) showed cytotoxicity (~95% death) and some toxicity to C. elegans (5-10% reduction in brood size). The toxicity depended on the flake size, and sheets >200 nm were non-toxic (50-300 µg/mL dose). In comparison, graphene made with sodium cholate as the exfoliant was cytotoxic at these dosages of 50-300 µg/L. 

    Table 1
     Table 1. Length and number of layers of graphene produced in different animal sera using a kitchen blender.

    Conclusions

    Based on the development of protein mediated graphite exfoliation, graphene production in sera, a protein-rich cell culture component, was contrived. Synthesis of graphene in six different animal sera and its implementation in material toxicity showed enhanced colloidal stability and decreased toxicity. Several inconsistencies in nanotoxicity of graphene is noted in the literature, primarily due to the aggregation of graphene and also due to the formation of protein coatings on the flakes. Graphene in sera is now well characterized and well defined by microscopy, spectroscopy and toxicity studies, which is suitable for biological applications. Graphene was prepared in several sera, and without further purification or sterilization it has been used for cell studies.  These samples and methods provide control in modulating graphene toxicity by using sera. Thus, serum suspensions of large flakes of graphene pacified with serum are more convenient for biological applications with encouraging results.

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