Hydrogels are characterised focusing on the contribution of the nanomaterial to the polymer network. Our results suggest that graphene is not a mere embedded nanomaterial within the hydrogels, rather it represents an intrinsic component of these networks, with a specific role in the emergence of these structures. Moreover, a hybrid hydrogel with a graphene concentration of only 0.2 mg mL−1 is used to support the growth of cultured brain cells and the development of synaptic activity, in view of exploiting these novel materials to engineer the neural interface of brain devices of the future. The main conclusion of this work is that graphene plays an important role in improving the biocompatibility of polyacrylamide hydrogels, allowing neuronal adhesion.In tissue engineering strategies, the design of scaffolds based on nanostructures is a subject undergoing intense research: nanomaterials may affect the scaffolds properties, including their ability to interact with cells favouring cell growth and improving tissue performance. Hydrogels are synthetic materials widely used to obtain realistic tissue constructs, as they resemble living tissues. Here, different hydrogels with varying content of graphene, are synthesised by  In general, 3D scaffolds have been designed to accomplish important features for implantable prostheses: biocompatibility, stability and homogeneity at the microscale, conductivity and controlled mechanical properties. Biocompatibility is in general defined as “the ability of a material to perform with an appropriate host response in a specific application”6, although new definitions, based also on cell function, are increasingly proposed7.Adapting the microenvironment of three-dimensional (3D) tissue-scaffolds to regulate neural cell adhesion or tissue growth is essential to design new therapies for neurological applications. In these developments, 3D systems are being highly investigated as they are showing, when compared 9. These nanomaterials represent biocompatible substrates, and they can be functionalised with polymers to modulate neuronal survival and growth10. Graphene has also attracted the attention of neuroscientists, demonstrating biocompatibility with neurons in 2D interfaces, being able to preserve the basal physiological level of neuronal activity13. Very interesting examples are related to 3D porous graphene structures14, which have been used as new scaffolds for neural stem cells in vitro. For example, Li and co-workers15 found that the graphene-foam not only supports cells growth, but also keeps them in an active proliferation state better than 2D graphene films.Recently, emerging carbon-based nanomaterials, with their unique physicochemical properties, have opened up additional and novel opportunities for neuronal tissue engineering developments. Among these, carbon nanotubes have been the most inspiring ones18. Hydrogels are the best candidates to play the role of extracellular matrix for 3D cell culture19, mainly due to their aqueous environment and their porous structure, which make it possible to link and maintain proteins in a functional state21. This fact allows improving cell culture development by anchoring, for example, growth factors to the polymer network, which makes these systems even more similar to the in vivo condition.Hydrogels - 3D polymer networks that possess high water contents - have been used to obtain more realistic tissue constructs because of their ability to mimic the nature of soft tissues23. In this direction, many efforts have been made to fabricate hybrid graphene-based hydrogels with enhanced properties, mainly using graphene oxide (GO)25, graphene oxide peroxide (GOP)26, or even reduced graphene (rGO) from GO28. However, most of these studies do not provide details about the role of the nanomaterial within the network, moreover GO sheets are insulating and its electronic properties differ substantially from those of graphene. As a matter of fact, there are almost no examples in literature of pristine graphene-based hydrogels29, which are mainly limited due to the difficulties to obtain pristine graphene aqueous solutions in a simple, scalable and cost efficient way.Hydrogels can be obtained from a wide range of materials; however, one of the main disadvantages of this kind of polymers is their low mechanical strength. Over the last years there has been a growing interest in reinforcing polymeric hydrogels with nanosized materials to improve and adequate their poor mechanical consistence. Remarkably, the nanomaterials embedded in a cross-linked polymer network not only make the system stiffer and tougher, but can provide novel features, adding synergistic benefits to the hybrid 3D materialset al.30 chemically modified a polysaccharide with pendant methacrylate groups, which acted as a physical exfoliant of graphite to produce multi-layer graphene flakes, followed by co-polymerization with methacrylic acid and a cross-linker. Toumia and co-workers31 dispersed graphite in an aqueous solution of a commercial amphiphilic surfactant containing aromatic moieties (PEG-PEPEMA) which was chemically grafted to a polyvinylalcohol (PVA) hydrogel network. Shim et al.32 probed the effects of different PVA and dextran polymers containing phenyl or pyrene units for the physical exfoliation of graphite in water and subsequent hydrogelation of the obtained suspensions with glutaraldehyde. Finally, Ahadian and co-workers developed a friendly aqueous route to graphite exfoliation down to few-layer graphene sheets by use of bovine serum albumin in water as exfoliant33, and used it to fabricate hydrogel nanocomposites made of methacrylated gelatin.With the latest advances in graphene research, one can find a handful of recent works that timely address this conceptual approach from top-down cleavage of graphite. Fan 2+ imaging experiments.Here, we present a new class of graphene-based hydrogels and we show their ability to support the growth of living primary neurons. The produced nanocomposites were compared to the hydrogels in the absence of nanomaterials and were fully characterised. The presence of graphene brought about a noticeable improvement in the properties of the hybrid materials and varying the graphene amounts allowed to study its role in the resulting features. Hippocampal neurons and astrocytes developed efficiently only in the graphene-doped hydrogels. In these graphene hybrids, the cultured neurons gave rise to active synaptic networks as observed by immunofluorescence microscopy and CaN,N′-methylenebisacrylamide (MBA), potassium peroxodisulfate (KPS) and melamine were purchased from Sigma Aldrich.Graphite powder was acquired from Bay Carbon Inc. (SP-1 reference). Acrylamide (Am), 34. In a typical experiment, a graphite/melamine mixture (1:3) (30 mg) was ball-milled at 100 rpm for 30 minutes using a Retsch PM100 planetary mill. The resulting solid mixture was dispersed in water (20 mL) to produce a dark suspension, which was then left to settle down for five days while some precipitate (mainly pure or poorly exfoliated graphite) segregated from the liquid. The liquid fraction with stable sheets in suspension was carefully extracted. Melamine was removed by washing the extracted liquid with hot water, leading to few-layer graphene sheets almost without defects. The obtained graphene dispersions were stable at room temperature for some weeks. The zeta potential of the dispersion was measured at 25 °C using Milli-Q water and CRISON buffer solution (pH = 7) by BIC 90Plus analyser.The exfoliation of graphite was carried out using a ball-milling procedure developed in our group−1) together with the MBA (0.2 mg mL−1). The solution was homogenised by stirring and mild sonication. Then, KPS was added  and polymerization took place at 75 °C for 1 hour. The resulting materials were immersed in water for at least four days, and the water was replaced every day in order to remove any unreacted monomer and initiator molecules.Pure polyacrylamide hydrogels were prepared using Am, MBA as cross-linker and KPS as initiator. Am was initially dissolved in ultrapure Milli-Q water (200 mg mL−1 and 2 mg mL−1).A similar process as the previously described for the preparation of AM was followed to synthesise the nanocomposite, adding in this case all the reactants to the aqueous graphene dispersion. Graphene water dispersions with different concentrations were used , which analysed 9 × 9 µm2 slices of sample and depth steps of 1 µm until reaching 10 µm. According to the dimensions of the probed volume and given that we registered 6200 spectra during the mapping, we end up with a pixel size of 0.13 µm3.The whole set of spectra obtained was fitted to the G-band region in three dimensions.Raman spectra were recorded with an InVia Renishaw microspectrometer equipped with a 532 nm point-based laser. Power density was kept in all cases below 1 mW/µm−1 (cell load = 100 N). The extent of the deformation was measured in terms of compression ratio which equals the ratio of the initial height of the sample to the final height of the sample. The extent of the deformation was also measured in terms of strain, which equals the ratio of the displacements of the samples. After holding the constant deformation, the deformed hydrogel was removed from the press. This behaviour was represented in the typical stress-strain curves from which it was possible to obtain the Young’s modulus. In this case that value was calculated between 15% and 20% of strain. Moreover, fatigue behaviour was studied using the aforementioned program in a dynamic way, reaching 100 compressive cycles with each sample. Creep experiments were conducted on hydrogels at ambient conditions with a Mecmesin Multitest 2.5-i dynamic mechanical analyser. Cylindrical disks of gels (×3) with a diameter of 1.5 cm and an initial thickness of 1.5 cm were molded. Hydrogel disks were uniaxially compressed between two plates. The deformed hydrogels were then held under constant applied load at 15% compressional strain for an extended period of time.Compressive tests were performed on hydrogels at 25 °C with a Mecmesin Multitest 2.5-i dynamic mechanical analyser. Cylindrical disks of gels (x6) with a diameter of 1.5 cm and an initial thickness of 1.5 cm were molded. Hydrogel disks were uniaxially compressed between two plates at the rate of 6 mm min37. All procedures were approved by the local veterinary authorities and the SISSA ethical committee, in accordance with the Italian law (decree 26/14) and the UE guidelines (2007/526/CE and 2010/63/EU). The use of animals was approved by the Italian Ministry of Health. All efforts were made to minimise suffering and to reduce the number of animal used. Hippocampi were isolated from the rest of the brain and upon enzymatic treatment and neurons were mechanically dissociated. Cells were plated on three different substrates: poly-L-ornithine-coated, pure polyacrylamide hydrogel (AM) and 0.2 mg mL−1 graphene hydrogels (AMG0.2). Before using for culturing, both hydrogels were sliced at 300–400 µm thickness and mounted on the glass coverslip  by a thin adhesive layer of Polydimethylsiloxane (PDMS)5. One hour prior to plating, hydrogels were treated with an air plasma-cleaner in order to facilitate cell adhesion and at the end sterilised with an UV lamp. Briefly, enzymatically dissociated hippocampal neurons37 were suspended in the culture medium consisting of MEM (Gibco), supplemented with glucose (35 mМ), Apo-Transferrin (1 mМ), HEPES (15 mМ), insulin (48 µМ), Biotin (3 µМ), Vitamin B12 (1 mМ) and Gentamicin (Gibco) (500 nМ), Fetal Bovin Serum . Cultures were incubated at 37 °C, in a humidified atmosphere with 5% CO2 in the same culture medium. Cultures were used for experiments after 8–10 days in vitro (DIV).Primary hippocampal cultures were prepared from postnatal (2–3 days) rats as previously reported2+ measurements, cultures were fixed in 4% formaldehyde  in PBS for 30 min, at room temperature. Cells were then permeabilised with Triton X-100 (1%) for 45 min, blocked with FBS (5%) in PBS (blocking buffer) for 45 min at room temperature and incubated with primary antibodies for 45 min. The primary antibodies used were: rabbit polyclonal anti-β-tubulin (1:250 dilution) and mouse monoclonal anti-GFAP (1: 500 dilution). After the primary incubation and PBS washes, cultures were incubated for 45 min with Alexa 594 goat anti-rabbit , Alexa 488 goat anti-mouse  and with DAPI  to stain the nuclei. Samples were mounted in Vectashield (Vector Laboratories) on 1 mm thick coverslips and imaged using a confocal microscope . Both 2D-polyornithine and hydrogels substrates were investigated at 20x magnification and serial confocal planes (z-stack) were acquired every 500 nm. The Z profile reconstructions of the images were performed offline using the image-processing package Fiji38.After Ca2+ measurements, cultures were loaded with cell permeable Ca2+ dye Oregon Green 488 BAPTA-1 AM (Molecular Probes); stock solution of the Ca2+ dye (4 mМ) was prepared in DMSO and cultures were incubated with a final concentration of 4 µМ for 30 min at 37 °C in the cell culture incubator. Samples were then placed in a recording chamber mounted on an inverted microscope (Nikon TE-200) where they were continuously superfused by a recording solution of the following composition (in mМ): 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose (pH adjusted to 7.4 with NaOH). The loaded cultures were observed with 40x objective (0.60 NA). Recordings were performed form visual fields (120 × 160 µm2) containing on average 6 ± 2 neurons. Prior to recording Ca2+ signals, we selected the cells by drawing regions of interest (ROIs) around cell bodies. Images were continuously acquired by a Till Photonics Till-Imago System, exciting the Ca2+ dye with a 488 nm wavelength light generated by a monochromator device equipped with integrated light source . Images were acquired continuously for 1800 s (200 ms exposure time) by a cooled slow-scan interline transfer camera  operating on 60 × 80 pixels binning mode. The imaging system was controlled by an integrating imaging software package  using a personal computer. In order to induce rhythmic bursts, bicuculline methiodide (a blocker of GABAA receptors) (20 µМ) was bath-applied after 10 minutes recording; at the end of each experiments, TTX (1 µМ)  was added to the recording solution to confirm the neuronal nature of the recorded signal. Recorded images were analysed off-line both with the Clampfit software  and Igor Pro Software . Intracellular Ca2+ signals were expressed as fractional amplitude increase (ΔF/F0 where F0 is the baseline fluorescence level and ΔF is the rise over baseline); we determined the onset time of neuronal activation by detecting those events in the fluorescent signal that exceed at least five times the standard deviation of the noise. We then computed the difference between consecutive onset times, to obtain the inter-event interval (IEI). All data are presented as mean ± standard deviation (SD) of the mean (n is the number of cells). Statistical significance for IEI was calculated with Student’s t-test .For CaAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).34. Graphene dispersions in water were obtained and thoroughly characterised  and N,N′-methylenebisacrylamide (MBA) were chosen as the initiator and the cross-linker respectively. Figure Graphene-based hydrogels are formed by −1) were synthesised. Figure A chemically cross-linked polyacrylamide gel (AM) prepared without graphene and a series of hybrid graphene-polyacrylamide gels  and the results show structural changes in the hydrogels with graphene compared to the sample without nanomaterial  are used in similar proportions. Moreover, we can exclude a relevant role of the residual melamine in the hydrogel structure, because the amount of melamine in the final gels is negligible. Hydrogels can be chemically or physically crosslinkedial Fig. . Three wial Fig. . The fir46, are ascribed to the different stretching (ν) and bending (δ) vibrations of the polyacrylamide skeleton, being the most intense the methylene and C-N stretchings, and bending of amino groups. Upon acquisition of Raman spectra from the hybrid graphene hydrogels, the characteristic features of graphene are visible, mainly the disordered carbon (D-band), the sp2 tangential mode (G-band), and the second-order vibration mode (2D-band). Raman spectroscopy was also employed to reveal the state of dispersion of graphene within the hydrogel matrix, throughout three-dimensional mappings at a micrometer scale . The final swelling degree decreases when increasing graphene content in the hybrid hydrogels Fig. . The mos48. Compressive tests were conducted on all the samples. For practical reasons, we only show the compressive Young’s modulus (E) for samples AM, AMG0.2 and AMG2, but, although not shown, samples AMG0.05 and AMG0.1 presented identical values in the elastic moduli as AMG0.2. These values are in the range of other materials already used for 3D neuronal growth50. The Young’s modulus of the hybrid prepared in the presence of a dispersion of 2 mg mL−1 of graphene is considerably higher in comparison with the other two hydrogels. The fatigue behaviour was studied only for AM and AMG2 gels, in order to evaluate if the introduction of graphene could deteriorate the gel and affect the fatigue response. In this case, the compressive toughness of the materials was measured from the area of the stress-strain curve of non-stopped 100 compressive cycles. It is evident that the incorporation of graphene in the hydrogel increases the toughness of the material  to address its potential interference with neuronal growth. The final content of melamine in this hybrid hydrogel is only 5.4 · 10−4 wt%, which is not comparable to the amount of cross-linker used to form the nanocomposite sample (0.1 wt%). The intrinsic role of graphene would be thus revealed, with potential interest in further scalability and applications. We cultured cells in three different conditions: in traditional 2D polyornithine-layered substrate (control)4 and in the hydrogels, cross-linked in the presence (AMG0.2) or in absence (AM) of graphene. After 10 DIV we compared neuronal growth by immunofluorescence techniques and confocal microscopy. As shown in Fig. 4. Despite repeated trials (3 different culture series) hippocampal cells when seeded on AM hydrogels did not adhere to the structure and, even more, barely grew. We detected only occasionally few clusters of small β-tubulin III-positive cells, virtually deprived of outgrowing axons, as shown in Fig. 4. The zeta-stack reconstruction revealed that hippocampal cells were able to grow within the porous niches present on the surface of the graphene-based hydrogels, but they did not migrate deeper in the structure4. This is further sustained by the reconstruction of Fig. We tested the 3D polyacrylamide scaffold efficacy to support neuronal cell growth and activity and we compared one of the nanocomposite sample (AMG0.2) to its blank AM counterpart. Although different in the pore size ranges, both materials have pores within dimensions known to permit cell survival in culture4. At 8–10 DIV neurons are usually mature and active and display synaptic signaling spontaneously, traditionally mediated by glutamate and GABAA receptor mediated-synaptic pathways36. With our imaging set up we sampled representative regions of 120 × 160 μm2. Neurons were stained with the membrane permeable Ca2+ dye Oregon green 488 BAPTA-1 and simultaneously visualised within the sampled area. On average, 6 ± 2 fluorescent cells were detected in the recorded field. In Fig. 2+ activity usually appeared as spontaneous bursts of activity fully blocked by TTX (1 µM) applications . TTX is a well-known blocker of voltage-gated fast Na+ channels54, responsible for the generation of action potentials. In our recordings, spontaneous Ca2+ activity was detected in 36% of control cells visualised in each field  and, similarly, in 25% AMG0.2 cells  of 32 ± 7 s (n = 21 cells) that is similar to that measured in AMG0.2 cultures  application. This antagonist of synaptic inhibitory components is known to potentiate rhythmic activity patterns56. In fact, in both groups, in respect to spontaneous activity, the addition of bicuculline significantly decreased the IEI average values 4. In Fig. 2+ episodes brought about by bicuculline in active cells that, on average, display IEI of 22 ± 4 s in control (n = 21 cells) and, similarly, of 16 ± 6 s in AMG0.2 cultures , therefore indirectly affecting neuronal growth.The main result obtained from our neurobiological studies is that the presence of graphene inside the structure of the hydrogel is crucial for allowing cell adhesion and the formation of mature and active synaptic networks. In fact, only sporadic clusters of few neurons can be found in the blank hydrogel, in the absence of proper axon formation. Ultimately, neurons only grew in the scaffold synthesised by using a graphene dispersion in water with a concentration of just 0.2 mg mL4. This is further suggested by the observed similarity in the activity patterns when recorded by calcium imaging in the two culturing conditions4. However, in both networks, episodes of calcium activity, whose neuronal and synaptic nature was supported by TTX experiments, were reflecting spontaneous synaptic activity. Based on these results, we are carrying out further experiments to adjust the pore size dimensions and interconnectivity in the graphene-based hydrogels in order to improve the growth and migration of neurons deeper within the sample, favouring by this way 3D neuronal network connections.The neuronal networks grown in control and AMG0.2 display basically similar Z projection (≈20 µm thickness), thus in the graphene-hybrid hydrogels, neurons did not develop in a genuine 3D geometryWe have designed hybrid graphene hydrogels as new 3D scaffolds for neuronal growth. Acrylamide was used as main monomer for the formation of the polymer networks and the radical polymerization was conducted in aqueous graphene dispersions of different concentrations. The scaffolds were thoroughly characterised, and the compressive moduli were measured, being higher for the sample that contains a noticeable amount of graphene. Moreover, no significant fatigue behaviour was observed in the studied samples, even in the hydrogel with the highest amount of graphene. The most important result observed from the neuronal studies is that neuronal networks are only visualised in the hybrid graphene hydrogel, but not in the scaffold without graphene. The mechanical properties of the scaffold do not seem to be critical for neuronal growth in the presence of graphene and thus an intrinsic beneficial role of graphene is revealed.Supplementary info


Glioblastoma Multiforme (GBM) is a lethal primary brain tumor with poor survival lifespan and dismal outcome. Surgical resection of GBM is greatly limited due to the biological significance of brain, giving rise to tumor relapse in GBM patients. Transactive response DNA binding protein-43 (TDP-43) is a DNA/RNA-binding protein known for causing neurodegenerative diseases through post-translational modification; but little is known about its involvement in cancer development. In this study, we found that nutrient deprivation in GBM cell lines elevated TDP-43 expression by a mechanism of evasion from ubiquitin-dependent proteolytic pathway, and subsequently activated the autophagy process. Exogenous overexpression of TDP-43 consistently activated autophagy and suppressed stress-induced apoptosis. The inhibition of autophagy in TDP-43-overexpressing cells effectively increased the apoptotic population under nutrition shortage. Furthermore, we demonstrated that HDAC6 was involved in the activation of autophagy in TDP-43-overexpressing GBM cell lines. The treatment with SAHA, a universal HDAC inhibitor, significantly reduced TDP-43-mediated anti-apoptotic effect. Additionally, the results of immunohistochemistry showed that TDP-43 and HDAC6 collaborated in GBM-tumor lesions and negatively correlated with the relapse-free survival of GBM patients. Taken together, our results suggest that the TDP-43-HDAC6 signaling axis functions as a stress responsive pathway in GBM tumorigenesis and combats nutrient deprivation stress via activating autophagy, while inhibition of HDAC6 overpowers the pathway and provides a novel therapeutic strategy against GBM. Glioblastoma (GBM), classified as Grade IV brain tumor by World Health Organization (WHO), is the most common and aggressive malignant primary brain tumor . GBM is TDP-43, also known as transactive response (TAR) DNA binding protein-43 (TARDBP), belongs to heterogeneous nuclear ribonucleoprotein (hnRNP) family and is a well-characterized RNA-binding protein (RBP). TDP-43 has been closely linked to the pathogenesis of neuronal degenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) upon post-translational modification , 6. BesiNutrient deprivation is often observed in solid tumors due to the rapid growth rate and ineffective blood supply in tumor tissue. Autophagy has been implicated as the key regulatory mechanism for cells to escape from cytotoxic stress. The role of autophagy in cancer therapy has been implicated to against cytotoxic, plenty of clinical trials have been launched targeting the inhibition of autophagy while the expression of autophagy-associated proteins, such as beclin-1 and LC3, are also connected with the poor prognosis of cancers –13. The In this study we investigated the mechanism of how TDP-43 promotes survival of the cells upon nutrition deprivation and contributes to the progress of GBM. We found that elevated expression of TDP-43 activates autophagy in stressful conditions and rescues cells from apoptotic process. Immunoprecipitation of TDP-43 revealed the increase of ubiquitination indicating the shortening of protein half-life of TDP-43 during starvation. Moreover, we discovered that HDAC6 acts as a downstream effector of TDP-43 in response to nutrient deficiency. In meanwhile, the inhibition of HDAC increases the apoptotic events even when TDP-43 is overexpressed. Concomitantly, we compared our findings to the clinical data and found that TDP-43 and HDAC6 are co-expressed and their high-expression reflected the better survival than that of others.p < 0.01), while TDP-43 also enhanced the formation of neurospheres of U87MG . To further assess the role of TDP-43 in tumorigenicity, we subcutaneously implanted the TDP-43-overexpressing cells into nude mice. In comparison to the empty vector control (Flag-control), the size of tumors was greatly increased in Flag-TDP-43 group , the TDP-43 was found to be involved in Warburg effect-associated pathways . In contrast, siTDP-43 enhanced the starvation-induced proportion of apoptotic cells in U87MG as compared to scrambled siRNA control . These data indicate that TDP-43 protects GBM cells from nutrient deprivation-induced cell death.Limited vascularisation caused by rapid tumor growth restricts local oxygen and nutrient supply and results in a stressful condition. However, the malignant neoplasms, including GBM, are able to survive this stress, although little is known about the detailed molecular mechanisms supporting such survival. Warburg effect is one of the most well-established mechanisms of tumor survival under nutrient shortage. In an s Figure . Nutriens Figure . In addis Figure . We alsos Figure . In meanp < 0.05). Western blotting further indicated that the endogenous autophagy marker LC3-II accumulated in nutrient-deprived U87MG cells with TDP-43 over-expression.  as a downstream effector of TDP-43 , we expep < 0.001; Figure hi/HDAC6hi. Collectively, we demonstrated that the co-elevated expression of TDP-43 and HDAC6 positively correlated with the high-grade glioma and GBM, suggesting the TDP-43/HDAC6 could be a potential marker of prognosis in GBM.Since our data suggested that TDP-43 and TDP-43/HDAC6 axis promotes GBM-related tumorigenesis, we therefore examined the expression patterns of TDP-43 and HDAC6 in GBM patient samples. First, we analyzed microarray dataset from TCGA Brain (Oncomine database) and found that expression levels of TDP-43 were significantly elevated in GBM specimens in comparison with the clinical white matter  Figure . SecondlATG7 mRNA that encodes an autophagy regulating factor, which is required to maintain autophagy system [Understanding of the molecular mechanisms of tumorigenesis in glioblastoma multiforme (GBM) is crucial for developing the effective therapeutic approaches and improving patient survival. In this study, we focused on expression of the nutrient deprivation-induced TAR DNA-binding protein TDP-43) and its downstream effector histone deacetylase-6 (HDAC6) to dissect their involvements in tumorigenesis of GBM. Our results indicate that TDP-43 expression is essential for tumor progression Figure  by promo and its y system . NotablyTDP-43 has been reported to be involved in repression of HIV transcription and regulation of alternative splicing of CFTR gene –27. BecaHistone deacetylases (HDACs) are involved in epigenetic modification through histone deacetylation. Eighteen HDACs were identified in mammals, among them class I HDAC family members that include HDAC1 and HDAC2, are localized in the nucleus and regulate some biological events in cancer cells, including apoptosis and proliferation. Unlike type I HDACs, HDAC6 is categorized as type IIB HDAC and is mostly located in the cytoplasm, where it is predominantly engaged in cytoskeletal regulation , 41. HDATo summarize our findings, we demonstrate that: (1) TDP-43 expression is required for tumor progression; (2) TDP-43 modulates autophagy in starved cells, which is in charge of tumor survival under nutrient deprivation; (3) increased stability sustains TDP-43 expression in starved cells; (4) HDAC6 expression mediates anti-apoptotic function of TDP-43. To our knowledge, this is the first demonstration that TDP-43 is involved in tumor progression via autophagy. These findings contribute to our understanding of the potential role of TPD-43 in cancer. In summary, TDP-43/HDAC6 functions as a stress responsive pathway in GBM tumorigenicity and promotes survival of nutrient-deprived GBM cells via activating autophagy Figure .The human GBM cell line, U87MG, and its derived TDP-43-overexpressed stable cell line were cultured in Dulbecco’s Modified Eagle’s Media  with 10 % fetal bovine serum , 150g/mL G418 , 100 units/mL penicillin, and 100 μg/mL streptomycin  under standard culture conditions . Subculturing was performed with 0.25% trypsin-EDTA . Media were replenished every two days.U87MG cells were seeded in 6-well plates  and were incubated for 24 hours. U87MG cells starved in nutrient deprivation condition for 6 days. After 6 days, U87MG cells were refreshed with fresh medium and cultured for 14 days. The cells were fixed with 9% formalin, and stained with 4% trypan blue (w/v) for 20 min. The stained cells were washed using PBS and counted.The cells were cultured in tumor sphere medium consisting of serum-free DMEM/F-12, N2 supplement, 10 ng/mL human recombinant bFGF, and 10 ng/mL EGF until sphere formation was observed after about 4 weeks of cultivation.5 cells was added, and the dishes were incubated at incubator for 4 weeks. Plates were stained with 0.005% (w/v) crystal violet for 1 hour and then a dissecting microscope was used to count the number of colonies.Each well (35 mm) of a six-well culture dish was coated with 1.5 ml bottom agar mixture  FCS, 0.6% (w/v) agar). After the bottom layer solidified, 1.5 ml top agar-medium mixture  FCS, 0.3% (w/v) agar) containing 1 × 10Protein samples were prepared with RIPA buffer  containing 1% protease inhibitor. Equal weights of total protein were separated by SDS/PAGE. After the proteins had been transferred onto a polyvinylidene difluoride membrane , the blots were incubated with blocking buffer (1 X PBS and 5% skim milk) for 1 hour at room temperature and then hybridized with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. The blots were obtained by X-ray film exposure, and the intensities were quantified by densitometric analysis . The primary antibodies used in this study were anti-Flag , TDP-43 , cleaved-caspase-3 , HDAC6  and beta-actin . LC3 (Novus NB100 2220).Total RNA was isolated from GBM cells using TRlzol  followed by phenol:chloroform purification and ethanol precipitation. Reverse transcription reactions were carried out using SuperScript III reverse transcriptase . Oligonucleotides were designed using Primer Express 2.0 . Oligonucleotide specificity was computer tested  by homology search with the human genome and later confirmed by dissociation curve analysis. The qRT-PCR was performed with power SYBR Green PCR Master Mix  according to manufacturer’s instruction. Signals were detected with 7900HT Fast Real-time PCR system . The expression level of each gene was normalized to endogenous beta-actin and experimental control through ΔΔCt method.The RNAi oligonucleotides targeting TDP-43 (siTDP-43) and the scramble sequence (scRNA) oligonucleotides were all purchased from Ambion. Transient transfection was carried out using INTERFERin siRNA transfection reagent  according to manufacturer’s instruction. Cell-based experiments were performed after 2-day incubation.Plasmid transfection was carried out using jetPEI DNA transfection reagent  according to manufacturer’s instructions.All procedures involving animals were performed in accordance with the institutional animal welfare guidelines of Taipei Veterans General Hospital. The GBM cell line U87-Flag and U87-TDP-43 were harvested, washed, resuspended in PBS and subcutaneously implanted into the dorsolateral side of the flank region of 8-week-old male BALB/c nude mice . After 14 days of subcutaneous inoculation tumor volume was measured by calipers.2O2. Sample were blocked in 5mg/ml BSA  for 30 mins before hybridizing with 1/100 diluted primary antibodies TDP-43 , cleaved-caspase-3 , HDAC6  overnight at 4°C. Signals were amplified by the TSA Biotin System  following the manufacturer’s instruction and the samples were counterstained with hematoxylin. The sections were examined under Olympus BX61 microscope , and three fields of view were randomly selected and photographed for evaluation. The relative staining index (rSI) represents the percentage of positive-expressed cells in the counting region.Tumor specimens from mice were fixed with 4% paraformaldehyde . Sections were deparaffinized and rehydrated, and subjected to antigen retrieval by boiling in 10 mmol/L (pH 6) citrate buffer  for 10 mins. Sections were cooled in PBS for 10 mins before treating with 3% Ht-test was used to compare the continuous variables between groups. The Kaplan-Meier procedure was used to calculate survival probability estimates. A log-rank test was used to compare the cumulative survival durations in different groups. The statistical significance level was set at 0.05 for all tests.The Statistical Package of Social Sciences software  was used for statistical analysis. An independent Student’s


The JAVELIN Solid Tumor clinical trials group recently reported the results of a multi-center open-label phase 1b study NCT01772004) evaluating avelumab in patients with progressive or platinum-resistant metastatic or recurrent non-small cell lung cancer (NSCLC) [004 evaluin vitro [in vitro.Among the five currently FDA-approved anti-PD-1/PD-L1 agents, avelumab is unique due to its unmodified fragment crystallizable (Fc) domain which may potentiate antibody-dependent cell-mediated cytotoxicity (ADCC) via interaction with CD16 (FcɣRIIIa) on natural killer (NK) cells . The impin vitro . Howeverin vitro . This strd line of therapy) and with other driver mutations . Multiple studies across multiple treatment modalities including immunotherapy have shown lower response rates with more advanced disease / later lines of therapy.With a median follow up of 8.8 months in the NSCLC dose-expansion cohort, the JAVELIN investigators enrolled 184 patients with stage IIIB or IV NSCLC with disease progression after prior platinum-based doublet therapy for metastatic disease across 58 academic centers . This stn = 22), and 1-year progression-free and overall survival rates were 18% and 36%, respectively. Ongoing follow-up has confirmed an additional 4 responses, raising the ORR to 14.1% (n = 26). Accounting for these factors, ORR was similar (12%-14.1% vs. 14%-20%) in this study compared with randomized prospective studies evaluating other PD-1 or PD-L1 inhibitors in the second-line advanced NSCLC setting [vs. 11.4%) [Objective response rate (ORR) was 12% . These findings are exploratory and hypothesis-generating at present but add to a growing compendium of literature attempting to clarify the appropriate use of PD-L1 expression as a predictive biomarker for patient selection. A preliminary exposure-response analysis of this cohort of NSCLC patients explored the relationship between PD-L1 expression and avelumab exposure (trough concentration after first dose) [To explore potential biomarkers of response to avelumab, clinical responses were stratified by PD-L1 expression. A proprietary assay for PD-L1 expression (anti-PD-L1 rabbit mAb clone 73-10) using a cutoff value of ≥1% on tumor cells showed a progression-free survival benefit for PD-L1 positive patients relative to PD-L1 negative counterparts (12.0 In consideration of this heterogenous, heavily pre-treated, PD-L1 non-selected advanced NSCLC patient population, avelumab demonstrated a toxicity profile and anti-tumor activity similar to other anti-PD-1 and PD-L1 mAbs .


Oxidative wet-chemical delamination of graphene from graphite is expected to become a scalable production method. However, the formation process of the intermediate stage-1 graphite sulfate by sulfuric acid intercalation and its subsequent oxidation are poorly understood and lattice defect formation must be avoided. Here, we demonstrate film formation of micrometer-sized graphene flakes with lattice defects down to 0.02% and visualize the carbon lattice by transmission electron microscopy at atomic resolution. Interestingly, we find that only well-ordered, highly crystalline graphite delaminates into oxo-functionalized graphene, whereas other graphite grades do not form a proper stage-1 intercalate and revert back to graphite upon hydrolysis. Ab initio molecular dynamics simulations show that ideal stacking and electronic oxidation of the graphite layers significantly reduce the friction of the moving sulfuric acid molecules, thereby facilitating intercalation. Furthermore, the evaluation of the stability of oxo-species in graphite sulfate supports an oxidation mechanism that obviates intercalation of the oxidant. Scalable graphene production from graphite via an intercalation-oxidation-reduction process is still hampered by low reproducibility and many lattice defects. Here, the authors show that reducing molecular friction by using highly crystalline graphite and mild oxidizing conditions is the key to high quality graphene. However, mainly high temperature processes are facilitated to produce graphene5. Up to now, it remains challenging to prepare the same quality of graphene wet-chemically utilizing chemical functionalization methods. The advantages of a wet-chemical process are its scalability and the possibility to introduce functional groups at the same time. Chemical modification can give new functions to graphene, which enable e.g., medical applications10.The outstanding electronic and mechanic properties of graphene and its chemical reactivity led to the development of graphene technology11. For improving the quality of the produced graphene, it is therefore highly desired to understand the underlying mechanisms in this process in more detail in order to be able to better control the individual synthesis steps. Key factors on the way to high-quality graphene are the proper formation of the intermediate graphite sulfate, a stage-1 graphite intercalation compound (GIC) with every layer of the graphite intercalated  of 0.3–0.05%14. This oxo-functionalized graphene was found to be suitable to establish a controlled chemistry of graphene where lattice defects play a minor role18. We term this product oxo-G1 (index = number of layers) in order to distinguish it from the known graphene oxide, which possesses an undefined amount of defects and consequently a disturbed order of the lattice.Conventional synthesis of graphene via graphite intercalation, oxidation, delamination into graphene oxide, and subsequent reduction 2)n has been reported, needs more clarification for improving the yield and the quality of the synthesized graphene21. The intercalation of graphite with sulfuric acid increases the layer distance to 7.98 Å24. This intercalation process is initiated by electronic oxidation of the graphite29. However, why the initial oxidation is needed and to what extend oxo-functional groups are introduced on the basal planes of the graphite scaffold at this stage, is currently unknown. Puzzling are also recent contradictory reports: while some groups observed the intercalation process to be reversible upon water addition24, we recently reported the formation of hydroxylated graphene under similar reaction conditions14. This contradiction unfolds the lack of our understanding of the overall graphene synthesis reaction.Nevertheless, how a proper stage-1 GIC is obtained that enables oxo-Gion Fig.  and the 1. Our oxo-G1 from the graphite with the highest crystallinity yields a graphene with an exceptionally low density of lattice defects down to 0.02% after reductive defunctionalization. Statistical Raman spectroscopy (SRS) and high-resolution transmission electron microscopy (HRTEM) prove the high lattice quality. Mechanistic ideas about the formation ability of different types of graphites to form stage-1 graphite sulfate are derived from ab initio molecular dynamics (MD) simulations, which provide an understanding why a high degree of crystallinity of graphite is mandatory. The simulations show that the friction of moving sulfuric acid molecules in the graphite intercalation compound is intimately coupled to the stacking and the electronic oxidation state of the graphite layers. Ideal AB stacking and initial oxidation significantly reduces friction and supports the spreading of the sulfuric acid molecules within the graphite. Oxo-species  are found to be unstable within the graphite intercalation compound below a formal oxidation state of about Here we reveal that crystallinity of the graphite is the decisive factor for obtaining a proper stage-1 GIC and subsequently oxo-G2SO4-GIC formation was described in the literature21, the origin of hindrance could not yet be elucidated.The formation of graphite sulfate was studied experimentally by immersing a high quality graphite crystal in a quartz glass cuvette with conc. sulfuric acid and ammonium persulfate as oxidant Fig. . The for30, and NG1 is the most crystalline graphite with 28% 3R-phase (ABC stacking) and crystallite size larger than 10 μm and 72% 2H-phase (AB stacking) with crystallite size of about 100 nm. NG2 also bears crystallinity with about equal amounts of 3R-phase (46%) and 2H-phase (54%) with however crystallite sizes of 100 nm and less. In contrast, TG bears no significant stacking order30. Raman spectroscopy of those graphite grades  according to the intensity ratio of the defect induced D peak and the G peak (ID/IG)32, which are for NG1 LD = 35 nm (ID/IG = 0.12), for NG2 LD = 36 nm (ID/IG = 0.11) and for TG LD = 19 nm (ID/IG = 0.38). The symmetrical shape of the 2D peak of TG also indicates the missing stacking order33.To shine more light on the intercalation and subsequent functionalization and graphene formation process three different grades of graphite were used: Sri Lankan natural graphite with 70–150 μm grain (NG1), natural graphite with 10 μm grain (NG2), and turbostratic graphite (TG). X-ray powder diffraction of those graphites was conducted in an earlier study−1 due to the high doping level. The 2D peak is almost absent in both NG-GICs as expected  of the D and G peak, respectively 11. However, few spectra of processed NG2 also exhibit characteristic spectral features of graphite  with lateral dimensions up to 10 μm is found for NG1, whereas for NG2 such flakes are only isolated from the supernatant and with lateral dimensions <3 μm. Apparently, mainly graphite-like particles are formed. In contrast, graphene was not found from TG. Consequently, we prepared dispersions of functionalized graphene from NG1 to investigate the potential use of this synthetic approach.Subsequent sonication of water purified samples of functionalized graphite leads to exfoliation and a deposition of flakes on Si / 300 nm SiO1 with an optical density of 0.5 could be obtained by iterative centrifugation and redispersion in a 1/1 mixture of H2O/MeOH assisted by ultrasonication  indicates a low degree of oxo-functionalization, which was estimated to θf = 0.04 in an earlier study14. Furthermore, graphene obtained after reduction with NaBH4 and stabilized by sodium cholate exhibits an absorption maximum at 266 nm, instead of 254 nm for oxo-G1 (θf = 0.5), indicating an improved quality of graphene . Moreover, the IG values of recorded Raman data let conclude that about 72% of the area are either single layer graphene (including areas of probed single layer graphene / substrate) with only 28% of few-layer graphene  technique. In Fig. 34. Although heteroatoms, such as nitrogen or oxygen, can’t be discriminated from carbon by contrast, the carbon framework of graphene is well resolved as it can be seen in Fig. 35. Although some areas of the carbon lattice are hidden by the adsorbed material, the periodic extension of the uncontaminated carbon framework gives evidence for a coherent hexagonal lattice.The interpretation of SRS results is further confirmed by HRTEM performed with atomic resolution after deposition of functionalized graphene on TEM grids by the LB technique. The carbon lattice was observed without additional cleaning or heating procedures, as we described in a recent HRTEM study36 and to probe how their mobility depends on the graphite stacking and the GIC oxidation state.The observation that micron-sized graphite grains with perfect crystalline stacking (NG1) are easier to intercalate and to oxidize than more defective graphite grades with smaller grains (NG2) and perturbed stacking (TG) is surprising. Intuitively one would expect that the reduced cohesion between the graphite layers and the increased layer separation in perturbed graphite makes it easier for the sulfuric acid molecules to enter the graphite lattice, widen the layer spacing and fill the available space. This suggests that for GIC formation and oxidation the initial ability of the sulfuric acid to penetrate the graphite lattice is less important, but the mobility of the sulfuric acid between extended graphite sheets is the more decisive factor. To explore this idea, a series of ab initio molecular dynamics (MD) simulations were performed in order to determine the interaction of confined sulfuric acid molecules with the graphite lattice22. The region between the graphite layers was filled with 8 sulfuric acid and one water molecules to reflect the composition of concentrated sulfuric acid (see Method section). Different stages of oxidation were obtained by adding one, two and three isolated pairs of hydroxyl groups to this unit cell, each in 1,2-trans configuration since this is the most stable arrangement of OH pairs , leaving behind a two-fold positively charged graphite layer see Fig. . Similar2SO4)2)n) of one positive charge per 24 carbon atoms19 and can be well understood from an analysis of the electronic structure  for our three configurations is shown in Fig. λ can be extracted from the plateau region, which is reached after about 0.4 ps. The comparison between the three simulations reveals that oxidation  shown in Fig. This result is corroborated by an analysis of the po was converted into a free energy surface via ∆A = −kBT In po from which a line scan  combined with large grain sizes as in NG1 efficiently reduces the corrugation of the free energy surface for the slip of the sulfuric acid molecules between the graphite sheets and supports successful stage-1 GIC formation. In contrast, any deviation  as in NG2 and TG leads to an increase in the free energy barrier and thus hinders the intercalation process.Finally, our observations on the stability of oxo-species in graphite sulfate suggest an alternative oxidation mechanism. Since oxo-species do not remain attached to the graphite sheets up to an oxidation state of ion Fig. . This prExperimental and theoretical results demonstrate that crystalline graphite with large grain size can act as a precursor for wet-chemically prepared graphene of high quality, as directly observed by HRTEM and quantified by statistical Raman spectroscopy. Friction of sulfuric acid confined between layers of graphite is significantly lowered with electronic oxidation, which initiates the intercalation process. Intercalation and oxidation reinforce each other: if intercalation is hindered by high friction, oxidation remains insufficient and the partial intercalation is reversible upon addition of water. Epoxides and hydroxyl groups are not stable at low oxidation levels, but become protonated and cleaved from the carbon lattice. Only at higher oxidation levels oxo-species remain attached to the graphite layers.Moreover, friction of confined sulfuric acid is lowest for AB-stacked graphene layers. Therefore, it is plausible that natural graphite, bearing grain boundaries and irregular stacking orders, is not an equally potent precursor for graphene production. Along the same line, turbostratic graphite is not forming a proper GIC (low oxidation degree) and thus reverts back to graphite upon water addition. Sulfuric acid, however, intercalates graphite of high crystallinity with large grains upon oxidation. The oxidation proceeds very likely outside the layer confinement by electron transfer, and charge compensation is accompanied by proton diffusion, leading to ers Fig. .The here presented route towards oxo-functionalized graphene and graphene on substrates or stabilized by surfactants in water can be expected to also lead to novel graphene derivatives with tailored functionality suitable for electronic or bio-sensing applications.42. UV–Vis spectroscopy was conducted on a Perkin Elmer—Lambda 1050.Graphite grades NG1 (grade 2012) and TG were obtained from Asbury Carbon. NG2 was purchased from Future Carbon. Large flake graphite was obtained from NGS Trading & Consulting GmbH. Sulfuric acid, hydroiodic acid 57% in water (HI), trifluoroacetic acid (TFA), and ammonium persulfate were obtained from Sigma-Aldrich® and used as received. Doubly distilled water was obtained from Carl Roth GmbH and used as received. Methanol was distilled in a solvent circulation apparatus before use. Centrifugation was accomplished by a VWR Micro 1814 table centrifuge. Sonication was performed using a Sonorex Digital P10 from Bandelin. LB films were prepared using a LB mini trough from KSV NIMA. AFM imaging was performed using a NT-MDT Solver Pro. Raman spectra were recorded with LabRAM HR Evolution from Horiba Scientific using an 532 nm laser line. Spectra were processed as reported elsewhereGraphite  was suspended in sulfuric acid . Over a period of three days ammonium persulfate  was added in small portions under stirring.g) and the supernatant was removed. The graphite sulfate was freezed by liquid nitrogen and doubly distilled water (1.5 ml) was added. The mixture was cooled for 16 h to 8 °C. The reaction mixture was purified by centrifugation and redispersion in water  until the supernatant was of neutral pH. Finally, the carbon material was taken up in 1/1 volume mixture of water/methanol. The dispersion was sonicated in a bath sonicator for 2 min. The oxo-G1 solution was obtained by centrifugation  and collecting the supernatant. Small particles were removed by centrifugation . Finally, larger particles were removed by centrifugation  leaving oxo-G1 in dispersion.Graphite sulfate (400 mg) was centrifuged  until the surface pressure reached 5 mN m−1. The substrate was then pulled up (2 mm min−1) to deposit flakes of oxo-G1 on Si/SiO2 (300 nm) substrates.The as-prepared oxo-G43, the Perdew-Burke-Ernzerhof PBE exchange-correlation functional44, Vanderbilt ultrasoft pseudopotentials45 and a plane wave basis set with kinetic energy cutoff of 30 Ry. To account for dispersion effects, the semi-empirical Grimme D2 correction was applied46. The MD simulations were carried out in the NVT ensemble using a Nose-Hoover thermostat set to 400 K in order to compensate for the known overstructuring of hydrogen-bonded liquids at room temperature in PBE47. An additional Nose-Hoover thermostat, a fictitious mass of 400 a.u. and a time step of 4 a.u. were used for the propagation of the electronic degrees of freedom. For obtaining converged results for the friction coefficient in the MD simulations, large supercells with lateral size of 24. The interlayer region was filled with 32 sulfuric acid and four water molecules for the neutral GIC simulations and eight hydrogen atoms were removed in the oxidized GIC(2SO4)2)n,21.The Car-Parrinello molecular dynamics simulations were carried out using the CPMD code48. The image acquisition time was 2 s and the dose rate for the nominally 300,000× magnified images (Fig. 6 e− nm−2 s−1. The diameter of irradiated area was approximately two times the camera width and the sample area imaged was changed after recording few micrographs with varying defocus; therefore, the maximum total dose per imaged area was about 108 e− nm−2, since the post-sample beam shutter was used. Vignettes were balanced using Gwyddion software.The TEM investigations were performed with a FEI Titan 80–300 transmission electron microscope fitted with a CEOS CETCOR third order spherical aberration corrector and a Gatan UltraScan 1000XP CCD camera. The microscope was operated at an electron acceleration voltage of 80 kV and the electron source extraction voltage was set to 2 kV reducing the primary electron energy distribution to 0.5 eV FWHMThe data that support the findings of this study are available from the corresponding authors upon request.Supplementary InformationPeer Review File


Fluorinated graphene, an up‐rising member of the graphene family, combines a two‐dimensional layer‐structure, a wide bandgap, and high stability and attracts significant attention because of its unique nanostructure and carbon–fluorine bonds. Here, we give an extensive review of recent progress on synthetic methods and C–F bonding; additionally, we present the optical, electrical and electronic properties of fluorinated graphene and its electrochemical/biological applications. Fluorinated graphene exhibits various types of C–F bonds , tunable F/C ratios, and different configurations controlled by synthetic methods including direct fluorination and exfoliation methods. The relationship between the types/amounts of C–F bonds and specific properties, such as opened bandgap, high thermal and chemical stability, dispersibility, semiconducting/insulating nature, magnetic, self‐lubricating and mechanical properties and thermal conductivity, is discussed comprehensively. By optimizing the C–F bonding character and F/C ratios, fluorinated graphene can be utilized for energy conversion and storage devices, bioapplications, electrochemical sensors and amphiphobicity. Based on current progress, we propose potential problems of fluorinated graphene as well as the future challenge on the synthetic methods and C‐F bonding character. This review will provide guidance for controlling C–F bonds, developing fluorine‐related effects and promoting the application of fluorinated graphene. The chemical modification endows graphene with many excellent properties, such as good dispersion in organic/water solvents, chemical activity on the surface via functional groups and tunable electronic properties, such as bandgap opening, charge transfer density and work functions. Despite numerous studies, GO and halogenated graphene continue to have several problems as follows: (1) GO has a variety of chemical bonds containing carboxyl, carbonyl, hydroxyl, lactone, and epoxide at graphene edge and basal‐plan. The amount and concentration of these functional groups are not controlled. (2) Halogenated graphene usually is a mixture of nanosheets with different degrees of substitution and different halogen‐carbon bonds. (3) GO and halogenated graphene show less chemical and thermal stability than graphene as a result of a great amount of defects or substituents on the surface, especially for brominated‐ and iodine‐doped graphene. The representative characteristics of halogenated graphene were shown in TableGraphene oxide (GO)Fluorinated graphene is regarded as the two‐dimensional basic structural element of fluorinated graphite first synthesized by Ruff et al. in 1934.x, x ≈ 0–1.12), which is a stable and wide‐bandgap nanosheet in which a certain amount of C atoms is covalently bonded to F atoms, becomes a rising star of graphene derivatives because of its outstanding properties, such as a large negative magnetic resistance (a factor of 40 in a 9 T field), a wide optical bandgap (3.8 eV) and a high room‐temperature resistance (>10 GΩ).[[qv: 4a]],Fluorinated graphene (CF1.0 is 2162 Wh kg−1).x battery shows high energy densities, good chemical stability, a long‐term shelf life (>10 years) and minimal (<10%) self‐discharge.x (x = 1) is 865 mAh g−1 with an average discharge potential between 4.5 and 5 V for a purely ionic C–F bond.Compared with other derivatives, fluorinated graphene shows many unique properties because of the formation of various types of C‐F bonds. First, because the F atoms has a higher electronegativity (4.0) than C (2.5), H (2.2), and O atoms (3.4), fluorinated graphene show great potential for using as an atomically thin insulator or a tunnel barrier based on the heterostructure.[[qv: 4a]] Second, because of the difference in electronegativity (1.5) between C and F atoms, fluorinated graphene exhibits several C‐F bonding characters from ionic, semi‐ionic to covalent bonds controlled by the fluorination conditions.FigureRecently, much progress has been made on the preparation and control of C‐F bonding characters, F/C ratios (the F/C ratio is defined as the mole ratio of fluorine to carbon) and configurations of fluorinated graphene. The brief roadmap of a synthetic strategy of fluorinated graphene is shown in 2TableThe methods for synthesizing fluorinated graphene or fluorographene are mainly classified into two groups: fluorination and exfoliation methods. Fluorination mainly include direct gas‐fluorination,[[qv: 4a]],[[qv: 12a]],[[qv: 13b]],[[qv: 21a]],2.12.1.12 gas to treat a graphene film at 70 °C  on the Si substrate using XeF2 gas at room temperature.[[qv: 12a]] Fluorographene showed a dominant stoichiometry of C1.0F1.0 and a high F/C ratio of graphene film on both the front and back surface because of the effective etching on the Si substrate by XeF2 gas. This effect was confirmed by fluorinated graphene (CF0.25) on Cu foils because the Cu substrate cannot be etched by XeF2  is another important fluorination agent to prepare fluorinated graphene because of its high reactivity. Fluorinated graphene with different F/C ratios was synthesized by Wang et al.[[qv: 22a]] GO was treated by F2 at a low temperature (from room temperature to 180 °C) , which was treated using 1 atm F2 at a high temperature of 600 °C. The resultant few‐layer fluorinated graphene showed a high F/C ratio (CF0.7). Interestingly, Sofer et al.[[qv: 22e]] also presented an easy and weighable method for the fluorination of Hummers GO and Staudenmaier GO in 20% F2/N2 (v/v) at elevated temperatures and pressures. The high resolution XPS results indicated that the F/C ratio was 17.8% and 5.61% for Hummers GO and Staudenmaier GO, respectively.Fluorine gas F is anoth2 is limited by poor controllability of the C–F bonding characters (semi‐ionic or ionic bonds) and F/C ratios, special equipment requirements and environmental hazards (high toxicity and corrosion). Thus, many other fluorine‐containing agents such as SF6, SF4 or MoF6 were also used for fluorinating graphene. Pumera et al.[[qv: 22d]] demonstrated the fluorination of GO using SF6, SF4 or MoF6. The surface elemental composition showed that GO synthesized by the Hummers method were thermally fluorinated using SF6, SF4 and MoF6 at 800 °C with different F/C ratios of 1.92%, 0.53%, and 0.26%, respectively. Additionly, GO synthesized by Staudenmaier method were treated by microwave in SF6 at 800 °C and 1000 °C and showed diffeirent F/C ratios of 4.25% and 0.49%, respectively. The results revealed that F/C ratios of GO could be tuned by different gaseous fluorine‐containing agents with the control of the temperature for the fluorination. The structural changes of GO also led to the changes of F/C ratios under the treatment of SF6.[[qv: 22d]] Despite recent progress, the fluorination using XeF2, SF6, SF4 or MoF6 is still far from up‐scale industrial production. Thus, exploring a low‐toxic fluorine‐containing gas (or mixed gas) for mild, selective and high efficient fluorination is important for preparing various fluorianted graphene in the future.Despite the high activity, the fluorination of graphene using F2.1.26,] CF4,] and F2,[[qv: 23h]] have been used. Baraket et al.[[qv: 23a]] synthesized fluorinated graphene using electron‐beam generated plasmas in Ar/SF6  at room temperature.[[qv: 23b]] Yu et al.[[qv: 23k]] also synthesized fluorinated reduced graphene oxide (RGO) using CF4 plasma at room temperature, and the F/C ratios (F/C ≈ 0.17–0.27) were controlled by the plasma exposure time. Recently, Wang et al.[[qv: 23i]] reported the fluorination of CVD‐grown single‐layer graphene using CF4 plasma. The results showed that F/C ratios of fluorinated graphene were tuned by the conditions of the plasma; however, the resultant fluorinated graphene and fluorographene consisted of a mixture of CFx (x ≈ 1–3), and the spatial distribution of F on graphene was highly inhomogeneous.[[qv: 23i]] K. I. Ho et al.[[qv: 23e]] presented a one‐step approach for the selective fluorination of graphene using CF4 plasma in a plasma‐enhanced chemical vapor deposition (PECVD) system. During the fluorination, F‐radicals preferentially fluorinated graphene at a low temperature (<200 °C), while the defect was suppressed by screening out the effect of ion damage  energies are lower than fluoride radicals. Desipte a simple and effective method, the plasma fluorination inevitably damages the carbon structure of graphene by severe ion bombardment at a relatively high temperature.[[qv: 25a]],High‐density plasma is important for fluorinating graphene with high F/C ratios because the fluoride‐containing ions (such as F2.1.33‐etherate[[qv: 24b]] and diethylaminosulfur trifluoride (DAST)] and hexafluorophosphoric acid (HPF6).[[qv: 24d]]Hydrothermal or solvothermal fluorination is another versatile method for preparing fluorinated graphene and fluorographene. The fluorination effect depends on fluorine precursors, such as hydrofluoric acid (HF),[[qv: 24c]] BF3‐etherate and alkyl thiol/alkyl amine on the gram scale.Wang et al.[[qv: 24c]] presented a convenient method to fluorinate dispersed GO using HF through a simple hydrothermal process. Note that some oxygen‐containing groups were substituted by F atoms during the hydrothermal reaction. In addition, the F/C ratios were controlled by varying the temperature, times and HF concentration. Similarly, Gao et al.[[qv: 24a]] reported the solvothermal fluorination of GO films through converting the oxygen‐containing groups  to C‐F bonds by treating GO with DAST in chloroform at 50 °C. More recently, Samanta et al.[[qv: 24b]] prepared fluorinated RGO with fluorine coverage of 38 wt% using anhydrous BFGO is an excellent nanosheet for the hydrothermal fluorianation because of many epoxide, hydroxyl, carboxylic and ketone functional groups on the surface The oxygen‐containing groups can be removed or substituted by the formation of C‐F bonds at high temperature using a suitable fluorination solvent. Thus, the hydrothermal fluorination shows great potential for fluoriated graphene with high F/C ratios. Unfortunately, the uniform distribution of C‐F bonds on fluorinated graphene by the hydrothermal fluorination has yet been reported.2.1.42/Si substrate. Active fluorine radicals, generated by the decomposition of CYTOP, reacted with the sp2‐hybridized carbon and formed C‐F bonds  source and laser irradiation. The fluoropolymer CYTOP decomposed under laser irradiation on the surface of a single‐layer graphene film on a SiOs Figure d. Gong e2.22.2.12.G) of the intercalation compounding process triggered by the F atoms, which is defined in Equation H and ΔS are the enthalpy and entropy for the intercalation of molecules or solvents respectively. Because of van der Waals attraction between two adjacent layers of fluorinated graphite, ΔH is generally expected to be positive; resulting in a small and positive ΔG, and thus the exfoliation is mainly affected by TΔS. At high temperature and pressure, the increase in ΔS leads to a decrease in ΔG, which indicates increasing driving forces. Thus, compared with graphite, fluorinated graphene is easily exfoliated by the intercalation of molecules with relatively weak van der Waals attraction and a large interlayer space. High‐yield single‐ or few‐layer fluorinated graphene with a specific F/C ratio is obtained.Sonochemical exfoliation of multilayer materials has been well researched because it is a versatile and nondestructive technique for preparing high‐quality two‐dimensional single‐ or few‐layer nanomaterials. Solution‐processed exfoliation has been employed for up‐scale production of two‐dimensional graphene and MoSFigureZborˇil et al.[[qv: 9a]] prepared fluorinated graphene (F/C = 1.00) by a single‐step liquid‐phase exfoliation. In this process, fluorinated graphene was exfoliated from commercial fluorinated graphite suspended in sulfolane at a 135 W ultrasonic bath for 1 h at 50 °C. Chang et al.[[qv: 26a]] reported an effective and low‐cost exfoliation to obtain single and few‐layer fluorinated graphene (F/C = 0.25 or 0.50) in ionic liquid. In this method, ionic liquid (1‐butyl‐3‐methylimidazolium bromide) intercalated into the interlayer of commercial fluorinated graphite by mixing and incubating. After intercalation, black colloidal dispersion of fluorinated graphene was obtained by ultrasonication. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images revealed that two‐dimensional fluorinated graphene showed 1–5 layers with 2–10 μm in edge size. Among many organic solvents, NMP is considered to be an important intercalated molecule to exfoliate fluorinated graphene because of its dipole moment value of 4.09 D Figure4a.],3‐hybridized C atoms connect to F atoms and (2) fluorine radicals react with graphene to form semi‐ionic C‐F bonds, in which the sp2‐hybridized C atoms connect to F atoms. C–F bonds change from ionic to semi‐ionic to covalent, accompanied by a decrease in F/C ratios by changing the fluorination conditions .[[qv: 14a]] Borini et al.[[qv: 25b]] reported oxy‐fluorianted graphene with semi‐ionic C‐F bonds through the electrochemical intercalation of graphite in hydrofluoric acid solution. Wang et al.[[qv: 19b]] found that C‐F bonds showed the partial transformation from semi‐ionic nature to covalence with the increasing F/C ratios of fluorinated grapehene, which was controlled by the exposure time in XeF2 atmosphere. This transformation was also found in the liquid‐phase exfoliation with the appropriate solvent such as chloroform. Feng et al.[[qv: 26c]] found the partial transformation of covalent to semi‐ionic C‐F bonds in fluorinated graphene exfoliated by chloroform due to the formation of C–H…F hydrogen bonds between chloroform molecules and F atoms of fluorinated graphite.[[qv: 22a]],[[qv: 33b]],2‐F bonds.[[qv: 33b]] Previous studies indicated that the partial transformation between ionic (semi‐inoic) and covalent bonds could be caused by the interaction between C‐F bonds and other molecules or materials. Importantly, the natrue of C‐F bonds have a significant impact on the properties of fluorinated graphene, such as work function,[[qv: 23g]] reaction activity,The fluorination of C–C bonds of graphene usually contains two competing reaction processes: (1) fluorine radicals react with graphene to form covalent C‐F bonds, in which the sp−1 in the FTIR spectra.[[qv: 19b]],[[qv: 22a]],[[qv: 25c]],],TableThe presence and percentage of covalent, semi‐ionic or ionic C‐F bonds in fluorinated graphene are investigated by X‐ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). According to previous studies, the characteristic peaks of semi‐ionic bonds between C and F atoms were observed at approximately 287–290 eV in the C1s XPS spectra Figure , 685–6883.2Precise control of the F/C ratio of fluorinated graphene is important for opening the bandgap, tuning electrical conductivity and optical transparency and understanding the structural transformation. Thus, beyond the aforementioned techniques in the second section, fluorination conditions, including the reaction temperature, the species of fluorination agents and catalysts, the type of carbon , the treated side and the sonochemical time, are utilized to tune the F/C ratios of fluorinated graphene.],[[qv: 24c]],[[qv: 26b]],4‐plasma treatment. Similar results were also observed in a recent study by Kuan‐I Ho et al.[[qv: 23e]] In addition, Wang et al.[[qv: 24c]] presented an easy, low‐cost and efficient hydrothermal‐process to tune F/C ratios of fluorinated graphene. The contents of each C‐F‐containing group  were dependent on the reaction temperature, time, and HF amount. An increase in the F/C ratios (from 0.11 to 0.48) was mainly attributed to the formation of the CF–CF2 group.[[qv: 24c]] Interestingly, Robinson et al. found that fluorine saturation coverage differed when graphene films were fluorinated by XeF2 on one or both sides. X‐ray photoelectron spectroscopy and Raman spectroscopy revealed that fluorine coverage saturates at 25% (C4F) for one‐side fluorination and at 100% (CF) for double‐side fluorination in XeF2 at room temperature.[[qv: 12a]] Gong et al.[[qv: 26b]] also reported that the F/C ratios decreased with increasing ultrasonication time in NMP, which might be attributed to the increasing stretching vibration energy of C–F groups gained from the sonic power facilitating the departure of fluorine.[[qv: 26b]]Yu et al.[[qv: 23k]] reported that F/C ratios (0.17–0.27) of fluorinated graphene were controlled by the time of CF3.3FigureFluorinated graphene and fluorographene consisting of weakly bound stacked two‐dimensional carbon monofluorides are a basic building block of fluorinated graphite.[[qv: 4a]],[[qv: 12a]],−1, which was twice the experimental value (100 ± 30 N m−1).[[qv: 4a]] The difference between the calculation and experiment might be attributed to a large number of structural defects in fluorographene because a certain portion of C atoms was not bonded to F atoms but formed dangling bonds.[[qv: 43a]] However, compared with fluorinated carbon nanotubes, the energy difference among various configurations of fluorographene is very small, and this result indicates that fluorographene is unlikely to be a pure single‐crystal form in a chair, stirrup, boat, or twist‐boat configuration.The first‐principles density functional theory (DFT) calculation showed that fluorographene with the chair configuration had a direct bandgap of 3.1 eV,4−1), good thermal stability below 400 °C, distinct nonlinear feature and high hydrophobicity. In this section, we discuss a variety of properties including bandgap, absorption or luminescence, stability, electronic conductivity, dspersibility, magnetic properties, tribological properties, mechanical or micromechanical properties, and thermal conductivity. These properties are significantly important for the application of fluorinated graphene.Fluorinated graphene shows many excellent properites such as wide bandgap of 3.1 eV, the highest theoretical specific capacity  using XeF2. The calculation indicated that the bandgap of fluorinated graphene increases with an increasing F/C ratio because of the interaction between the p‐orbital of F and the π‐orbital of C. The formation of sp3 bonds led to a large change in charge densities and scattering centers in the conduction band  showed a large bandgap of 3.07 eV.[[qv: 12a]] Liu et al.0.031, CF0.056, and CF0.125) were polar covalent bonds because of the high electronegativity of F atoms, and thus, they exhibited a metallic behavior. This behavior could be changed by increasing F/C ratios. CF0.25 and fluorinated graphene (CF1.0) had wide bandgaps of 2.92 eV and 3.13 eV, respectively, according to the generalized gradient approximation (GGA) calculations. Interestingly, fluorinated graphene (CF0.5) in which C atoms bonded to F atoms on one side also exhibited metallic behavior ascribed to the exchange splitting of the dangling C‐pz orbital with a coupling with an impurity state induced by F atoms. The results indicate that the bandgap of fluorinated graphene is greatly influenced by F/C ratios.Robinson et al.[[qv: 12a]] prepared fluorinated graphene films (on one side) with fluorine coverage of 25%  had a much narrower indirect bandgap than that of monolayer fluorinated graphene. Additionally, the bandgap of C4F nanosheets was further decreased by increasing the number of stacked layers because the conversion from insulator to semiconductor based on the dipole‐dipole interaction between two C4F layers induce a subtle interlayer polarization.Based on the density‐functional GGA calculation, the bandgap can also be controlled by different configurations and layers of fluorinated graphene and fluorographene. Specifically, a chair configuration shows a bandgap of 3.10 eV, while the bandgaps of the stirrup, boat, and twist‐boat configuration are 3.58, 3.28, and 3.05 eV, respectively.[[qv: 12b]] In the chair configuration, F atoms are alternately distributed on the plane. One F atom locates above the carbon layer, while the other one is under the same layer. Thus, the chair configuration has more symmetry than the stirrup configuration. In addition, the stirrup configuration is more significant for the conduction state because charge density follows the chain characteristic.[[qv: 12b]] Li et al.4.22. The absorption coefficient decreased after fluorination by SF6[[qv: 23c]] and CF4 plasma,[[qv: 23e]] which was in agreement with other fluorinated carbon materials.[[qv: 14b]],Fluorine‐substitution on carbon atom dramatically changes the optical properties of graphene including the absorption band, photoluminescence and transparency. Robinson et al.[[qv: 12a]] found that graphene film was optically transparent in the visible region after treatment by XeF2 at 70 °C  below the bandgap because of phonon‐assisted radiative recombination across the bandgap where the C–F vibration mode was excited when the electron‐hole pair recombined. Analogously, two accompanying peaks at 2.88 eV and 2.73 eV were also observed in low‐degree fluorinated graphene (fluorination for 1 day)  of fluorinated graphene have attracted attention because it not only yields insight into understanding electronic properties but is also crucial for advanced semiconductor devices and energy harvesting. Jeon et al.[[qv: 13a]] reported the room‐temperature PL spectra of graphene and fluorinated graphene dispersed in acetone using 290 nm (4.275 eV) excitation Figure d. The re[154–156] Samarakoon et al.[[qv: 12b]] reported the in‐plane absorption spectra of graphene and fluorographene calculated by GW‐BSE along with the random phase approximation (RPA) and GW‐RPA, respectively. RPA was regarded as the result of the DFT level. As shown in FigureOptical properties of ground‐state fluorinated graphene were also predicted by theoretical calculation based on DFT.154–1 Samarako4.3FigureD band. In contrast, fluorographene shows a high stability below 400 °C .[[qv: 4a]],[[qv: 12a]],[[qv: 22c]] The removal of both C and F atoms in fluorographene was observed by a prolonged annealing time at high temperature (≈450 °C).[[qv: 4a]] In addition, according to XPS data, fluorinated graphene, prepared using XeF2 gas on SiO2, Au, and Cu substrates, lost approximately 50–80% of the initial F/C ratios over 10 days until the F/C ratios were not changed.A below 200 °C. Fluorographene became weakly conductive, and the effective resistivity ρ = V/I decreased to ≈1 GΩ at 350 °C. The results indicated that the thermal stability and chemical inertness of fluorographene were similar to Teflon.[[qv: 4a]],[[qv: 13b]]Compared with the instability of GOn + nN2H4 → 4C + 4nHF + 2nN2. Radek et al.[[qv: 9a]] provided a pathway for defluorination using KI in DMF. In this process, fluorinated graphene transformed to metastable graphene iodide, which quickly decomposed to graphene and iodine at just 150 °C: CF+ KI → KF + [CI]; [CI] → C + 1/2I2. Additionally, Lee and co‐workers[[qv: 14d]] reported that ionic C‐F bond was selectively reduced by acetone treatment at a low temperature with the equation 2C2F(semi‐ionic) + CH3C(O)CH3(l) → HF + 2C(s) + C2F + CH3C(O)CH2(l).In addition, fluorographene showed a good chemical stability in many liquids such as water, acetone, and propanol, and under ambient conditions except for strong reductants.[[qv: 4a]],[[qv: 9a]] It was found that fluorinated graphene could be reduced by hydrazine, potassium iodide, ultraviolet irradiation and alkylamine compounds.[[qv: 9a]],[[qv: 12a]],More recently, new fluorinated graphene derivatives were prepared by the covalent modification of fluorinated graphene.4.42 hybridized C atoms with a pz orbital forming a π‐conjugated bond. Fluorination is widely used to chemically tailor the electrical conducitivity because it enables the transition from metallic/semiconducting to an insulating nature controlled by different F/C ratios.Single‐layer graphene shows high electron mobility because of its spI–V curves.[[qv: 4a]],[[qv: 22c]],[[qv: 23i]] Different from C–C bonds of graphene, every C atom in fluorographene with sp3 hybridization is bound to an F atom. Thus, fluorographene is an insulator because of the disappearance of π‐conjugated bonds. Wang et al.[[qv: 23i]] investigated the typical I–V characteristics of fluorinated graphene prepared by CF4 plasma. Fluorinated graphene showed a linear I–V curve with a short fluorination time (<10 min) with a resistance <10 MΩ because a low amount of F and sp3 C atoms were regarded as defects in an sp2 hybridized C network. Thus, the π‐conjugated network of graphene is preserved. However, this network was destroyed by a long fluorination‐time (>10 min) using CF4 plasma. Fluorinated graphene underwent the transition from semiconductor to insulator, resulting in a nonlinear curve with a resistance >1 GΩ. Compared with graphene, the resistance of fluorinated graphene showed a sharp increase by more than 7 orders of magnitude (from 10 kΩ to >100 GΩ) when the fluorine coverage is a few tenths of a percent.[[qv: 23i]],2 hybridized carbon network.−1, respectively.Fluorographene, the thinnest two‐dimensional insulator, shows a distinct nonlinear feature of 4.5z orbitals to form pseudohydrogen bonds with the hydroxy group of ethanol.[[qv: 13a]] The pseudohydrogen bond has been demonstrated to facilitate the dispersion of graphene with the π bond  in ethanol.[[qv: 13a]],[[qv: 26b]],Because of the presence of C‐F bonds, fluorinated graphene and fluorographene are highly hydrophobic and difficult to disperse or solubilize in most solvents because of its low surface free energy.[[qv: 21b]],z orbitals such as phenylethylene (PS), NMP, and THF than others with nonhybridized pz orbitals.[[qv: 26b]] Specifically, in a homogeneous solvent, a free pz orbital acted as an electron acceptor and formed pseudo‐hydrogen bonds with (CnF)x–F groups, thus resulting in an increase in dispersion of fluorinated graphene.[[qv: 13a]],[[qv: 26b]] Recently, the dispersion of fluorinated graphene in water was improved by fluorosurfactants in which perfluorinated units were adhered on the surface of fluorinated graphene and cationic or anionic units provided static repulsion.z orbitals, such as CH3CN, chloroform, and DMF.[[qv: 24e]]Gong et al.[[qv: 26b]] studied the dispersibility of fluorinated graphene in a variety of organic solvents. It was found that fluorinated graphene showed much better dispersion in solvents with a large closed conjugated system formed by p4.6T.x samlpes with x increasing from 0.1 to 1 was described by the Brillouin function. z = gJμBH/kBT, g was the g‐factor, J was the angular momentum number, N was the number of spins and kB was the Boltzmann constant. The number of spins N increased monotonically with x up to ≈0.9, and then showed some decrease for fluorographene. The maximum M achieved by the fluorination of graphene was one order of magnitude higher than that achieved by irradiation. Unfortunately, the concentration of magnetic moments was only ≈0.1% of the maximum hypothetically possible magnetism of one moment per carbon atom because F adatoms have a strongly towards clustering.[[qv: 73a]] Tang et al.[[qv: 73b]] reported that small F clusters that could be preferably formed around the vacancies in RGO produced a lot of magnetic edge adatoms. And such fluorinated RGO have a high magnetization of 0.83 emu g−1, a high magnetic moment of 3.187 × 10−3μB per carbon atom and a high efficiency of 8.68 × 10−3μB per F adatom. Recently, fluorinated GO was used as an efficient magnetic resonance imaging (MRI) contrast agent by Ajayan et al.Graphene obtained by sonochemical exfoliation of high‐purity HOPG shows a strongly diamagnetic response and no sign of ferromagnetism over a wide range of temperature, 4.73 configuration.[[qv: 76c]],[[qv: 77a]] Hou et al.Generally, graphene shows good tribological performance due to its high chemical inertness, extreme strength, easy shear capability on its densely packed and atomically smooth surface.4.84.8.1E) and intrinsic strength (σ) because of the presence of C‐F bonds. Nair et al. measured the E and σ of fluorographene using AFM.[[qv: 4a]] Fluorographene exhibited a lower E (100 ± 30 N m−1) and a lower σ (≈15 N m−1) than of graphene .[[qv: 4a]],E and σ arised from longer sp3 hybridized C–C bonds in fluorographene than sp3 hybridized C–C bonds in graphene.[[qv: 4a]] Interestingly, the elastic deformation σ/E of fluorgraphene showed litttle change in comparison of graphene because of the absence of structural defects during fluorination.[[qv: 4a]] The mechanism of controlling specifc mechanical properties including the strength, modules and deformation has yet been understood.Fluorination usually affects mechnical properities of graphene such as Young's modulus  were first used as the cathode in lithium primary batteries by Watanabe et al. in 1972.x was considered to be one of the ideal cathode materials for lithium primary batteries because of a variety of unique properties, such as high energy density, high average operating voltage, long shelf life, stable operation ability and wide operating temperature. Subsequently, Li/CFx batteries were first commercialized by Matsushita Electric Co. in Japan in 1975.x batteries have the highest theoretical specific capacity  in primary battery systems.[[qv: 14c]],[[qv: 18c]] With an ultrathin two‐dimensional layer‐structure, fluorinated graphene and fluorographene are regarded as the most promising CFx to achieve the theoretical capacity because of their tunable F/C ratios and C‐F bonding characters, favorable diffusion kinetics of lithium ions and large specific surface area. Recently, many studies focused on the performance of Li/CFx batteries using fluorinated graphene or fluorographene as the cathode material.[[qv: 17b]],],Fluorinated carbon materials , excellent rate capabilities (305 mA h g−1 at 5 A g−1), and outstanding cycling stability (capacity retention of ≈95% at 5 A g−1 after 2000 cycles) as the anode material for lithium ion batteries.−1 at 50 mA g−1 and excellent cycle performance for 50 cycles  (865 mAh g−1)  because of the high electronegativity of F atom (χ = 3.98) and the strong C‐F covalent bonds  at the edges.Feng et al. reported that lithium primary batteries using fluorographene exhibited a remarkable discharge rate because of good Lite Figure11a.],5.3Although fluorinated graphene is a semiconductor with a wide bandgap and shows UV‐fluorescence,[[qv: 12a]],[[qv: 13a]],5.4Fluorinated graphene can also be used for applications in supercapacitors,[[qv: 24e]] electrochemistry−1, which was much better than GO. The results were also confirmed by charge/discharge curves.[[qv: 24e]]Zhao et al.[[qv: 24e]] prepared solid supercapacitors using fluorinated graphene as an electrode material. Cyclic voltammetry measurements showed that fluorinated graphene prepared in dichloromethane exhibited the highest specific capacitance at 106.6 F gkobs0) at 2.69 × 10−3 cm s−1 and 4.37 × 10−3 cm s−1 in [Fe(CN)6]4–/3− and Eu2+/3+ redox probes, respectively. And the overpotentials of ascorbic acid and uric acid oxidations decrease with the increasing F/C ratios. And the fluorographite with F/C ratio of 0.75 provided a response to uric acid at 18.46 μA mM−1, which more sensitive than that to ascorbic acid (2.15 μA mM−1).Pumera et al.−1, synthesized by oxidizing the basal plane of fluorinated graphite. This method allows for unique, accessible, carbon‐based amphiphobic coatings.[[qv: 21b]]C‐F bonds drastically reduce the surface energy of graphene, resulting in a change in wetting behavior. Mathkar et al.[[qv: 21b]] reported an amphiphobic coating of fluorinated GO with a low surface tension of 59 dyn cm6In this review, we have given an overview of synthetic methods, structures and properties of fluorinated graphene that can be utilized for applications in high‐energy storage, unique biological response and magnetic resonance imaging, fluorinated graphene quantum dots, supercapacitors, electrochemistry and amphiphobicity. We have emphasized the importance and significance of controlling C‐F bonding characters, F/C ratios and configurations of fluorinated graphene, fluorographene and F‐GQDs by fluorination (gas or liquid phase) or exfoliation. The selective fluorination enables graphene with different two‐dimensional configurations for various properties including wide bandgap, blue luminescence, excellent electrochemistry, high stability and self‐lubricating. For example, an increase in the F/C ratio enlarges the bandgap of fluorographene, while a low F/C ratio usually ensures charge transport based on π‐conjugated structures. The covalent C‐F bonds in gas fluorination are crucial for thermal and chemical stability, while semi‐ionic and ionic bonds endow fluorinated graphene with a higher discharge potential for lithium batteries. Moreover, thermal conductivity, magnetic properties and luminescence of fluorinated graphene are not well developed because of a complicated fluoro‐carbon structure.Although significant progress has been made, additional challenge for uniform up‐scale synthesis/production, target‐oriented fluorination, the homogeneity of C‐F bonding character, solution processability and removal of other fluorides in applications must be addressed. It is very difficult to tune C‐F bonding precisely at the specifc microstructure and/or chemical structure because of the strong fluorination and the complicated chemical and microstructures  of graphene. A versatile, low‐cost and safe method of fluorinating graphene has not yet been found, resulting in the limitation of a wide range of use in commercial applications. Furthermore, because the mechanism of the formation of different C‐F bonding character is still unclear, fluorinated graphene is often composed of a mixture of various types of covalent, semi‐ionic and/or ionic C‐F bonds with different ratios. As a result, fluorinated graphene with different structures  needs to be separated and/or purified before the use in advanced electronic devices. Thus, chemical methods or strategies for selectively fluorinating graphene are of paramount importance.To date, there is a huge number of opportunities and challenges for designing and synthesizing fluorinated graphene with different structures such as core–shell, nanoporous spheres, nanocages and topologically nontrivial assemblies. The investigation of diffenent fluorinated graphene will put insightful understanding of their properties. On the basis of controlling the selectivity of plasma‐treatment, gas‐ and/or mild fluorniation, the change in F bonding nature, bandgap or electronic interaction of fluorinated graphene with the increasing F/C ratios and/or the specific C‐F bonds will be investigated. This result will further illustrate the effect of C‐F bonding character and configuration on thermal conductivity, self‐lubricating and optical properties.In addition to controllability and uniformity, multifunctional fluorinated graphene will be an interesting subject of intense and fruitful research in the future.A systematic study of thermal conductivity, magnetic property and luminscence will provide more special application in fluorinated graphene and F‐GQDs as well as other fluorinated carbon materials. Besides, the electrochemical properties of fluorinated graphene will be further improved to maxmize the energy density and power density by optimizing the microstructure, C‐F bonding character, the interfacial wettability and cooperation with additives. By the integration of chemical groups, polymer chains and/or functional nanoparticles, fluorinated graphene and its composites will show great potential for secondary batteries , super‐insulating materials, light emitting diodes (LED) and display materials. Based on much effort focused on the controllability of structures and optimized properties, in the future, fluorinated graphene will exhibit excellent performance in flexible nanoelectronics, energy conversion/storage, special protective coatings, and tissue engineering.