Easy and Versatile Synthesis of Bulk Quantities of Highly Enriched 13C-Graphene Materials for Biological and Safety Applications

The preparation of bulk quantities of 13C-labeled graphene materials is relevant for basic investigations and for practical applications. In addition, 13C-labeled graphene materials can be very useful in biological and environmental studies, as they may allow the detection of graphene or its derivatives in cells or organs. In this paper, we describe the synthesis of 13C-labeled graphene materials (few-layer graphene, FLG, and graphene oxide, GO) on a tens of mg scale, starting from 13C-labeled methane to afford carbon fibers, followed by liquid-phase exfoliation (FLG) or oxidation (GO). The materials have been characterized by several analytical and microscopic techniques, including Raman and nuclear magnetic resonance spectroscopies, thermogravimetric analysis, X-ray photoelectron spectroscopy, and X-ray powder diffraction. As a proof of concept, the distribution of the title compounds in cells has been investigated. In fact, the analysis of the 13C/12C ratio with isotope ratio mass spectrometry (IRMS) allows the detection and quantification of very small amounts of material in cells or biological compartments with high selectivity, even when the material has been degraded. During the treatment of 13C-labeled FLG with HepG2 cells, 4.1% of the applied dose was found in the mitochondrial fraction, while 4.9% ended up in the nuclear fraction. The rest of the dose did not enter into the cell and remained in the plasma membrane or in the culture media.

. XRD of a) pristine and b) graphitized 12 C carbon nanofiber.  Here, we show the characterization of the exfoliation of non-graphitized 12 C-CNF using glucose and melamine at different conditions (30min, 1 and 2hr) for melamine and, (2, 4 and 5hr) for glucose (Table S1).
With this purpose, we use the next system of labelling in this apart: i C-(G)FLG-n-t Where: i is the type of carbon ( 12 C or 13 C); G is for graphitized nanofibers, (without G is used when the starting material is not graphitized) FLG means Few Layers Graphene and, n depend on the exfoliating agent used (1 for Glucose and 2 for Melamine) and, t represents the time used in the exfoliation process. Table S1. Experiments related to exfoliation of carbon nanofibers using glucose and melamine as exfoliating agent.
All the different treatments were analyzed principally by Raman spectroscopy.
In Raman spectroscopy there are three principal bands for graphene nanomaterials (D, G and 2D bands). The D band is related to the defects on the sample, meanwhile the G band accounts for graphitization of the sample, therefore the intensity ratio between these two bands (ID/IG) can quantify the density of defects in the graphene flakes. 1, 2 Finally, the 2D band can be used to determine the number of layers of our graphene material through its f ull width at half maximum (FWHM) 3, 4 , a narrow 2D bands indicates a low number of layers 5 Using glucose as exfoliating agent, the relation of intensities I2D/IG of the samples corresponding to 2h and 4h of treatment show very similar values, while after 5h there is a slight increment in this value (Fig. S6). The I2D/IG ratio are around 0.5-1 indicating the presence of few-layers graphene. 6 Meanwhile, the relation ID/IG is minimum for the sample of 4h and the maximum value is for 5h of treatment. By other part, with respect to the FWHM, the lowest value corresponds to the synthesis of 4h, however here it is important to mention that this band is not relevant to quantify the number of layers due to the narrow band of the pristine nanofibers. But, in general we can corroborate that 4h of treatment are the best condition for the exfoliation of carbon nanofibers using glucose.
On the other hand, in the synthesis of graphene with melamine (Fig. S6), it is possible to observe similar values of I2D/IG (around 0.3-0.7) indicating again a structure of few layers graphene in all the samples. The relation ID/IG shows lower values that the ones observed in glucose samples, which indicates the presence of few defects in the samples prepared using melamine. Finally, the FWHM, with a range between 52 and 71cm -1 , has the lowest value for the sample of 30min indicating a good exfoliation in this treatment . Moreover, the final yield of whole exfoliation was considered. 4h and 5h of treatment give similar yields. As already discussed 5 h of treatment produce a better exfoliation so we considered this time as the best condition for our experiment. The same idea was assumed in the choice of melamine, where 2hr of synthesis are the best condition to get a major yield in the exfoliation of graphene (Table S2). Figure S6. Raman spectra (I2D/IG, ID/IG bands, FWHM, 2D and G position band) of 12 C nanomaterials at different times using glucose and melamine as exfoliating agent. After the observation, we stablished the best condition of exfoliation at 5h for glucose and 2h for melamine, and we tried this conditions to exfoliate 12 C-GFLG. Figure S7. a) Raman spectra and b) Thermogravimetric Analysis of 12 C nanomaterials: Graphitized carbon nanofibers ( 12 C-CNF) and, FLG prepared by exfoliation of graphitized carbon nanofibers using glucose and melamine ( 12 C-GFLG-1 and 12 C-GFLG-2) as exfoliating agents.