Deep eutectic solvents (DESs) have received attention in various applications because of their distinctive properties. In this work, DESs were used as functionalizing agents for graphene due to their potential to introduce new functional groups and cause other surface modifications. Eighteen different types of ammonium- and phosphonium-salt-based DESs were prepared and characterized by FTIR. The graphene was characterized by FTIR, STA, Raman spectroscopy, XRD, SEM, and TEM. Additional experiments were performed to study the dispersion behavior of the functionalized graphene in different solvents. The DESs exhibited both reduction and functionalization effects on DES-treated graphene. Dispersion stability was investigated and then characterized by UV–vis spectroscopy and zeta potential. DES-modified graphene can be used in many applications, such as drug delivery, wastewater treatment, catalysts, composite materials, nanofluids, and biosensors. To the best of our knowledge, this is the first investigation on the use of DESs for graphene functionalization.The online version of this article (doi:10.1186/s11671-015-1004-2) contains supplementary material, which is available to authorized users. It is comprised of spal sheet . Graphenal sheet , 4. Howeal sheet , 6. ThusFunctionalization is a process that includes the addition of new functional groups to the surface of the carbon through a chemical or physical attachment. According to this connection, functionalization is classified into two types, i.e., covalent and non-covalent functionalization –9. With Generally, the most common conventional materials used for functionalization are strong inorganic acids, such as sulfuric acid, nitric acid, and mixtures of the two acids. Highly-volatile organic solvents also have been used in this respect. However, due to the associated risks and precautions of using such chemicals, it is highly essential to develop safer and more eco-friendly alternatives .et al. used an IL, namely 1-(3-aminopropyl)-3-methylimidazolium bromide, as a functionalizing agent to modify graphene oxides [In the past decade, there was increased interest in ionic liquids (ILs) as new "green" solvents because of their unique physicochemical properties , 18. Zhae oxides . The rese oxides  by usingIn 2003, special analogues of ILs were introduced and defined as deep eutectic solvents (DESs) . These sIn this study, selected ammonium- and phosphonium-based DESs were used as novel functionalizing agents for graphene. In addition, the DESs were compared based on the structure of the graphene and the improvement in dispersibility. Changes on the structure of the surface were identified by FTIR, Raman spectroscopy, XRD, STA, and TEM. The dispersibility and the stability of the suspension of the DES-functionalized graphene were investigated and characterized using UV–vis spectroscopy and zeta potential analysis.Methyltriphenyl-phosphonium bromide (purity ≥98%), N,N-diethylethanolammonium chloride (purity ≥98%), sucrose, urea (purity ~99.5%), ethanol, (purity ≥99.9%), ethylene glycol (purity 99%), diethylene glycol (purity 99%), and triethylene glycol (purity 99%) were purchased from Merck . Choline chloride (2-hydroxyethyl-trimethylammonium) (purity ≥98%) and malonic acid (purity 99%) were provided by Sigma-Aldrich. Anhydrous D(+)-glucose, D(−)-fructose (purity >98.0%) and glycerol (purity 99.8%) were obtained from R&M Chemicals, and potassium permanganate was obtained from Univar (purity 99%).2/g.Sixty-nanometer flakes of graphene nanopowder (AO-4) grade were obtained from Graphene Supermarket (USA) with the following specifications: purity 98.5%, lateral particle size ~ 3–7 microns, 60 nm average flake thickness, and specific surface area <15 mTable et al. method [All solid materials were dried at 60 °C under vacuum before they were used. All of the DESs listed in Table . method , 27. SalFresh DES samples were used for all experiments and analyses to avoid any possible contamination because of humidity or structural changes over time. Eighteen types of DESs were prepared from different ammonium and phosphonium salts and different HBDs. Table 4 was prepared at 70 °C for ease of solvation. Graphene was oxidized by the following procedures: 1) 200 mg of graphene were mixed with 7 ml of the previous permanganate solution inside a 20-ml glass vial; 2) the mixture was subjected to ultrasound waves using an ultrasonic bath at 70 °C for 3 h; 3) graphene was collected by a filtration process under vacuum using filter paper with a pore size of 0.45 μm; 4) the graphene was washed several times with 0.01M HCl and distillated water until a clear, transparent product was obtained; and 5) the collected cake was dried for 3 h at 100 °C.Graphene nanopowder flakes were dried at 100 °C under a vacuum for 3 h to eliminate any water that was present on the surface of the carbon. A 1-M solution of KMnO4. Graphene was collected and dried in air at 100 °C for at least 3 h and then transferred to a clean-air desiccator where it was allowed to cool to room temperature. Then, the graphene samples were ready for characterization.In a procedure similar to the previous chemical treatment of the graphene flakes, functionalization was conducted ultrasonically in a 20-ml glass vial that contained 200 mg of oxidized graphene and 7 ml of DES. The suspension was sonicated for 3 h at 60 °C and then filtered under vacuum. The retained carbon was washed with water until a transparent effluent was produced that had the same pH as the influent. The experiments included 18 different DESs, and, for comparison, one experiment was conducted with the pristine graphene without prior treatment with KMnO2 environment and at a heating rate of 5 °C/min. Raman spectroscopy (Renishaw System 2000 Raman Spectrometer) and X-ray diffraction tests (XRD) also were performed in this study.The FTIR measurements of the carbon samples before and after the treatment were conducted using a Perkin Elmer FTIR spectrometer with a range of 450–4000 wavenumbers and a four-time scanning repetition. All DESs and their individual components were analyzed under the same conditions. Thermogravimetric analysis (TGA) and Differential Thermogravimetry (DTG) were performed simultaneously for all samples using the Simultaneous Thermal Analyzer . The tests were performed in an NIn order to obtain a fair idea of the successful modification of graphene, a series of dispersibility tests was conducted using solvents with different polarities, e.g., distillated water, acetone, and n-hexane . First, UV–vis spectroscopy was conducted using PerkinElmer-Lambda  to charaBefore identifying the structural changes on the surface of the carbon, a preliminary investigation was conducted to recognize the functional groups of DES as functionalizing agents. Therefore, FTIR spectra were studied for all of the selected DESs and their individual components. The spectrum of each DES, which is a combination of two or more components, was interpreted based on the well-known and recognized peaks of DES components. Figure The details of the FTIR spectra are provided in Additional file Additional file 2O from the raw materials that were used, since all of the HBDs had this functional group. The C–H, C–O, and C-C-O functional groups were identified in all glycolic-based DESs. They seemed to be the dominant groups that existed when the four glycols were involved.Table 2. However, the remaining four groups were found in minute quantities in some types of DESs. Having urea as part of the mixture enabled the appearance of N-H and C–N+ bonds from a ChCl source. Thus, three ChCl-based functional groups out of four can be detected with the presence of U as an HBD.Among all of the functional groups that existed in ChCl, only five of them can co-exist after the formation of the DESs Table . The proOnly three functional groups were identified in the N,N-based DESs Table . C-H gro3 functional group was detected in the DEG-based DESs, as illustrated in Table The aromatic ring in MPB was clearly identified in MPB-based DESs as =C-H and C=C groups. The bond between this ring and the phosphorus atom also was found in all of the DESs based on MPB salt as was the case in the salt itself. However, the P-CH-1 and other oxygen bridges, such as C–O–C, C–O–H, and C–O, between 1463–1020 cm-1 [-1 and 657 cm-1 might correspond to the O–H bond with stretching and bending vibrational modes [2 asymmetric and symmetric stretching at 2915 cm-1 and 2850 cm-1 [-1 and 672 cm-1, which were expected to be for the stretching and bending vibrations of the C–H bond, respectively. The C=C from unoxidized sp2 CC bonds was found at 1618 cm-1 [-1 was caused by the absorption of CO2 from the air [According to the FTIR results shown in Fig. 020 cm-1 , 37, whial modes . However850 cm-1  and at 3618 cm-1 , 40, whi the air . These p4, the peak shifted to 226 nm, and a shoulder peak appeared at 384 nm, indicating the n–π* transition of carboxyl groups [The p-Gr and o-Gr were dispersed in water and scanned spectrophotometrically in the wavelength range of 200–700 nm. Figure l groups , 42, 43.4. Further information about the impact of pretreatment with graphene is discussed in the Raman spectroscopy analysis, XRD test, and STA sections.The aqueous dispersions of p-Gr and o-Gr were characterized further by dynamic light scattering (DLS). The study was conducted using the standard model of a spherical particle Fig. . Graphen-1 represented −NH2 and −NH− stretching vibrations, whereas the in-plane stretching of –NH was evident at ~1605 cm-1 [FTIR analyses for a series of DES-modified graphenes were conducted to characterize the functionalization process. Figure 605 cm-1 , 45.Fig.4 accounted for the changes in the functional groups obtained from DES on the carbon surface. To prove this, an experiment was conducted in which pristine graphene was treated directly with DES 5. The FTIR spectrum of the sample that was obtained was identical to the spectrum for untreated graphene  covalent bonds between free radicals or dienophiles and C=C in the skeleton of pristine graphene, and 2) covalent bonds between an organic group and the oxygen-containing functional groups in graphene oxide [Given the above observations, it is apparent that at any rate, the treatment of graphene with KMnOene Fig. . Cleaninne oxide . Thus, tA convenient comparison between all carbon spectra was conducted, and the results are summarized in Additional file In order to confirm the presence of oxygen-containing functional groups and other groups, the thermal stabilities of the graphene samples were examined using TGA and DTG.In the following sections, samples of DES-functionalized graphene oxide are divided into four groups to facilitate their comparisons. The samples functionalized by DES 1 to 5, which were based on ChCl salt , are represented in Group 1. The samples modified by DES 6 to 9, based on N,N salt , are in Group 2. Gr 10, 11, 12, and 13, modified by MPB-based DES, are in Group 3. The rest of the sample were treated with natural DES 14–18 (provided in a previous study ), and GrThe tests were conducted in a nitrogen atmosphere. Figure Based on their TGA and DTG curves, nearly all of the oxidized and DES-functionalized graphene samples were found to have three degradation steps. Therefore, Table The rate of maximum weight loss in o-Gr within the temperature range of 90–280 °C occurred at 225 °C; however, the rate decreased after DES functionalization. The second degradation was observed in various temperature ranges for the different groups of graphene samples. In Group 1 , this range was 280–580 °C, while it was narrower in the other groups. The presence of oxygen-containing functional groups accounted for o-Gr’s having less thermal stability than p-Gr. Moreover, some samples showed less thermal stability than o-Gr due to the functionalization effect and the introduction of new functional groups, such as in Gr 1, 2, 4, 10, 13, and 17. By contrast, the total weight loss was decreased noticeably in most samples, indicating the deoxidation effect of DES as a result of the elimination of functional groups and/or their replacement with other groups from the DES source.In the four groups that were studied, it was observed that the graphene samples treated with Gly-containing DESs had higher weight loss than others from the same group . In addition, the presence of ChCl salt in the DES increased the weight loss of the treated samples to a greater extent than samples treated with DESs based on other salts. Therefore, ChCl-based DESs are considerably more effective in providing a higher level of functionalization than N,N- and MPB-based DESs.The final estimate concerning the role of DESs in eliminating and/or adding new functional groups was determined through the analysis of data from Raman, XRD, and FTIR spectroscopy as well as thermogravimetric results, which are discussed in the following sections.-1, 1580 cm-1, and 1340 cm-1 refer to G′, G, and D bands, respectively. The most intense peak among all of the spectra was found for the G band, a result that was similar to that of a previous study that used the same AO-4 grade graphene nanopowder [2-bonded carbon atoms in the hexagonal lattice of the graphitic structure. However, the D band reflects the presence of defects or disorders caused by sp3 hybridized carbon in the lattice, while G′ is caused by second-order, zone-boundary phonons that are related to the assembly of the multilayers [D/IG provides an indication of degree of oxidation and/or covalent functionalization [Raman spectroscopy was performed to study the changes in the structure of the graphene after each treatment. The Raman spectra for each group of graphene samples were studied individually and compared with p-Gr and o-Gr. An overall comparison is provided in Additional file nopowder . Generaltilayers . The intlization . The applization , 55.D/IG value due to the deformation caused by the introduction of oxygen-containing functional groups. This deformation was not as significant as it was for common o-Gr produced by Hummers’ method starting from graphite. The reason was the use of a non-destructive ultrasonic method in the pretreatment and the low deformation level of p-Gr. The G′ band shifted to a lower frequency , reflecting the simultaneous effects of functionalization and deoxidation.Gr 18 was found to have the highest deformation level, which can be attributed to the effect of its high acidity based on ChCl:MA. However, considerable increases in ID/IG ratio is expected after oxidation, some researchers have reported that the reduction effect resulted in a lower ratio [D/IG is expected. Conversely, the restacking of the graphene layers can result in a decrease in the ratio [D/IG, especially after the chemical reduction of o-Gr produced by Hummers’ method. However, this is in disagreement with other studies [Although an increased Ier ratio –60. Nevehe ratio . Reducti studies , 62.D/IG ratio is increased. In contrast, this ratio could be either increased or decreased with reduction. Two possible reduction pathways could be proposed, i.e., 1) the decrease in ID/IG indicates the healing effects that are caused when the sp2 double bonds are restored in graphene and 2) constant or increased ID/IG values indicate that the sp3 structure has been preserved, which could the result of the addition of different functional groups accompanied by the reduction reaction.In the case of oxidation, the ID/IG. Thus, ChCl:U was expected to produce a deoxidation effect as a result of the replacement of some of the oxygen-containing functional groups with other groups from DES (as detected by FTIR spectroscopy). The alkalinity of this type of DES could be responsible for this effect, which would be in agreement with previous findings that alkaline solutions are good deoxygenating agents for o-Gr [D/IG ratio were reported for Gr 1, 4, 10, and 13, reflecting a lower level of reduction than occurred in the other samples. It was anticipated that Gr 18 would undergo elimination of the functional group and deformation of its structure because it presented the highest ID/IG with a lower weight loss than o-Gr.The reduction effect in this study was confirmed by the TGA and XRD results. It was assumed that a greater reduction level would be accompanied by less weight loss (from TGA) than occurred for o-Gr. However, reduction also can occur in parallel with functionalization, i.e., replacing oxygen groups with other groups from DES. Figure for o-Gr . The deo4, a prominent diffraction peak appeared at 2θ = 11.16°, reflecting the interlayer spacing of 0.79 nm . Thgg’s law . In suchgg’s law .Table As shown in Additional file For Gr 1 modified by ChCl:Gly, the first peak in the XRD pattern appeared at 2θ = 18.22°, reflecting a preserved form of graphene oxide that was partially reduced by the DES. The prominent peak at 2θ = 26.64° was observed as an indication of the reduced form of graphene.The intensities of the peaks of all DES-treated samples were lower than those in p-Gr. This behavior frequently has been reported to be the result of functionalization, especially when it is accompanied by peak shifting to less than 2θ degrees –71. FiguIt can be expected that the presence of new functional groups from the DES sources contributed to changing the structure such that it differs from that of p-Gr. For example, if the reduction reaction took place and new hydrophobic functional groups were added, the π-π stacking between the layers would be enhanced to a higher level than the stacking in p-Gr. In contrast, the addition of hydrophilic functional groups (which replace the existing oxygen groups) would result in a lower stacking level than that of a fully-cleaned surface. To prove this, dispersibility tests were conducted in aqueous solutions. The improvement or deterioration in graphene dispersibility compared to o-Gr and p-Gr confirmed the roles of the hydrophilic or hydrophobic functional groups that possibly were attached after treatment with DES.A few samples were selected at random for the tests to provide a general vision of the impact of DESs on the structure of graphene. Figure The particle sizes of graphene modified by DES 5 and 18 were investigated similarly in p-Gr and o-Gr . The first dispersibility test included the preparation of graphene suspensions with relatively high concentrations, i.e., 1 mg/ml, and the test continued for 24 h. These conditions were used to accelerate the precipitation, thereby expediting the comparisons of the treated and untreated samples. Figure Note that many parameters affect the duration of a stable dispersion. In a previous study on the aqueous dispersion of graphene, Ding et al.  found thFurthermore, dispersibility in acetone was monitored for the five types of modified graphene, i.e., Gr 1–5 Fig. . Disperset al. reported similar stability results in several polar solvents [Dispersibility tests in different polar and non-polar solvents can be used to determine the nature of the surface and the exact hydrophilicity and hydrophobicity levels of the graphene nanosheets. The stability of the suspension in a non-polar solvent, i.e., n-hexane, was very short for the samples that were tested before and after treatment with DES. Conversely, stability in water was better for all samples, and it lasted for a longer period of time in some cases. Zhang solvents . By treaIn a further investigation, a suspension was made from poorly dispersed graphene in distilled water . The resulting mixtures were stable for longer periods of time than other suspensions, exceeding 24 h, as shown in Fig. Solvation in water generally is caused by the hydrogen bonds between the graphene sheets and the water molecules , but wheAs mentioned before, the aim of this section was to show the differences in the behaviors of the dispersions among all of the graphene samples. The stability of their dispersion can be prolonged if various conditions, such as sonication time, concentration, and temperature, are adjusted properly to optimum levels. Choosing a suitable type of dispersant with or without the addition of dispersing aids also is important when stability over time is required .4 led to instantaneous homogenization immediately after water was added to o-Gr. This phenomenon was similar to mixing water with a soluble black pigment powder, and it resulted from the higher surface energy of o-Gr [Changes in the surfaces of the graphene after treatment with DES sometimes resulted in greater decrease in the stability of the dispersions of DES-functionalized graphene than oxidized graphene due to the reduction effect. To explain this behavior, a small experiment was conducted to determine how graphene particles reacted when immersed in an aqueous solution. The first moments after water was added to the graphene samples were monitored for p-Gr, o-Gr, and Gr 1 Fig. . Fast ag of o-Gr .Fig. 17FHowever, Fig. 4 was for the graphene that was functionalized by DES 5 based on ChCl:U. However, not all graphene functionalization processes led to better dispersibility. This can be attributed to changes on the surface of the carbon after each treatment due to the manipulation of functional groups that have different hydrophilicity and hydrophobicity properties. It also confirmed the reduction effect caused by the DESs, which control the presence level of oxygen-containing functional groups in the modified samples. These groups led to electronegativity with the different values of electron density responsible for the negative values of zeta potential. However, according to Riddick [Zeta potential was measured for graphene dispersions in water (0.01 g/100 ml), and the results are presented in Fig.  Riddick , a zeta 2 bonds commonly are present on the surface of the carbon in samples that have zeta potential values less than 20 eV. Hydrophobic affinity for these functional groups was anticipated to influence the solubility of graphene in water. In general, the deterioration of dispersibility can be explained by (a) the effect of the removal of some hydrophilic functional groups from the surface of the carbon caused by the DESs, e.g., the deoxidization effect mentioned earlier in Section 3.5; (b) the addition of new functional groups that could have an adverse effect on the stability of the dispersion in water; and (c) the physical adsorption of DES materials occurring on active sites on the surface of the carbon through irreversible, non-covalent modification [Dispersibility improvements in Gr 5, 7, and 9 can be ascribed to the simultaneous functionalization and reduction effects resulting from the respective DESs. In some cases, the zeta potential values decreased after the DES treatment. However, the overall difference between pristine and DES-modified graphene indicated substantial improvements, especially for Gr 5, 7, and 9. Treating with DES 3, 4, 6, 11, 14, and 16 changed the surface chemistry such that their dispersion stability in water was diminished. Based on FTIR analyses of these samples, −C-H and -CHfication , which hfication . Figure 4 further, pristine graphene flakes were treated directly with some types of DES without prior purification. For the flakes treated with DES 1 and DES 5, there were no changes in the values of the zeta potentials. This is also in accordance with the FTIR results, which confirmed the need for pretreatment with KMnO4.To investigate the role of DES and the prior treatment with KMnOModified graphene can be produced and used in various functional applications. Graphene with more hydrophobic affinity can be used as additives in polymer-based composites . In addiThe use of DESs as functionalizing agents for graphene-based materials was investigated on graphene oxide. Chemical changes on the surface of the graphene were studied using FTIR spectra. The thermal stability obtained from simultaneous TGA/DTG analyses showed various stability changes when compared with pristine and oxidized graphene. Raman spectroscopy and XRD results also identified chemical and structural changes. Different levels of reduction were achieved after DES treatment, and simultaneous functionalization/reduction was reported in some cases. Further investigations of the structural changes were performed using SEM, TEM, and particle size analyses. The dispersibility of the treated graphene also was assessed and characterized based on zeta potential values. The results provided convincing indication of the changes on surface chemistry that occurred after DES treatment. However, considerable improvements in the dispersion stability of the graphene/water systems were recorded for graphene treated with DES 5, 7, and 9 compared to graphene oxide. The introduction of new hydrophilic functional groups was responsible for this behavior. The different reducing abilities of the DESs used were responsible for the different levels of agglomeration during the dispersibility tests. However, each modification process was an independent case that could be interpreted based on various aspects. The as-functionalized graphene may have different electrical, mechanical, or optical properties than the original pristine graphene. Thus, it is important to extend this study and test the functionalized graphene in various potential applications.


Idiopathic pulmonary fibrosis (IPF) is an age-related fatal disease with unknown etiology and no effective treatment. In this study, we show that primary cultures of fibroblasts derived from lung biopsies of IPF patients exhibited (i) accelerated replicative cellular senescence (CS); (ii) high resistance to oxidative-stress-induced cytotoxicity or CS; (iii) a CS-like morphology (even at the proliferative phase); and (iv) rapid accumulation of senescent cells expressing the myofibroblast marker α-SMA. Our findings suggest that CS could serve as a bridge connecting lung aging and its quite frequent outcome -- pulmonary fibrosis, and be an important player in the disease progression. Consequently, targeting senescent cells offers the potential of being a promising therapeutic approach. It was Élie Metchnikoff who first proposed that “aging is the replacement of noble elements by ignoble ones” . More spIPF is a progressive lung disease of indefinite etiology in which the pathological hallmark is the heterogeneous alveolar and peribronchial accumulation of scar tissue in the lungs of affected individuals, dominated by an exaggerated non-regenerative repair called “usual interstitial pneumonia” (UIP) -8. The iin vitro repair response [IPF is marked by an accumulation of fibroblasts and myofibroblasts along with excessive production of type I collagen rich matrix . As suchresponse , resistaresponse , an alteresponse .Recently, the pathogenesis of IPF was linked to the phenomenon of cellular senescence (CS)  – an ackGiven the importance of fibroblasts in the pathogenesis of IPF, we further address the issue by characterizing fibroblasts derived from lung biopsies of IPF patients in terms of proliferation rate, morphology, and expression of α-SMA (a myofibroblast marker), with a special focus on the connection of these characteristics to replicative and stress-induced CS. Here, we show that IPF fibroblasts are predisposed to CS and acquire some CS-like features far before reaching cell growth-arrest.While initially replicating in a rate similar to normal human pulmonary fibroblasts (N-PF), regularly passaged IPF-derived pulmonary fibroblast (IPF-PF) cultures reached CS much faster than N-PF Fig. . A clear2O2 than N-PF , with irregular shape and a diffused distribution of actin within the cell , a known marker of myofibroblasts. Lung fibroblasts of early passages derived from both healthy donors and IPF patients showed no immunostaining for α-SMA Fig. . HoweverAging has a significant impact on wound healing  and coulin vivo, both for fibroblasts and other cell types, and the amount of senescent cells was found to increase with age [reviewed in The phenomenon of cellular senescence (CS) has recently been implicated in the pathogenesis of age-related diseases , IPF incIn order to receive further insight into the links between CS and IPF, we investigated pulmonary fibroblasts derived from IPF patients, in light of well-established features of senescent cells. We found that in comparison to normal human pulmonary fibroblasts, the IPF fibroblast cultures displayed (i) some CS-like morphological features  even in the proliferating stage, and also (ii) a high resistance of proliferating cells to oxidative stress-induced CS and cytotoxicity; (iii) despite this, the IPF fibroblasts showed an accelerated entry to replicative CS accompanied by an accumulation of senescent cells with features of myofibroblasts (high expression of α-SMA).in vitro conditions, our findings together with recent works in the field, could shed light on the progressive course of IPF and why aging is a predisposing factor for pulmonary fibrosis. First, although the IPF cells of early passages proliferated at the same rate as normal cells and did not stain for the CS marker SA-β-gal (or the myofibroblast marker α-SMA), they exhibited some morphological features similar to senescent cells such as cell hypertrophy and irregular morphology, as well as resistance to death stimuli. In other words, this could indicate that IPF fibroblasts are predisposed to CS, long before they differentiate into myofibroblasts. The observed changes in cell size and shape could in part be attributed to a low density of actin and its weak co-localization with the cell membrane -induced cell death  , and thihttp://ClinicalTrials.gov Identifier: NCT01687946).As part of the investigative framework of the large collaborative EU project RESOLVE (2), enabling a systematic analysis of 17 different wound healing conditions, 30 patients with proven histopathology of UIP and a clinical diagnosis of IPF underwent combined clinical and biological analysis. This included sequential pulmonary function tests (PFT) and taking of lung biopsies during repeated bronchoscopies. Classification of patients as either limited or advanced UIP was based on the progress of disease in lung function using PFT values currently approved for assessment of IPF progression  . Biopsie2 in mesenchymal growth medium until reaching confluence. Primary cultures of human pulmonary fibroblasts from both IPF patients and normal cells  were grown in standard conditions in Dulbecco's modified Eagles medium (DMEM) . Cells were passaged upon 75-80% confluence and counted. Senescence was induced either by serial passaging (for replicative CS) or by H2O2  treatment for 2 hours at varying concentrations (for stress-induced CS) in FCS-free DMEM. Cell survival and cytotoxicity was measured by neutral red assay.Two transbronchial lung specimens were cut into approximately 2×2×2 mm cubes immediately after removal, and were transferred into a cell culture flask and allowed 5-10 min for adhesion. Cell cultures were incubated at 37°C, 5% COThe SA-β-gal assay was carried out using the Sigma Aldrich SA-β-galactosidase detection kit, according to the manufacturer's protocol. α-SMA was detected as previously described  using on2. DiD (2.85 μM) in serum-free DMEM  was added per well. Cells were incubated for 30 min followed by two washes with PBS containing Ca & Mg, fixated with 4% PFA for 20 min in room temperature, and then washed twice with PBS. Blocking of non-specific binding with 5% BSA for 30 min was performed, and again washed with PBS. Five μl of Phalloidin stock solution was diluted in 200 μl PBS with 1% BSA for each well. One μl of DAPI was added per each well . After 20 min of incubation, wells were mounted with 200 μl PFN. To avoid photobleaching, all the above procedural steps were performed in the dark. Cells were visualized using the Olympus IX81 confocal microscope. For ImageStreamX analysis, the cells were suspended in 1.5-ml Eppendorfs, and the same protocol was used for staining with fluorescent dyes.Twenty thousand cells were seeded in Ibidi 8-well slides in 200 μl of complete medium and incubated for 24 hours at 37 °C and 5% COThe statistical package for the social sciences  software was used for the statistical evaluation of the results. Statistical evaluation was carried out using factorial analysis (ANOVA) to test for differences between the control and the experimental groups. Values of p < 0.05 were considered statistically significant. Statistical analysis of ImageStreamX results was performed with the IDEAS® software  according to their guidelines.


We theoretically and numerically analyze thermal invisibility based on the concept of scattering cancellation and mantle cloaking. We show that a small object can be made completely invisible to heat diffusion waves, by tailoring the heat conductivity of the spherical shell enclosing the object. This means that the thermal scattering from the object is suppressed, and the heat flow outside the object and the cloak made of these spherical shells behaves as if the object is not present. Thermal invisibility may open new vistas in hiding hot spots in infrared thermography, military furtivity, and electronics heating reduction. The realization of electromagnetic invisibility cloaks12378111314152318et al.35363839414243The concept of invisibility has been extended to other realms of physics. Cloaks capable of hiding objects from acoustic waves2324283032In this paper, we propose to use the concept of scattering cancellation to generate the invisibility effect for heat diffusion waves. The peculiarity of our cloak is that, unlike earlier designs, we consider both static and time-harmonic dependence ), it is possible to suppress the dominant multipolar scattering in the quasistatic limit.Following the procedure described in the previous section, with these new boundary conditions, one can show that the In conclusion, we have proposed an original route towards designing thermal cloaks based on the scattering cancellation technique. This technique is inspired by the plasmonic cloaking, which makes use of shells with induced negative polarization to suppress scattered electromagnetic fields. And contrary to invisibility cloaks based on transformation optics, SCT offers simple cloaking designs .One may envision that using this design may further make the thermal cloaking closer to its practical and feasible realization. We believe that such a structured cloak could be manufactured within current technology, having in mind some potential applications in invisibility, sensing and thermography. The range of industrial applications is vast, and our proof of concept should foster research efforts in this emerging area of thermal cloaks and metamaterials.Analytical methods based on scattering Mie theory of spherical thermal scatterers are used to obtain the results presented in M.F. and P.-Y.C conceived the idea of this study. M.F. performed numerical simulations and wrote the manuscript. P.Y.C., H.B., C.A., S.G., and A.A. contributed to the analysis of the results and reviewed the manuscript. S.G. and A.A. supervised the project.


Research in cancer immunology is currently accelerating following a series of cancer immunotherapy breakthroughs during the last 5 years. Various monoclonal antibodies which block the interaction between checkpoint molecules PD-1 on immune cells and PD-L1 on cancer cells have been used to successfully treat non-small cell lung cancer (NSCLC), including some durable responses lasting years. Two drugs, nivolumab and pembrolizumab, are now FDA approved for use in certain patients who have failed or progressed on platinum-based or targeted therapies while agents targeting PD-L1, atezolizumab and durvalumab, are approaching the final stages of clinical testing. Despite impressive treatment outcomes in a subset of patients who receive these immune therapies, many patients with NSCLC fail to respond to anti-PD-1/PD-L1 and the identification of a biomarker to select these patients remains highly sought after. In this review, we discuss the recent clinical trial results of pembrolizumab, nivolumab, and atezolizumab for NSCLC, and the significance of companion diagnostic testing for tumor PD-L1 expression. Non-small cell lung cancer (NSCLC) is by far the leading cause of cancer related mortality in the US and worldwide , 2. It iNSCLC was not traditionally considered to be an immunotherapy responsive tumor type when the earliest clinical trials employed interleukin-2, vaccines, and interferons . More rend line setting) and pembrolizumab . We will also explore the ongoing effort and challenges to identify a PD-L1 assay that can select patients who will benefit from these drugs.The most successful immune checkpoint inhibitors so far are monoclonal antibodies which bind to either PD-1 or PD-L1 and prevent their interaction at the tumor-immune interface. The depth and durability of responses in NSCLC have revolutionized the conceptual approach to lung cancer treatment. Still, fewer than a quarter to half of patients, even in highly selected cohorts, have experienced a clinical benefit while on anti-PD-1 or anti-PD-L1 therapies. In this review, we will describe the recent data leading to the FDA approvals of nivolumab  as of 2015 . All patThree phase II and III clinical trials were initiated to evaluate squamous and non-squamous tumors independently. For squamous NSCLC, a phase II single arm study enrolled 117 heavily pre-treated patients with unresectable disease, the vast majority of whom were prior or current smokers . The bes2 every 3 weeks) in terms of the primary endpoint OS  and ORR . It is important to mention that 34 % of patients had previously received paclitaxel, though this is unlikely to fully account for the differential activities. Consistent with the earlier studies, 9 patients on nivolumab (6.5 %) experienced a delayed response following an initial pseudo-progression. Nivolumab was much better tolerated than docetaxel, with grade 3 or 4 AEs in just 7 % of nivolumab treated patients vs 55 % for docetaxel. The most common immune-related toxicities were hypothyroidism, colitis, pneumonitis, nephritis, and rash, each occurring at a rate of about 4–8 % , and there were no immune-related deaths.CHECKMATE 017 and CHECKMATE 057 were parallel, randomized controlled phase III trials for advanced, platinum-refractory squamous and non-squamous NSCLC, respectively, and are summarized in Table CHECKMATE 057 enrolled exclusively patients with non-squamous NSCLC and patients were randomized to docetaxel or nivolumab . Again, Two clinical trials have studied pembrolizumab in patients with advanced NSCLC. KEYNOTE 001 was a large phase I trial with a NSCLC expansion cohort that enrolled 495 patients who were treated with pembrolizumab 2 mg/kg or 10 mg/kg every 2–3 weeks . The trip < 0.0001; 2 mg/kg: 10.4 months, HR 0.71, p = 0.0008). There was a non-significant trend toward longer survival in the 10 mg/kg cohort compared with the 2 mg/kg cohort. When stratified by PD-L1 positivity, defined as a proportion score (PS) ≥50 % staining of the tumor, the survival benefits were more pronounced , with median survivals of 17.3 and 14.9 months. In the subgroup analysis, both squamous and non-squamous histology favored pembrolizumab treatment, consistent with results from the nivolumab clinical trials. Similarly, EGFR mutated tumors seemed to have no survival advantage with pembrolizumab over docetaxel, though the number of patients (n = 86) was small. Pembrolizumab was more tolerable than docetaxel, with fewer grade 3–5 AEs (14 % vs 35 %) and fewer drug discontinuations (4.7 % vs 10 %). There were six (0.9 %) pembrolizumab-attributed deaths, three due to pneumonitis, two pneumonia, and one myocardial infarction, and five (1.6 %) docetaxel-attributed deaths. Based on the unprecedented survival achieved in the PD-L1+ population, the FDA approved pembrolizumab for second line therapy but only for tumors with a PD-L1 PS ≥50 %.KEYNOTE 010 was a randomized phase III trial analogous to CHECKMATE 017 and 057, comparing pembrolizumab at two doses, 2 mg/kg and 10 mg/kg every 3 weeks, to docetaxel in 1034 patients [n = 25) and 14 % of negative patients (n = 51) had an objective response. In CHECKMATE 057, PD-L1 positivity at ≥5 % strongly correlated with objective response (34 % vs 14 % for PD-L1 negative) as well as predicted an OS benefit compared with docetaxel  [In the multi-tumor phase I trial, a non-commercial anti-PD-L1 murine monoclonal antibody (5H1) was used to measure PD-L1 expression by IHC in tumor biopsies , 18. Eac< 0.001) . Meanwhior intercalating immune cells is counted manually and the PS is reported as a percentage . This automated assay is performed on formalin-fixed, paraffin-embedded (FFPE) tissue using similar conditions for heat-based antigen retrieval and staining as the 28–8 assay. After staining is completed, the percentage of membranous PD-L1 staining of neoplastic In KEYNOTE 001 for NSCLC, it was evident early in the trial that increasing efficacy correlated with PD-L1 positivity by this IHC assay . FollowiAt the time of analysis, both PFS and OS were considerably longer for the group with a PD-L1 PS ≥50 % , while PFS and OS were similar for the groups with a PS ≤1 % or 1–49 % (~10 and 40 % at 1 year). The duration of response, however, was no different between groups, suggesting that even patients with PD-L1 “negative” tumors could attain a durable, meaningful benefit albeit at a much lower frequency than the PD-L1 “positive” tumors.In KEYNOTE 010 the same assay was used with a threshold for PD-L1 high (PS ≥50 %), intermediate (PS 1–49 %), or low (PS ≤1 %) tumors, roughly a third of patients fell into each category and those with PS ≤1 % were excluded from the trial. As noted earlier in this review, patients with a higher PS were much more likely to have an objective response to pembrolizumab (30 %), however responses were still observed in 10 % of those with a PS 1–49 % and the OS subgroup analysis still favored pembrolizumab over docetaxel . Interestingly, this group with intermediate PD-L1 expression did not have a PFS advantage over docetaxel (HR 1.04), a potential indicator that atypical immunologic anti-tumor responses are more common in this subset.Several anti-PD-1  and anti-PD-L1 antibodies  are in various stages of clinical development for NSCLC and other cancers. Like nivolumab and pembrolizumab, these agents are designed to block the interaction of PD-1 with PD-L1 and most have been modified to have no Fc-mediated antibody dependent cellular cytotoxicity.p = 0.04), while safety was similar to other anti-PD-1 agents (11 % with treatment related grade 3 or 4 AEs). Responses lasted a median of 14.3 months (vs 7.2 months for docetaxel), while neither the ORR nor PFS were higher in the atezolizumab arm, confirming that traditional radiographic criteria are imprecise measures of benefit from immunotherapy.A phase II randomized trial (POPLAR) with atezolizumab was recently published. In this trial, 287 patients with previously treated advanced or metastatic NSCLC were randomized 1:1 to docetaxel or atezolizumab (given at a flat dose of 1200 mg IV every 3 weeks) [Importantly, enrollment was stratified by PD-L1 expression using a novel IHC assay  and companion test for pembrolizumab Dako 22C3 pharmDx) are now FDA approved for use in NSCLC, while the complementary test for atezolizumab (Ventana SP142) is approved for urothelial carcinoma. The performance characteristics of the companion tests for atezolizumab and durvalumab (Ventana SP263) are still being assessed in NSCLC , was formed with the task of creating a resource to compare the performances of the four major PD-L1 companion assays. The ultimate goal of this collaboration is to establish cross-platform standards for PD-L1 positivity analogous to those for ER, PR, and HER2 testing. Results of the pilot phase of this “Blueprint project” were presented at the AACR Annual Meeting in 2016, and included 39 lung cases reviewed by 3 pathologists . Tumor cThere are other biological limitations to PD-L1 detection in tumor biopsies. Most traditional cancer biomarkers evaluate fixed elements that do not vary tremendously with time such as gene mutations or proteins directly involved in cellular growth signaling or replication. Examples include estrogen and progesterone receptor expression, HER2 gene amplification, or mutations in EGFR, KRAS, etc. Alternatively, the PD-L1 gene is not typically mutated or amplified and its expression is dynamic both in space and over time in response to a constantly evolving immune response. There is concern that sampling error could result in false negative tests, though some recent case series have suggested a reasonable concordance between both synchronous (same time but different location) and metachronous (different time) specimen in the range of 75–90 % –40. PD-LAlternative biomarker approaches have focused on quantifying and qualifying tumor infiltrating lymphocytes (TILs) or on identifying tumor neoantigens, which are fragments of mutated proteins displayed in the major histocompatibility complexes (MHC) of tumor cells and which are probably critical to the anti-tumor immune response. Several studies now have found that the mutational load, as well as the number of predicted neoantigens , are better predictors of response to checkpoint inhibition than PD-L1 IHC, TILs, or clinical variables –47. WhilThe recent FDA approvals of nivolumab and pembrolizumab represent the earliest phase of a major paradigm shift in treating NSCLC. So far, it appears that both anti-PD-1 antibodies, as well as the therapeutic anti-PD-L1 antibodies in development, will have comparable efficacy and toxicity, with responses in approximately 15–20 % of unselected NSCLC patients and serious autoimmune toxicities in 5–10 % of patients. In the absence of head-to-head to comparisons or clear biological differences between these agents, it is not possible to recommend one treatment over another.Due to the low toxicity of checkpoint inhibitors and the theorized synergy with other treatment modalities , 50, comIn the current clinical realm of FDA-approved immunotherapies, the role of PD-L1 testing is largely prognostic for patients beginning treatment with either nivolumab or pembrolizumab given that none of the assays can conclusively identify non-benefiting patients. In light of the preliminary results from Blueprint, it is not possible to recommend one assay over another. From a practical standpoint some clinicians may choose the 22C3-based assay to have optional access to pembrolizumab in the event of a positive assay. Meanwhile many laboratories have begun reporting both tumor and immune cell positivity analogous to the atezolizumab companion assay by Ventana, however the meaning of these separate scores is still very much uncertain. As more treatment options and combinations become available, measures of immune activation including PD-L1 expression will clearly become more relevant, particularly as the clinician factors the line of therapy and weighs the potential benefit and toxicity of combination immunotherapy in PD-L1 positive versus negative tumors. Specifically, it remains to be shown whether PD-L1 negative patients should be treated concurrently with an anti-CTLA-4 antibody (such as ipilimumab or tremelimumab) as appears to be the evolving strategy for melanoma. It seems PD-L1 expression represents one lens and sensitivity and specificity is likely to be more robust as multiple lenses are used in the analyses.AE, adverse event; FFPE, formalin-fixed, paraffin-embedded; GI, gastrointestinal; HR, hazard ratio; IC, immune cell; IHC, immunohistochemistry; MHC, major histocompatibility complexes; NSCLC, non-small cell lung cancer; OR, odds ratio; ORR, overall response rate; OS, overall survival; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand 1; PFS, progression-free survival; PS, proportion score; RECIST, response evaluation criteria in solid tumors; TC, tumor cell; TILs, tumor infiltrating lymphocytes


Neurotrophin-4 (NT-4) and brain derived neurotrophic factor (BDNF) bind to the same receptor, Ntrk2/TrkB, but play distinct roles in the development of the rodent gustatory system. However, the mechanisms underlying these processes are lacking.in vivo, that single or combined point mutations in major adaptor protein docking sites on TrkB receptor affect specific aspects of the mouse gustatory development, known to be dependent on BDNF or NT-4. In particular, mice with a mutation in the TrkB-SHC docking site had reduced gustatory neuron survival at both early and later stages of development, when survival is dependent on NT-4 and BDNF, respectively. In addition, lingual innervation and taste bud morphology, both BDNF-dependent functions, were altered in these mutants. In contrast, mutation of the TrkB-PLCγ docking site alone did not affect gustatory neuron survival. Moreover, innervation to the tongue was delayed in these mutants and taste receptor expression was altered.Here, we demonstrate, We have genetically dissected pathways activated downstream of the TrkB receptor that are required for specific aspects of the taste system controlled by the two neurotrophins NT-4 and BDNF. In addition, our results indicate that TrkB also regulate the expression of specific taste receptors by distinct signalling pathways. These results advance our knowledge of the biology of the taste system, one of the fundamental sensory systems crucial for an organism to relate to the environment. It is well known that the rodent gustatory system develops under tight control of two neurotrophins, brain derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), preferentially activating their high affinity receptor TrkB ,2. Each Bdnf/Nt4 double null mutants mirrors that of Trkb null mice, it has been suggested that BDNF and NT4 mediate their effects through TrkB [Since the gustatory system phenotype of ugh TrkB -10. Uponugh TrkB ,12.In order to dissect the contributions of the SHC and PLCγ docking sites of TrkB in taste system development, and at the same time to determine which of these sites mediate the effect of BDNF and NT-4 in this system, we have studied mouse lines in which Y515 or Y816 have been mutated either singly or in combination -15. InteTrkb greatly impairs the development of the gustatory system [Bdnf or NT-4, which acts primarily through TrkB receptors, affects distinct aspects of gustatory system development according to their different spatio-temporal expression patterns [SHC, TrkBPLC) or double (TrkBD) point mutations in two specific phosphorylation sites of TrkB [TrkbS/S and Trkb+/+, TrkbP/P, TrkbD/D, and TrkbW/W (see Methods). Neuronal counts at E12.5 revealed a significant loss (~20%) of neurons in TrkbS/S mutants compared with wild-type littermates  revealed them to be phenocopies of Trkb null mutants, showing 80% loss at E12.5 and 90% loss by E14.5 .These results indicate that signalling from the TrkB-SHC docking site largely regulates survival of taste neurons first by transducing NT-4 signalling, followed by the later onset BDNF signalling. Whilst signaling from the TrkB-PLCγ docking site is dispensable for geniculate neuron survival, this site nonetheless supports it, at least to a limited extent, since all TrkB-dependent taste neurons are lost in mice carrying both point mutations  mice revealed that the amount of all neural buds  in TrkbP/P and TrkbD/D animals were similar, and both were significantly reduced compared with controls . These results suggest that, at this stage of development, the TrkB-PLCγ site has a greater influence on the establishment of innervation in the most anterior region of the tongue.Geniculate ganglion lingual afferents innervate and support taste buds in the fungiform papillae that reside in the anterior two thirds of the tongue . This prese ages . We therese ages . Quantifs Figure A,B. Anals Figure A,B, wherTrkbP/P and TrkbD/D point mutants is restored at a later stage, we examined fungiform taste bud innervation at birth (P0). While TrkbD/D animals showed an amount of innervated taste buds comparable to that seen at E16.5, likely reflecting the loss of the geniculate neurons in these mice . Analysis of taste bud innervation in TrkbS/S mutants, as expected from the loss of geniculate neurons in these animals, revealed significantly fewer innervated taste buds both in the anterior two thirds of the tongue and in the tip of the tongue when compared with control littermates . On the other hand, the TrkbS/S mutant mice showed significantly reduced fungiform taste bud innervation compared with control littermates , which are considered to be the anatomical substrate for taste detection. These cells transduce gustatory information upon activation of specific TRs located on their surface and convey taste information to gustatory afferent fibres ,26. TherIn order to determine whether mutation of the SHC or PLCγ docking sites in TrkB would affect any particular TR combination and eventually functionality of the mature taste buds, we examined the lingual expression of different TRs known to transduce different taste modalities in the different point mutant mice. In particular, RNA was extracted from the anterior two thirds of tongues of 2- to 3-month-old mice and subjected to quantitative RT-PCR analysis for GPCRs T1R1 (Tas1r1), T1R2 (Tas1r2), T1R3 (Tas1r3), and T2R5 (Tas2r105), as well as PLC-β2 and TRPM5, which are downstream effector and transduction channels, respectively, of the sweet, umami, and bitter taste pathways. In addition the expression of PKD2L1, a putative sour TR, was quantified.TrkbP/P mutant mice and their respective controls   Figure L. Togeth) Figure L. A simis Figure M. Analyss Figure N. In cons Figure N. Howeves Figure N. These Understanding and advancing the biology of the taste system is undoubtedly important as this system, together with the olfactory system, is fundamental for an organism to relate to the environment. Herein, we have genetically dissected pathways activated downstream of the TrkB receptor that are required for specific aspects of the taste system regulated by the two neurotrophins NT-4 and BDNF. In addition, we have reported that both TrkB docking-sites modulate the expression of specific TRs. These results advance our knowledge in the biology of the taste system in general, and in particular underpin the mechanisms underlying neurotrophin-signalling control of the gustatory system development in rodents.All animal procedures conformed to the UK legislation Animals (Scientific procedures) Act 1986 and to the University of Edinburgh Ethical Review Committee (ERC) policy as well as the University of Oxford ERC policy.TrkbSHC, TrkbPLC, and TrkbD, and the control line, TrkbW, which is a direct control for the knock-in strategy used to generate the TrkbPLC and the TrkbD strains, have been previously described [TrkB signalling point mutant lines, escribed -15. The http://rsb.info.nih.gov/ij/). Total numbers of geniculate ganglion neurons per animal were estimated using previously described methods [Embryos were collected at stages E12.5 and E14.5 using timed matings. Their heads were dissected and fixed in ice-cold 4% paraformaldehyde (PFA) and left at 4°C overnight. Tissues were then dehydrated by incubation in a saline solution of increasing alcohol concentrations, cleared in xylene and embedded in paraffin. P4 pups were transcardially perfused with 4% PFA. Their heads were post-fixed in the same solution at 4°C for 3 days, decalcified in 5% formic acid in PBS, dehydrated, and embedded in paraffin. Serial transversal sections of either 7 μm (E12.5 and E14.5 tissues) or 8 μm (P4 tissues) were mounted on SuperFrost Plus Slides (VWR) and stained using hematoxylin and eosin. Bright-field images of the whole geniculate ganglion were taken from each section with an Axio Scope (Carl Zeiss) microscope using 40×/0.75 NA plan-apochromat objective, and analysed using ImageJ software  for 30 minutes at room temperature. Sections were then washed 2 × 15 minutes in 0.1 M PB and blocked overnight at 4°C in blocking solution  and the next day they were incubated in primary antibody solution (200 μL per slide). Serial tongue sections spaced 100 μm, were co-immunostained using the following antibodies: anti-β III tubulin  and either anti-P2X3  for E16.5 tissues, or anti-cytokeratin 8  for P0 samples. Slides were covered with parafilm and left at 4°C for 5 days. Following primary antibody staining, the sections were washed 4 × 15 minutes in 0.1 M PB and incubated in secondary antibody overnight at 4°C. The conjugated secondary antibodies used were Alexa-488 anti-mouse  and Alexa-555 anti-rabbit  for E16.5 samples; or FITC anti-rat  and Alexa-555 anti-mouse  for P0 samples. Sections were washed with 0.1 M PB, and mounted in Vectashield. Tissues were imaged with a Zeiss LSM710 Meta confocal microscope. For E16.5 and P0 tissues, a plan-apochromat 20×/0.8 M27 objective was used, and the whole section was imaged with a 3 × 7 tile scan and 4-μm Z-steps. This imaged the whole section of the tongue, so that the innervation into the neural/taste buds could be analyzed. Adult tissues were imaged with a plan-apochromat 10×/0.45 objective and 8-μm Z-steps through a 4 × 6 tile scan.Embryos at E16.5, pups at birth (P0), and adult mice (3 months old) (n = 3 in each case) were perfused with ice-cold 4% PFA. Dissected tongues were post-fixed in 4% PFA at 4°C overnight, then washed in PBS and placed in 30% sucrose at 4°C overnight. Tissues were then embedded in OCT mounting medium (VWR) and serially sectioned in the sagittal plane at 50 μm. Sections were washed 4 × 15 minutes in 0.1 M phosphate buffer (pH 7.4) (PB), followed by an antigen retrieval procedure for adult sections. In this case, each slide was subjected to 10 minutes of 200 μL of proteinase K in 0.1 M PB at 20 μg/mL. This was followed by a wash in 0.1 M PB 2 × 10 minutes and washed in quenching solution . cDNA synthesis was performed using Superscript VILO cDNA synthesis kit (Invitrogen) as per the manufacturer’s instructions. Quantitative PCR was performed using UPL assays (Roche) and the CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad). Gene specific primers were designed using the Universal ProbeLibrary System Assay Design Centre (Roche) and their sequences were as follows: Tas1r1/T1R1 (Forward (For) 5’-actgctgcttcgagtgcat-3’ , Reverse (Rev) 5-acaaggctggcaggtgtg-3’), Tas1r2/T1R2 , Tas1r3/T1R3 , Tas2r105/T2R5 , Plcb2 , Trpm5 , and Pkd2l1 . The threshold cycle of Ct value was calculated using the CFX Manager™ software (Bio-Rad). Ct values (threshold cycle) of each gene were normalized to that of Tbp (ΔCt), Actb, or Cytokeratin-8 in the same sample, as indicated in Figure t-tests or two-way ANOVA followed by Fisher’s Protected Least Significant Difference post-hoc tests. For qRT-PCR analysis, unpaired Student’s t-tests were used. Differences were considered significant when P <0.05.Sample sizes were based on pilot experiments. Statistical analysis was performed using StatView and GraphPad Prism software. Mean values were compared using two-tailed Student’s +/+: Genotype denoting homozygous wild-type sequences encoding for TrkB; TrkbS/S: Genotype denoting a homozygous point mutation in the genomic sequence encoding for Y515 (the SHC docking site) of TrkB; TrkbW/W: Genotype denoting a homozygous insertion of wild-type cDNA sequence encoding for Y816 of TrkB; TrkbP/P: Genotype denoting a homozygous point mutation in inserted cDNA sequence encoding for Y816 (the PLCγ docking site) of TrkB; TrkbD/D: Genotype denoting homozygous point mutations in inserted cDNA sequence encoding for Y515 (the SHC docking site) and for Y816 (the PLCγ docking site) of TrkB; TROMA-I: Antibody recognizing Cytokeratin-8; TRP: Transient receptor potential; TRPM5: Transient receptor potential cation channel, subfamily M, member 5; TUJ1: Antibody recognizing neuron-specific class III beta-tubulin; Y: Tyrosine.BDNF: Brain-derived neurotrophic factor; E: Embryonic day; GPCR: G protein-coupled receptor; NT-4: Neurotrophin-4; P: Postnatal day; P2X3: P2X purinoreceptor 3; PKD2L1: Polycystic kidney disease 2-like protein 1; PLC-β2: Phospholipase Cβ2; PLCγ: Phospholipase Cγ; SHC: Sh2-domain containing protein; T1R1: Taste receptor, type 1, member 1; Tbp: TATA binding protein; TR: Taste receptor; TRC: Taste receptor cell; TrkB: Tropomyosin-receptor kinase B; TrkbThe authors declare that they have no competing interests.JK performed most of the experiments. JMH managed the genetically modified strains. CV performed the RT-PCR data. LM contributed to the experimental plans, provided theoretical input and supervision, and wrote the manuscript. All authors read and approved the final manuscript.


A heat-initiated chemical reaction was developed to functionalize CVD-grown graphene at wafer scale and the reaction was universally extended to carbon nanotubes, and other precursors that could be thermally converted to active radicals. The chemical reaction can occur in absence of oxygen and water vapor when the temperature is above the decomposition temperature of the reactants. The chemical reaction was also found to be substrate-dependent due to surface doping and inhomogeneity. A large-scale graphene pattern was demonstrated by combing with microfluidic technique. This heat-initiated solid-phase chemical reaction provides a facile and environmentally friendly approach to functionalize carbon nanomaterials with various functional groups. Raman and X-ray photoelectron spectroscopy (XPS) measurement showed that the absorbed benzoyl peroxide (BPO) molecules on graphene decomposed above 75 °C to active radicals, which further reacted with directly adhered graphene in nitrogen atmosphere. The chemical reaction is highly sensitive to temperature, oxygen and water vapor. The chemical functionalization could be controlled by heating time and be tuned by substrate via surface doping and inhomogeneity. It is found that this heat-initiated solid-phase chemical reaction could be applied to carbon nanotubes and other molecules that can be thermally converted to active radicals. A large-scale graphene pattern was also demonstrated by combing with microfluidic technique.2 substrate via polymethyl methacrylate (PMMA)-mediated method. Single-layer graphene was identified by micro-Raman spectroscopy equipped with a 532 nm laser line. As shown in 2g vibrational mode of sp2 bonded carbon atoms and is observed at ~1580 cm−1, and the 2D band at ~2670 cm−1 which is a second-order vibration caused by the scattering of phonons at the zone boundarySingle-layer graphene films were grown on 25 μm-thick copper foil using low-pressure CVD. Unless stated, for subsequent characterizations and functionalization, graphene was transferred onto SiO−1 emerged and gradually became the most prominent feature of the Raman spectra. The D-band is originated from the A1g mode breathing vibrations of six-membered sp2 carbon rings, and becomes Raman active after neighboring sp2 carbon atoms are converted to sp3 hybridization−1 significantly weakened, while the G band around 1580 cm−1 was broadened due to the presence of a defect-induced D’ shoulder peak at ~1620 cm–1. These observations strongly suggest that covalent C-C bonds were formed and thus a high degree of structural disorder was generated by the transformation from sp2 to sp3 configuration due to reaction with BPO. Indeed, after 25 min of reaction the Raman spectra looked more like that of graphene oxide or highly disordered carbon-based materials. The Raman signatures of graphene did not change any more after 25 min of reaction. It is probably because BPO molecules adhering directly to graphene were completely decomposed within 25 min, since a second experimental procedure on the functionalized graphene always led to a slight stronger D band  are taken as examples, respectively. −1 and G band weakened a lot, indicating that the SWNT was functionalized by BPO. Note that the functionalization of SWNTs was performed in air atmosphere. It is known that due to the extremely small curvature and hence large strain, SWNTs are not as inert as graphene and hence are expected to react with phenyl radials faster than graphene. The increased reaction rate between phenyl radicals and carbon nanotubes is believed to be higher than that between phenyl and graphene and is speculated to be comparable to that between phenyl radicals and oxygen, leading to a more-likely-occurred functionalization of SWNTs. The above two experiments demonstrate that the heat-initiated chemical reaction is a universal method in chemical functionalization of carbon nanomaterials by various functional groups.2 and hexagonal boron nitride (hBN) followed by functionalization with BPO at 80 °C for 25 min. 2, copper and hBN substrates before BPO functionalization. The peak indicated by asterisk is from hBN. The integrated intensity ratio between 2D band and G band of graphene on SiO2, copper and hBN (I2D/IG) are 3.62, 4.02 and 5.74, respectively. As reported, I2D/IG is an indicator of doping level, where a larger I2D/IG corresponds to a lower doping level2 is the highest.Thirdly, to better utilize the heat-initiated chemical reaction and to tune the chemical functionalization, reactions between BPO and graphene on various substrates were performed. Graphene is a type of materials, in which all atoms are on surface, making their properties to be highly allergic to adsorbed molecules323335362, copper and hBN substrates after BPO functionalization. For stronger peaks, the Raman spectra of graphene after functionalization on copper were collected after transferring the functionalized graphene onto SiO2, which would not change the relative intensities between the peaks. After functionalization a prominent D band appeared on SiO2 substrate, indicating the significant formation of sp3 bonds. On hBN substrate, a mild D band appeared, indicating a slight covalent functionalization. On copper substrate, a very small D band appeared, meaning a sparse formation of sp3 bonds. As shown in 3 bonds could form when phenyl radicals attack graphene. Phenyl radical is a highly oxidative species, whose reactivity increases for increasingly n-doped graphene and is negligible for p-doped graphene2, hBN and copper35402 is the most p-doped, large electron and hole fluctuations2 surface, resulting in the reaction to likely occur. Graphene on hBN is the least p-doped but has small electron-hole fluctuations due to its atomic flatness within large area3 bonds.Finally, graphene pattern can be prepared by combing heat-initiated chemical functionalization and microfluidic technique. Polydimethylsiloxane (PDMS) stamp, which was prepared from compact disk (CD), was gently pressed against graphene sample. BPO solution was led into the tunnels between PDMS stamp and graphene. After drying, graphene was separated from PDMS to obtain BPO pattern on graphene . ChemicaIn conclusion, a heat-initiated chemical reaction was developed to functionalize CVD-grown graphene at wafer scale and the reaction was universally extended to carbon nanotubes, and other precursors that can be thermally converted to active radicals. The chemical reaction could occur in absence of oxygen and water vapor when the temperature is above the decomposition temperature of the reactants. The chemical reaction was also found to be substrate-dependent due to surface doping and inhomogeneity. A large-scale graphene pattern was demonstrated by combing with microfluidic technique. This heat-initiated solid-phase chemical reaction provide a facile and environmentally friendly approach to functionalize carbon nanomaterials with various functional groups.4 and 4.00 sccm H2 at a total pressure of ~100 mTorr. For subsequent characterizations and functionalization, the CVD-grown graphene was transferred onto other substrates as follows. PMMA solution  was spin-coated onto the graphene-covered copper foil at 3000 rpm for 1 min and then baked at 100 °C for 30 min to form a ~250 nm-thick thin film The PMMA film was detached by etching the copper away with 2.0 M FeCl3 aqueous solution and then attached to silicon substrate with 300 nm thick silicon oxide (SiO2) and hBN flakes. Finally, PMMA film was dissolved in hot acetone, leaving the transferred graphene anchored on target substrates.Single-layer graphene was grown on copper foil at temperature of 1000 °C by low-pressure CVD using a mixture of 0.15 sccm CH2 substrate was pressed onto the tape. After peeling off the tape, a few hBN flakes could be left on the SiO2 surface.Thin hBN flakes were prepared by micromechanical cleavage of commercially available hBN crystals . The crystals have an average size of ~45 μm. A tiny amount of hBN powder was placed on a stripe of scotch tape, which was then repeatedly folded onto itself. Subsequently, a SiOThe graphene samples were immersed into 10 mM reactants’ (i.e. BPO and laurel peroxide) acetone solution, and were then taken out drying for chemical reaction. The heat-initiated chemical reaction was carried out in a high-pressure stainless-steel container with the outlet connecting with a pump and the inlet connecting with a nitrogen cylinder. To this end, the graphene samples were placed into the container, followed by sealing with a flange cover. The container was filled with pure nitrogen by repeating pumping and inflating with nitrogen 5 times. The container was then put into a preheated oven to initiate the reaction. Afterwards, the graphene samples were rinsed thoroughly in acetone to remove BPO residues for subsequent characterization.−1. The laser spot size is ~1 μm. The spectral resolution is ~2 cm−1. The functionalized graphene on copper was transferred onto SiO2 substrate for enhanced Raman signals. Atomic force microscope (NT-MDT) in tapping mode was used for height and phase imaging of graphene pattern and SWNTs. XPS spectra were recorded using a Thermo Scientific K-Alpha spectrometer at pressures lower than 5 × 10−9 mBar.Raman spectroscopy (B&W-Tek) with laser excitation energy of 532 nm (2.33 eV) was used to evaluate the graphene quality and the functionalization results. The spectra were recorded with an acquisition time of 1 s by averaging 10 scans over the range 1200–3000 cm2 alcoholic solution, used as the catalyst, was spin-coated onto quartz surface. The growth was performed at 870 °C with a flow of methane (1000 sccm) and hydrogen (140 sccm) for 30 min. SWNTs were then transferred onto a SiO2 substrate with markers . The 0.3 mM CoCl markers  for locaGraphene pattern was fabricated by combining heat-initiated chemical reaction and microfluidic technique. PDMS stamps were prepared by using inexpensive commercially available CD, where the polycarbonate surface was used as masters after peeling off aluminum layer with tweezers. A bubble-free mixture of elastomer and curing agent with the ratio of 5:1 was poured onto the polycarbonate surface and was then baked at 120 °C for 60 min. After peeling off the master, cured PDMS stamp was available for subsequent microfluidic experiment. To this end, PDMS stamp was pressed against the graphene sample, and slowly put into BPO solution with one end of tunnels in the solution and the other end above the solution. After 5 min, the assembly was taken out and was dried overnight. After separating the PDMS stamp, patterned BPO molecules on graphene were obtained for subsequent heat-initiated chemical reaction.How to cite this article: Gao, G. et al. Heat-Initiated Chemical Functionalization of Graphene. Sci. Rep.6, 20034; doi: 10.1038/srep20034 (2016).


Utilizing nanomachining technologies, it is possible to manipulate the heat transport in graphene by introducing different defects. However, due to the difficulty in suspending large-area single-layer graphene (SLG) and limited temperature sensitivity of the present probing methods, the correlation between the defects and thermal conductivity of SLG is still unclear. In this work, we developed a new method for fabricating micro-sized suspended SLG. Subsequently, a focused ion beam (FIB) was used to create nanohole defects in SLG and tune the heat transport. The thermal conductivity of the same SLG before and after FIB radiation was measured using a novel T-type sensor method on site in a dual-beam system. The nanohole defects decreased the thermal conductivity by about 42%. It was found that the smaller width and edge scrolling also had significant restriction on the thermal conductivity of SLG. Based on the calculation results through a lattice dynamics theory, the increase of edge roughness and stronger scattering on long-wavelength acoustic phonons are the main reasons for the reduction in thermal conductivity. This work provides reliable data for understanding the heat transport in a defective SLG membrane, which could help on the future design of graphene-based electrothermal devices. Because of the limited temperature sensitivity of Raman frequency shift, it is rather difficult to accurately measure the difference in the thermal conductivity of graphene caused by nano-scale defects. Alternatively, electrical methods could provide higher measurement accuracy and the temperature sensitivity is independent of the sample’s defect. The thermal conductivities of supported graphene and suspended graphene with different layers have been measured by using a microresistance thermometer910Recently, the Raman thermometry method has been used to measure the thermal conductivity of suspended single-layer graphene (SLG)67in-situ T-type sensor method: (a) complete and flat graphene; (b) graphene with nanoholes induced by a focused ion beam (FIB) radiation; (c) graphene with edge scrolling. The highest λ value of type (a) graphene is found to be about 2300 Wm−1 K−1 near room temperature considering the contact thermal resistance. The Raman spectrum and scanning electron microscope (SEM) image were used to test the quality of fabricated graphene sample. Although no obvious polymeric residues or impurities were found on the suspended graphene, nano-scale residues may still remain after the MEMS process. In the in-situ measurement means that the experiment was performed in an electron beam (EB)/FIB dual-beam system and the data of the same SLG sample were collected uninterruptedly before and after FIB radiation. By comparing these data, the influence of defect on thermal conductivity could be precisely captured. λ values of type (b) and type (c) SLGs are reduced by about 42% and 38%, respectively, compared with the type (a) SLG. The underlying mechanisms are explained using the theory of lattice dynamics. Several phonon scattering mechanisms are discussed in detail.In the present study, we report thermal conductivities of three types of suspended SLGs measured electrically by an 2 gas etching to remove the Si beneath the sensor. The etching depth was about 5 μm. During the etching process, the graphene was fully protected by EB-resist and SiO2 layer on the upper and lower surfaces, formed as a sandwich structure. The Raman spectrum of graphene sample is given in the 2 plasma etching. The O2 plasma caused unavoidable defect to the edges of graphene ribbon, thus a small D-band peak was found afterwards. Similar phenomenon was observed elsewhere in SLG4 C atomsWe developed a new method for suspending SLG ribbon connected to a microsensor. The fabrication details are given in the method section and in-situ measurement procedure, the reduction in thermal conductivity was accurately measured.2 plasma radiation. In order to avoid any possible influence to the measurement accuracy, the experiment was conducted according to the following steps: (1) the T-type sensor with SLG was placed on a Peltier heating/cooling stage in FEI Versa 3D EB/FIB dual-beam system. A four-wire method was used for resistance measurement; (2) the thermal conductance of Au sensor connected with SLG was measured under different temperature and electrical power conditions; (3) EB was turned on to take SEM images of SLG  and left about 40 nm thick SiO2 layer below the suspended Au sensor and SLG. The high-magnification SEM image of the cross-section of film sensor is given in the 2 sandwich structure. The reported interfacial thermal resistance for Au-Ti/graphene/SiO2 is 4 × 10−8 m2 KW−1 As described in the method section, the final step is to remove the protection layers of SLG. We carefully controlled the wet etching time for removing SiOλ values measured by Raman and electrical methods2 layers during the gas etching processes. Defects and polymeric residues come from the last step of removing the protection layers. We optimized the wet etching parameters (time and temperature) to minimize the residues on SLG sample. However, nano-scale polymeric residues may still remain on the graphene surface. We discussed about the quality of graphene based on the Raman spectrum in the λ value extracted from the measured electric field data in ref. −1 K−1 at room temperature, which is at the same level of our data. In this work, the SLG sample was carefully suspended and cleaned after removing the SiO2 layer beneath SLG. The authors also found that the clean samples mostly approached the intrinsic properties of graphene.λ values of the other samples are relatively smaller because of the following reasons: smaller width, edge scrolling and nanohole defect. Comparing with SLG5 and SLG6, SLG2 and SLG3 have smaller widths. λ is decreased by about 15%. SLG4 has smaller width and two nanoholes. λ is decreased by about 36%. SLG1 has smaller width and obvious edge scrolling. λ is decreased by about 43%. More detailed analysis is given in the following sections. Considering the uncertainties of sensor calibration, finite-element analysis, temperature fluctuation of stage and size measurement, the total uncertainty of λ is estimated to be about 10%.Except for SLG5 and SLG6, + ions in a shape of 100 nm line on SLG5 and SLG6. Under the condition of lowest current 1.6 pA and 30 kV accelerating voltage, the exposure dose is estimated to be about 2.3 × 10−3 pA/nm2. The thermal conductance of sensor was measured three times after 1s, 3s FIB radiation and complete cut-off of SLG.Perfect material properties of graphene come from its unique 2D crystal structure. On the other hand, it is possible to manipulate the properties of graphene by designing and changing its 2D structure. EB/FIB dual-beam system provides us a powerful tool for doing such nano-machining on graphene. In the experiment, we focused GaIn the framework of lattice dynamics, the thermal conductivity of SLG can be calculated askB, ωs, τs, q and T are the Boltzmann constant, reduced Planck constant, phonon frequency, relaxation time, wave vector and temperature, respectively. δ = 0.35 nm is the interplanar spacing of graphite. vs = dωs/dq is the group velocity. The subscript s stands for six different phonon polarization branches, including three acoustic branches  and three optical branches . ωs, vs and τs are mode-dependent variables, which are determined for each phonon wave vector and phonon branch. A full phonon dispersion relation from a valence-force field method is used for calculating λ. All six phonon branches have been taken into accountwhere τs is determined by the following equation:The relaxation time τU,s and τB,s are the relaxation times of phonon Umklapp scattering and boundary scattering. These two relaxation times are given aswhere γs, M are the Gruneisen parameter, average phonon velocity and mass of a graphene unit cell, respectively. ωs,max = ωs (qmax) is the maximum cut-off frequency. d is the width of graphene ribbon. p is a specularity parameter describing the roughness at the graphene edges.where λ values of SLGs (six samples and SLG5 after 1 s FIB radiation) and the theoretical calculation results through equation γLA and γTA are chosen to be 1.80 and 0.75, respectively, as recommended in Ref. 23λ of SLG. For Umklapp scattering, the minimum cut-off frequency ωs,min = ωs (qmin) plays an important role in determining λ. The acoustic phonons near the center of 1st Brillouin zone have long life time and high group velocity, contributing significantly to λ. Theoretically speaking, the one-atom thick SLG has no long-wavelength limit, because there is no coupling between the in-plane and cross-plane phonon modesωs,min approaches zero, which leads to an erroneous calculation resultωs,min is about 0.9 THz and 0.6 THz for LA and TA branches, respectively, which is much smaller than the value of 4 THz in bulk graphite2728λ, the following conclusions are obtained:λ when comparing the results of SLGs 5, 6 and SLGs 2, 3. Judging by their SEM images, there are no nanoholes or obvious edge scrolling. The specularity parameter p is between 0.85 and 0.90, indicating that the edge roughness is at the same level. Based on equation τB is longer for wider SLGs, leading to a larger λ.The width has a notable effect on λ by about 42% when comparing the results of SLGs 4, 5-1s FIB and SLG6. It is noted that the width of SLG4 is almost the same as that of SLG5 after 1s FIB radiation. However, λ of SLG4 is higher. This finding indicates that the nanoholes created by FIB induce more limitation on λ than the naturally generated ones. This is reasonable because some additional damage to the lattice structure of graphene could be caused during FIB radiation. λ of SLG6 after 3s FIB radiation is further decreased to 470 Wm−1K−1.The defect of nanoholes decreases λ by about 38% when comparing the results of SLG1 and SLGs 2, 3. These SLGs have similar widths. The specularity parameter p of SLG1 is 0.53, which is much smaller than the value of SLG3, 0.85. Apparently, the scrolled edges are more “rough” and cause stronger phonon scattering. Furthermore, the scrolled graphene is no longer single layer. The interlayer phonon scattering will decrease the thermal conductivity of graphene. It is found that the minimum cut-off frequency ωs,min is slightly higher for SLG1, indicating that more long-wavelength phonons, which are the main contributors to λ, are “cut off” because of the interlayer coupling.The defect of edge scrolling decreases λ. An increase of edge roughness and stronger scattering on long-wavelength acoustic phonons are believed to be the main reasons behind the experimental observations.In summary, we have developed a new method for preparing micro-sized suspended SLG. Thermal conductivities of six suspended SLG samples with different widths and defects were measured using a T-type sensor. FIB was employed to create nanoholes in the suspended SLG. Both edge scrolling and nanoholes have remarkable deterioration effect on 2/Si substrate using a traditional PMMA method. Then the graphene layer was cut into micro ribbons by electron beam (EB) lithography and O2 plasma etching. After that, a pattern of T-type sensor was drawn by EB lithography on 300 nm thick EB-resist layer, which was spin-coated onto the graphene ribbon. 100 nm Au film was deposited by EB physical vapor deposition (PVD) method. After lift-off process in 45 °C ZDMAC solution, the T-type sensor was created and the graphene ribbon was located in the middle of the sensor. Subsequently, 600 nm thick EB-resist layer was spin-coated and patterned by EB lithography on the chip as a protection layer for graphene. After that, the SiO2 layer not covered by EB-resist was removed by 60 s reactive ion etching (RIE). The graphene ribbon was sandwiched between the EB-resist and SiO2 layer. Then the T-type sensor connected with graphene ribbon was suspended from the substrate after 20 s XeF2 gas etching. The etching depth was about 5 μm. Finally, the EB-resist and SiO2 layer were removed by ZDMAC solution and buffered hydrofluoric acid, followed by sufficient DI water rinse. A supercritical point dryer was used to release the device from water to avoid possible damage caused by the surface tension. It is worth noting that we carefully controlled the etching time to minimize the defects and chemical residues on graphene. Please refer to the In order to achieve micro-sized free-standing graphene sample for electrical measurement, we developed a new fabrication method. A SLG grown by chemical vapor deposition (CVD) method was transferred onto SiOHow to cite this article: Wang, H. et al. In-situ measurement of the heat transport in defect-engineered free-standing single-layer graphene. Sci. Rep.6, 21823; doi: 10.1038/srep21823 (2016).


Spin and pseudospin in graphene are known to interact under enhanced spin–orbit interaction giving rise to an in-plane Rashba spin texture. Here we show that Au-intercalated graphene on Fe(110) displays a large (∼230 meV) bandgap with out-of-plane hedgehog-type spin reorientation around the gapped Dirac point. We identify two causes responsible. First, a giant Rashba effect (∼70 meV splitting) away from the Dirac point and, second, the breaking of the six-fold graphene symmetry at the interface. This is demonstrated by a strong one-dimensional anisotropy of the graphene dispersion imposed by the two-fold-symmetric (110) substrate. Surprisingly, the graphene Fermi level is systematically tuned by the Au concentration and can be moved into the bandgap. We conclude that the out-of-plane spin texture is not only of fundamental interest but can be tuned at the Fermi level as a model for electrical gating of spin in a spintronic device. Potential electronic applications of graphene rely on controlling its spin-dependent properties. Here, the authors use spin-resolved photoemission spectroscopy to demonstrate how Au-intercalation produces gapped one-dimensional quasi-freestanding graphene on Fe(110) with tunable Fermi surface spin texture. Graphene is a remarkably promising material for applications in electronics358−5 eV)121415This requires three ingredients—lifting the spin degeneracy, enabling the spin currents and switching spin currents electrically. Ferromagnetic exchange is negligible in carbon and cannot create any substantial lifting of the spin degeneracy at the Fermi surface of graphene. Also the spin–orbit coupling is extremely weak . We choose this system because it is known that graphene grows on Fe(110) with an anisotropic moiré pattern with giant (∼1.5 Å) height corrugationEg) at the Dirac point (Eg∼230 meV). We attribute this behaviour to the reduced in-plane symmetry at the graphene–Au interface since we observe that the graphene also becomes 1D patterned as evidenced by a 1D arrangement of Dirac cone replicas in the band structure. Apparently this cannot occur unless the geometrical structure of the Au layer takes on the two-fold symmetry of the Fe(110) substrate. The bandgap, in turn, can be moved to the Fermi level since in this system the charge doping of the graphene changes with the concentration of the intercalated Au. Furthermore, we measured the photoelectron spin polarization directly. This measurement reveals a giant (ΔOS∼70 meV) spin–orbit splitting of the Dirac cone and a hedgehog-like out-of-plane reorientation of the spin within the gap, in line with recent theoretical predictionsIn addition to rendering the graphene quasi-freestanding after intercalation of Au, the Au arrangement creates a significant bandgap  ref.  as a staπ-band shifted by ∼2 eV to higher binding energy as compared with free graphene (π-band undergoes a shift back toward lower binding energy (ΔEπ∼1.2 eV). This apparent decoupling does not contradict hybridization with Au 5d states which is very pronounced as indicated in Similar to the cases of graphene on Ni(111) and on Co(0001) ref. , graphengraphene . Intercax- and y directions. This, in turn, indicates that intercalation of Au strongly enhances the 1D of the graphene structure. The role of the Au is revealed by the additional hexagon that appears around the graphene spot  and ] aG is inversely proportional to the graphene lattice constant and allows to determine the moiré periodicity dG before and after intercalation of Au. Analysis of a large set of LEED data reveals that after intercalation of Au dG changes by a factor ∼0.7 which gives (27±2) Å for the real-space periodicity of the 1D modulation of the Au-intercalated graphene. The periodicity of the Au-derived pattern can correspondingly be extracted from dAu and gives a value of (18±2) Å.D=2π/Δk=2π/(0.24 Å−1)=26 Å perfectly agrees with the periodicity of the 1D graphene moiré determined from the LEED pattern in et al.The observation that the dimensionality of the Au-intercalated graphene is reduced from 2D to 1D is also confirmed by the observation of characteristic 1D replicas of the Dirac cone in photoemission band mapping. While no replicas are seen in the ARPES dispersion probed along the direction uin zone , clearlyn zone  and positive (k+) wave vectors , we observe an out-of-plane spin polarization, which reverses between upper and lower gapped Dirac cone  Rashba-type spin–orbit splitting due to electronic hybridization with the Au. As a result of interplay between this giant Rashba effect and broken sublattice symmetry of graphene, the gapped Dirac cone reveals a hedgehog out-of-plane reorientation of electron spins. These findings indicate that the intercalation of strongly modulated graphene layers provides versatile possibilities for manipulation of electronic configurations and spin textures of graphene Fermi surfaces.In summary, we have investigated the intercalation of strongly corrugated moiré-type graphene on Fe(110) with Au. We demonstrated that the intercalation of Au decouples graphene from Fe and turns it quasi-freestanding. The intercalated Au forms a 1D superstructure at the interface and makes the quasi-freestanding graphene electronically 1D. Furthermore, the contact to Au introduces a bandgap of ∼230 meV in the Dirac cone. This bandgap can be moved to the Fermi level because the charge doping of graphene depends systematically on the Au concentration. Using spin-resolved photoemission we have shown that the Dirac cone of Au-intercalated graphene/Fe(110) exhibits a giant  substrate was prepared as Fe overlayer on top of W(110). Fe was deposited on perfectly clean W(110) at low deposition rate (0.5 ML min−1) and subsequently annealed at 800 K for 5 min to improve the crystallinity of the Fe film. The amount of deposited Fe was verified by an oscillating quartz microbalance and varied between 20 and 40 MLs for different sample preparations. The W(110) substrate was initially cleaned by repeated cycles of annealing in oxygen  followed by a short flash of the sample up to 2,300 K in ultra-high vacuum environment. Cleanliness of the W substrate was verified by the observation of surface-derived states in photoemission from 4f core level and from the valence bandAll sample preparations were done −6 mbar for 5–10 min. The successful synthesis of graphene depends strongly on the partial hydrocarbon pressure and sample temperature. In the case of insufficient control over these parameters, an Fe surface carbide was forming.Graphene on top of Fe(110) was synthesized by chemical vapour deposition of either ethylene or propyleneFor preparation of intercalated graphene/Fe(110), the Au was deposited on top of graphene and the sample was subsequently annealed at 800 K for 5 min. The amount of deposited Au was controlled by means of a quartz microbalance.Extensive characterizations of the sample by ARPES and LEED at all stages of its preparation are reported in 2 and RGBL-II. Photon energies ranging from 30 to 100 eV and linear (s+p) polarization of light were used. The endstations were equipped with ARPES spectrometers Scienta R8000/R4000. The RGBL-II has a combined detector which comprises 2D channelplate for ARPES and Mott-type spin-detector for spin-resolved measurements. The spin detector is able to acquire both in-plane and out-of-plane components of photoelectron spins and can be operated simultaneously with ARPES acquisition of the band structure.Photoemission experiments were performed at UE112-PGM1, UE112-PGM2, U125/2-SGM beamlines at synchrotron light source BESSY II using endstations ARPES 1T=25 K or at room temperature was used. Base pressure in all experimental chambers was below 4 × 10−10 mbar.Overall resolutions of ARPES measurements were 5 meV (energy) and 0.3° (angular). Resolutions set for spin-resolved ARPES were 80 meV (energy) and 1.5° (angular). For positioning and angular orientation of samples in the ARPES experiment, a cryomanipulator operated either at How to cite this article: Varykhalov, A. et al. Tunable Fermi level and hedgehog spin texture in gapped graphene. Nat. Commun. 6:7610 doi: 10.1038/ncomms8610 (2015).Supplementary Figures 1-9, Supplementary Notes 1-8 and Supplementary References.